Particles 2006 Medical/Biochemical Diagnostic, Pharmaceutical, and Drug Delivery Applications of Particle Technology
13-16 April 2006 Wyndham Resort Hotel, Orlando, Florida
3D Model of an Immunoliposome by Dr. R. Rezka, MDC, Berlin Courtesy of Dr. Reto A. Schwendener 2 TABLE OF CONTENTS
Sponsors 4 International Organizing Committee 5 Exhibitors 6 General Program Schedule 9 Maps of Meeting Spaces 11 Technical Program 12 Abstracts of Oral Program 33 Abstracts of Poster Program 212 Author/Speaker/Presenter Index 273 List of Preregistered Conferees 278 Notes 304
3 Sponsors
Particles Conference
Division of Colloid and Surface Chemistry of the American Chemical Society
European Association of Pharma Biotechnology
American Association of Pharmaceutical Scientists
4 International Organizing Committee
J. M. Asua (Spain) V. Babak (Russia) J. R. Baker (USA) F. Boury (France) D. Burgess (USA) K. Caldwell (Sweden) R. Duncan (UK) M. El-Sayed (USA) N. Garti (Israel) K. Johnston (USA) H. Kawaguchi (Japan) J. H. Kim (Korea) C. M. Lehr (Germany) S. Margel (Israel) H. Moehwald (Germany) B. Moudgil (USA) R. Mueller (Germany) F. Nielloud (France) T. Nilsen (USA) R. Pfeffer (USA) D. Poncelet (France) M. C. Roco (USA) P. Rogueda (UK) J. L. Salager (Venezuela) S. Simoes (Portugal) P. T. Spicer (USA) S. Svenson (USA) J. Texter (USA), General Chair
5 Exhibitors
CPS Instruments, Inc. 3755 Glenn Ridge Road Furlong, PA 18925, USA Telephone +1 215 794 2250 http://www.cpsinstruments.com/
FEI Company 5350 NE Dawson Creek Drive Hillsboro, OR 97124, USA Telephone +1 503 726 7500 Fax +1 503 726 2615 http://www.fei.com/
Horiba Instruments, Inc. 17671 Armstrong Avenue Irvine, CA 91614 Telephone +1 949 250 4811 http://www.horibalab.com/
Inotech Biosystems International, Inc. 15713 Crabbs Branch Way, Suite 110 Rockville, MD 20855, USA Telephone +1 301 670 2850 http://inotechintl.com
6 Liquidia Technologies, Inc. P.O. Box 110085 Research Triangle Park, NC 27709, USA Telephone +1 919 991 0835 Fax +1 919 991 0827 http://www.liquidia.com/
L.U.M. GmbH Rudower Chaussee 29 12489 Berlin, Germany Telephone +49-(0)30-6780 6030 Fax +49-(0)30-6780 6058 http://www.lum-gmbh.com
Microtrac
nd 12501-A 62 Street North Largo, FL 33773, USA Telephone +1 727 507 9770 http://www.microtrac.com
NOF Corporation 75 Aero Camino Blvd., Suite B Santa Barbara, CA 93117, USA Telephone +1 727 846 0866 Fax +1 727 846 0865 http://www.nof.co.jp/english/business/life/index.html
7 Particle Sizing Systems 75 Aero Camino Blvd., Suite B Santa Barbara, CA 93117, USA Telephone +1 727 846 0866 Fax +1 727 846 0865 http://www.pssnicomp.com
Surface Measurement Systems, Ltd. 5 Wharfside, Rosemont Road, Alperton, Middlesex HA0 4PE United Kingdom Telephone +44 (0) 208 795 9400 Fax +44 (0) 208 795 9401 http://www.smsuk.co.uk/index.php
Sympatec GmbH System-Partikel-Technik, Am Pulverhaus 1 D-38678 Clausthal-Zellerfeld Germany Telephone +49 5323 717 0 http://www.sympatec.com/
8
General Program Schedule
Registration – Registration is outside of Citrus Ballroom in Prefunction Area
Saturday, May 13
(1800 – 1930) – Opening Informal Reception & Exhibition (Citrus Ballroom)
Sunday, May 14
(0800 – 0850) - Plenary 1 (Citrus Ballroom)
(0900 – 1230) – Technical Sessions: A1(Magnolia), B1 (Biscayne-Siesta), C1
(Jasmine), D1 (Oleander A), E1 (Oleander B), and F1 (Marathon-Cedar)
(1200 – 1400) – Luncheon/Exhibition (Citrus Ballroom)
(1400 – 1730) – Technical Sessions: A2 (Magnolia), G1 (Jasmine), H1
(Biscayne-Siesta), D2 (Oleander A), I1 (Oleander B), and J1 (Marathon-Cedar)
(1800 – 1930) – Poster Session (P1)/Reception/Exhibition (Citrus Ballroom)
Monday, May 15
(0800 – 0850) - Plenary 2 (Citrus Ballroom)
(0900 – 1230) – Technical Sessions: A3 (Magnolia), K1 (Marathon-Cedar), L1
(Biscayne-Siesta), D3 (Oleander A), E2 (Oleander B), and M1 (Jasmine)
(1230 – 1400) – Luncheon/Exhibition (Citrus Ballroom)
9 (1400 – 1730) – Technical Sessions: A4 (Magnolia), B2 (Biscayne-Siesta), C2
(Jasmine), D4 (Oleander A), I2 (Oleander B), and N1 (Marathon-Cedar)
(1800 – 1930) – Poster Session (P2)/Reception/Exhibition (Citrus Ballroom)
Tuesday, May 16
(0800 – 0850) - Plenary 3 (Citrus Ballroom)
(0900 – 1230) – Technical Sessions: A5 (Magnolia), G2 (Jasmine), O1
(Biscayne-Siesta), D5 (Oleander A), E3 (Oleander B), and J2 (Marathon-Cedar)
(1200 – 1400) – Luncheon/Exhibition (Grand Ballroom D)
(1400 – 1730) – Technical Sessions: A6 (Magnolia), K2 (Marathon-Cedar), H2
(Biscyane-Siesta), D6 (Oleander A), I3 (Oleander B), and M2 (Jasmine)
Conference Ends
10
Maps of Meeting Spaces
Contiguous meeting spaces at Wyndham. Conference activities use the Floral Ballroom on the left, the Citrus Ballroom (center) through the Biscayne-Siesta room in the Key Rooms to the right. Registration is outside of the Citrus Ballroom (center).
The Citrus Ballroom is the venue for all three Plenary Lectures, the luncheons, and the receptions. The Marathon-Cedar and Biscayne-Siesta room to the right are used for some of the technical break-out sessions.
All four parts of he Floral Ballroom are used throughout.
11 Technical Program
Technical Program
Sunday, May 14, 2006, Morning
Plenary 1 (Sunday 0800-0850, Citrus Ballroom) Session Chair: Diane Burgess, University of Connecticut
0800 1. Francis Szoka, Jr.; Targetted bioresponsive nanolipid particles
Session A1 (Sunday 0900-1210, Magnolia) Nanoparticles & Hydrogels Session Chair: Carlos Gamazo, Universidad de Navarra
0900 2. Hans Bäumler; Carrier for nanoparticles - Targeted delivery of water insoluble drugs 0925 3. Giulio Paciotti; Developing tumor targeted controlled release drug vectors on colloidal gold nanoparticles 0950 4. Dan V.Goia; Preparation and characterization of electrostatically stabilized dispersions of gold nanoparticles for medical and biological applications 1005 Break 1025 5. Arianna Friggeri; Low molecular weight hydrogelators as oral drug delivery systems 1040 6. Kazunari Akiyoshi; Nanogel engineering for polymeric drug delivery 1105 7. Michael Giersig; Cell growth and cell manipulation based on 2 and 3 dimensional organization of nanosized materials of different morphology 1130 8. Julie Straub; Porous microparticle technology for imaging and drug delivery 1155 9. Satoshi Seino; Development of magnetic nanocarrier consisting of gold and iron oxide for biomedical applications 1210 End of Session
Session B1 (Sunday, 0900-1200, Biscayne-Siesta) Emulsions Session Chair: Tommy Horozov, University of Hull
0900 10. Irwin Gruverman; Keynote - Optimizing drug delivery - Efficient formulation development and scaleable manufacturing methodology 0930 11. David Driscoll; Lipid injectable emulsions: Pharmacopeial and safety issues 0955 12. Maria-Teresa Celis; Droplet size distribution of liquid-liquid systems from UV-VIS spectra 1010 Break 1030 13. Lynn Gold; Applications of Vitamin E based formulations 1055 14. Takasi Nisisako; Generation of monodisperse double emulsions in a planar microfluidic network 1120 15. Robert Hoerr; Applying nanocomposite coatings on medical devices using electronanospray 1145 16. Csilla Kollar; Silicone based controlled release dressing for accelerated proteolytic debridement 1200 End of Session
12 Technical Program
Session C1 (Sunday, 0900-1230, Jasmine) Controlled Release Session Chair: Nily Dan, Drexel University
0900 17. Heike Bunjes; Supercooled smectic nanoparticles – Development and characterization of a novel drug delivery systems 0925 18. Brian G. Amsden; Sustained and active protein delivery from photo-cross-linked biodegradable elastomer milli-cylinders 0950 19. Katarina Edsman; Pharmceutical application of catanionic complexes - sustained drug release from gels 1015 Break 1030 20. Ponisseril Somasundaran; Modified polyacrylic and polysaccharide nanoparticles for controlled extraction and release 1055 21. Dieter Trau; Enzymatic reactions in microcapsules 1110 22. Sabine Fischer; Bio goes Nano: The TNO Planomers® technology, developed to improve commodity plastics by using nanotechnology, opens new possibilities for biomedical materials like nanocomposites for tissue engineering or hybrid materials for drug delivery 1125 23. Irwin C.Jacobs; Development of formulations and equipment for spray drying of high value active ingredients for the pharmaceutical industry 1150 24. Kenneth K. S.Lau; All-dry encapsulation of fine pharmaceutics for controlled drug release 1205 25. Vince Rotello; Keynote - Drug, protein, and DNA delivery with glutathione-mediated release using gold nanoparticles 1230 End of Session
Session D1 (Sunday, 0900-1200, Oleander A) Diagnostics Session Chair: Todd Krauss, University of Rochester
0900 26. Shlomo Margel; Keynote - Synthesis and characterization of nano and micron-sized particles of narrow size distribution for medical imagin applications 0930 27. Yuzhuo Li; Dynamic NMR characterization of nanoparticles for medical imaging applications 0950 28. Terry Matsunaga; Perfluorocarbon nanobubbles as diagnostic and therapeutic agents 1010 Break 1030 29. Marc Herold; Molecularly imprinted nanoparticles for analytical and preparative separation of amino acid derivatives 1045 30. Reyad Sawafta; Nanomagnetic particles in rapid diagnostics 1105 31. John Forsayeth; Real time visualization of nano-liposomal delivery into primate brain by magnetic resonance imaging 1125 32. Jean-François Berret; Controlled clustering of superparamagnetic nanoparticles using block copolymers : Design of new contrast agents for magnetic resonance imaging 1140 33. Karin Caldwell; Multilayered nanoparticles: Characterization and bioanalytical use 1200 End of Session
Session E1 (Sunday, 0900-1230, Oleander B) …Somes Session Chair: Laurence Navailles, Centre de Recherche Paul Pascal, Bordeaux
13 Technical Program
0900 34. David Thompson; Keynote - Comparative performance of transfection complexes derived from a family of bisvinyl ether cationic lipids 0940 35. Martin Caffrey; Lipidic cubic phases: Applications in membrane protein crystallogenesis and in uptake and delivery 1010 Break 1030 36. Alexander Couzis; Spatially addressable protein arrays based on proteoliposome self- assembly into microwells 1100 37. Daryl Drummond; Use of novel intraliposomal stabilization strategies for forming highly stable and long circulating nanoliposome constructs for camptothecins and vinca alkaloids 1125 38. Jan A.A.M. Kamps; Liposomes targeted to activated endothelial cells selectively affect glomerulonephritis in mice 1140 39. Shinichi Kaneda; TRM-484 steroid included in long circulating cationic lipsosomes actively targeting inflammatory tissues 1205 40. Dan Luo; DNA-based hydrogels, lipsosomes and nanobarcodes 1230 End of Session
Session F1 (Sunday 0900-1230, Marathon-Cedar) Particle Characterization Session Chair: Frank Etzler, Boehringer-Ingelheim Pharmaceuticals
0900 41. P. Stephen Williams; Characterization of magnetic nanoparticles using quadrupole magnetic field-flow fractionation 0930 42. Hélène Dihang ; Colloidal stability of emulsions and nanoparticles in pharmaceutics 0955 43. Frank M. Etzler; Particle size analysis: Compartive results from several techniques 1010 Break 1030 44. A.P. Tinke; Particle size and shape characterization of nano- and submicron suspensions 1100 45. Natasha Starostina; AFM capabilities in characterization of particulate matter from Angstoms to microns 1115 46. Mike Bogan; Femtosecond pulse vacuum-ultraviolet-free electron laser diffraction scattering of polystyrene nanoparticles 1130 47. Chak K. Chan; Understanding the efflorescence of supersaturated droplets using fluorescence spectroscopy 1200 48. Tim B. Van der Wood, Three dimensional characterization of pharmaceutical powders by scanning white light interference microscopy 1215 49. Dietmar Lerche; Potential of microcapsules made from plant pollen for application in chromatography 1230 End of Session
Sunday, May 14, 2006, Afternoon
Session A2 (Sunday, 1400-1730, Magnolia) Nanoparticles & Hydrogels Session Chair: Hans Bäumler, Charité - Universitätsmedizin Berlin
14 Technical Program
1400 50. S. Iwamoto; Novel methods of producing nano- and microsized particles composed of natural polyelectrolytes 1430 51. Andreas Taubert; Peptide and polymer assembly-controlled nanoparticle formation 1455 52. Kent Coulter; Development of nanoengineered platelets 1510 Break 1530 53. Esko Kauppinen; Gas-phase synthesis and coating of multifunctional nano- and microparticles for drug delivery 1555 54. Yuichi Yamasaki; DNA condensation mechanism revealed under fluorescence video-microscopy 1620 55. Andrew Loxley; Novel nanoparticle technologies for cost-effective, targeted, non-systemic delivery of therapeutic agents 1635 56. Tamoaki Hino; Silk microspheres prepared by spray-drying of an aqueous system 1700 57. Igor Sokolov; Synthesis and applications of arrays of silica nanotubes 1715 58. Carlos Gamazo; Use of flagellin and mannosamine nanoparticles as bioadhesive carriers for mucosal vaccination 1730 End of Session
Session G1 (Sunday, 1400-1700, Jasmine) Particle Toxicology Session Chair: Roberta Brayner, Université Paris 7-Denis Diderot
1400 59. Vicki Colvin; Structure-function relationships of nanoparticles and their toxicity - A general introduction to nanomaterials and their uses in medicine 1440 60. Hong Yang; Nanoparticle-induced biological responses: Effect of size, shape and surface chemistry 1510 Break 1530 61. Daniel J. Watts; Phytotoxicity of some manufactured nanoparticles 1600 62. Janos Szebeni; Complement-mediated hypersensitivity reactions to nanoparticulate drugs and solvents systems 1630 63. Natalia Varaksa; Aqueous dispersions of carbon nanotubes for biological testing and applications 1645 64. Roberta Brayner; Toxicological impact studies based on Escherichia coli bacteria in an ultrafine ZnO nanoparticles colloidal medium 1700 End of Session
Session H1 (Sunday, 1400-1725, Biscayne-Siesta) Photodynamic and EM Therapy Session Chair: Ross W. Boyle, University of Hull
1400 65. Hubert van den Bergh; Keynote - Photodynamic therapy in the chorioallantoic membrane model: Combination therapy using asprin enhances photodynamic selective drug delivery and influence of the size of TCPP-loaded nanoparticles on phototoxicity 1430 66. Wei Chen; Nanoparticle self-lighting photodynamic therapy for cancer treatment 1455 67. Min Hu; Time-resolved spectroscopy study of the photothermal properties of gold nanocages 1510 Break 1530 68. Gereon Huettmann; Laser irradiated gold nanoparticles: A new tool for selective mainpulation of cells and biomolecules
15 Technical Program
1555 69. Naoto Oku; Angiogenic vessel-targeted cancer therapy 1620 70. Robert Ivkov ; Development of tumor targeting magnetic nanoparticles for cancer therapy 1645 71. Cornelus F. van Nostrum; Methacrylamide-oligolactates as building blocks for targeted biodegradable polymeric micelles to deliver photosensitizers 1700 72. Leanne B. Josefsen; Porphyrin-nanoparticle sensors for detecting cellular response(s) to oxidative stress 1725 End of Session
Session D2 (Sunday, 1400-1715, Oleander A) Diagnostics Session Chair: Yuzhou Li, Clarkson University
1400 73. Roger Leblanc; Keynote - Quantum dots as immunoassay probes 1430 74. Hedi Mattoussi; Development of FRET-based QD-bioconjugate sensors for detecting enzymatic activity 1455 75. Detlef Müller-Schulte; Giant magnetic quantum dots as individually addressable high sensitive optical bar codes for diagnostics and bioanalytics 1510 Break 1530 76. Yongxian Wang; Synthesis of ultra-stable bare CdS nanocrystals with strong luminescence by hydrothermal synthesis 1545 77. Todd D. Krauss; Colloidal semiconductor quantum dots in selective labeling of pathogenic bacteria 1610 78. Robert M. Tsikhudo; A generic approach to biomolecular functionality on gold and silver nanoparticles 1635 79. David E. Cliffel; Epitope presentation on gold nanoparticles for immunoassay development 1700 80. Volker Mailänder; Surface modification of particles enhances intracellular uptake 1715 End of Session
Session I1 (Sunday, 1400-1710, Oleander B) Micelles, Dendrimers & Conjugates Session Chair: Michael Giersig, CAESAR Research Center, Bonn
1400 81. Vladimir Torchilin; Keynote - Polymeric micelles: Potential as pharmaceutical nanocarriers 1440 82. Rex P. Hjelm; Self-assembly of surfactant aided delivery systems 1510 Break 1530 83. Afsaneh Lavasanifar; Polymeric micelles for tumor targeted delivery of P-glycoprotein inhibitors 1555 84. Christine Allen; Engineering copolymer materials and micelles for targeted delivery of hydrophobic drugs 1620 85. Francois Ravenelle; Nanodelivery using block copolymers: A Propofol solution 1645 86. Kent Jørgensen; Tumor triggered activation and release of drugs from lipid based drug and prodrug nanocarriers 1710 End of Session
16 Technical Program
Session J1 (Sunday, 1400-1730, Marathon-Cedar) Gene Delivery Session Chair: David Thompson, Purdue University
1400 87. Mizuo Maeda; Non-crosslinking aggregation of DNA-carrying nanoparticles for gene diagnosis 1430 88. Jon Preece; Gluatathione degradable and pH modulatable polycation/DNA nanoplexes for gene delivery 1455 89. Rumiana Koynova; Lipid phase control of DNA delivery 1510 Break 1530 90. Diane Burgess; Gene delivery and quantum dot assisted intracellular trafficking 1600 91. Wee Beng Tan; Multifunctional nanoparticles for imaging assisted gene therapy 1630 92. Roberta Carbone; Cluster assembled nanostructured TiO2 film mediates efficient and safe retroviral gene transduction in primary adult human melanocytes for ex-vivo gene therapy 1645 93. Harald Petry; JC virus-like particles: Size dependent in vitro DNA packaging and gene delivery 1700 94. Pauline Li ; Amphiphilic core-shell nanoparticles with poly(ethyleneimine) shells as potential gene delivery carriers 1730 End of Session
Sunday, May 14, 2006, Evening
Poster Session P1 (Sunday, 1800-1930, Citrus Ballroom) Session Chair: John Texter, Eastern Michigan University
General Papers
274. Venisetty R. Kumar; Pharmacokinetics and antileishmanial activity of mannose grafted amphotericin B lipid nanospheres 275. Venisetty R. Kumar; Formulation and quality control of gastroretentive tablets of ranitidine hydrochloride 276. Kevin A. Heitfeld; Smart membranes for flavor delivery 277. Ganesan Alasundaram; Increased osteoblast functions on Ti nanopatterned with peptides 278. Daniel J. Burnett; Correlation of polymer energetics and drug release rates 279. Daniel J. Burnett; Investigating the kinetics of moisture-induced crystallization of amorphous lactose
Nanoparticles & Hydrogels
280. Ginger Denison Rothrock; Scalable fabrication of monodisperse, shape-specific organic nanoparticles for use as delivery vectors 281. Virginia Saez-Martinez; Chemical functionalization of poly(nitrophenyl acrylate-co-methacrylamide) microgels with pH-sensitive groups and a targeting ligand 282. Chul Ho Park; Nanoparticle preparation of ?–lipoic acid susceptible to polymerization 283. Boon Sing Ng; Synthesis, functionalization and transport properties of mesostructured particles
17 Technical Program
284. Anika A. Odukale; Molecularly imprinted hydrogel microspheres for phosphate removal 285. Michihiro Nakamura ; Synthesis and characterization of silica nanoparticles with high fluorescence 286. Nobuyuki Morimoto; Enzymatic synthesis and characterization of nanogels by self-assembly of hydrophobized poly(L-lysine)-amylose conjugates 287. Yoshitake Masuda; Invited Poster - I. Control of crystallization for medical applications: I - Control of nucleation and growth of magnetite particles for cancer heat immunotherapy; II - Spherical particle-assembly by two-solution method for drug delivery system 288. Eun-Jung Kim; Solid lipid nanoparticles to overcome multi-drug resistence in cancer 289. Liisa T. Kuhn; Effect of nano-sizing calcium phowphate/cisplatin coordination complexes on in vitro drug release 290. Jianhua Hu; Biodegradable polymer aggregates prepared by amphiphilic poly(hydroxyethyl aspartamide-co-propyl aspartamide) grafted with (lactide) for drug deliverly
Diagnostics
291. Sudip Nath; Synthesis, characterization and localized surface plasmon resonance study of silver organosol 292. Wee Beng Tan; Water-soluble fluorescent LaF3:Ln3+ nanocrystals for bio-applications 293. Igor Sokolov; Fluorescent silica beads for detection of cervical cancer 294. Jaswinder Sharma; DNA-templated self-assembly of two dimensional and periodical gold nanoparticle arrays 295. Anna Galperin; Synthesis and characterization of polymeric microspheres of narrow size distribution containing iodine for x-ray imaging applications 296. E. Sócrates. T. Egito; Incorporation of magnetic particles in polymer systems of xylan 297. E. Sócrates. T. Egito; Magnetite particle dissolution at gastric pH 298. Detlef Müller-Schulte; Composite thermoresponsive microgel core decorated with magnetic nanoparticles 299. Detlef Müller-Schulte; Magnetic nanoparticles – Polyelectrolyte interaction: A layered approach for biomedical application 300. Arnold J. Kell; Functionalized superparamaganetic nanoparticles as pathogen capture probes 301. Marc Herold; Core-shell nanoparticles with covalently linked TNF for cell signalling investigations 302. Marc Herold; Novel nanoparticles with activated ester surface prepared by emulsion polymerization of polymerizable surfactants Inhalation 303. Janne Raula ; Production of novel multicomponent pharmaceutical nano and microparticles with an aerosol flow reactor method 304. Philippe Rogueda ; Size and concentration monitoring of HFA suspensions 305. David P. Brown, Dispersion of novel inhalation drug powders with nanocrystalline L-leucine coating
Characterization
18 Technical Program
306. Andrew Parker; Quantitative hardness and surface characterization by atomic force microscopy for use in early stage screening of pharmaceutical formulations 307. Ben Lich; Particle size distribution characterization of sub-micron suspensions using electron microscopy 308. Dietmar Lerche ; Methods for characterization of micro- and nano-sized particles and capsules 309. Sarah Hook; Characterizing bicontinuous cubic nanoparticles by cryo field emission scanning electron microscopy 310. Wolfgang Fritzsche; Characterization of metal deposition on nanoparticles at single particle level 311. Lizandra Castro; Adhesion forces between hybrid colloidal particles and concanavalin A 312. Amber Clausi; Investigation of the particle size distribution and zeta potential of aluminum hydroxide adjuvant following freeze drying and freeze thawing
Super Critical Fluid Processing
313. A.R. Sampaio de Sousa; Incorporation of hydrophilic compounds in solid lipid particles using supercritical fluid technology 314. Ana Rita C. Duarte; Acetazolamide controlled release from microparticles prepared by SAS
Particle Toxicology
315. Giovana R. Tofoli; Pharmacological and histological evaluation of hydroxypropyl—cyclodextrin-bupivacaine inclusion complex 316. Tammy Oreskovic; In vitro testing of vascular smooth muscle cells exposed to unmodified multiwalled carbon nanotubes 317. Regine Hedderich; NanoCare - Chances and risks of manomaterials
Monday, May 15, 2006, Morning
Plenary 2 (Monday 0800-0850, Citrus Ballroom) Session Chair: Shlomo Margel, Bar Ilan University
0800 95. Sandy Asher; Particle Array Sensors for Biochemical Diagnostics
Session A3 (Monday, 0900-1210, Magnolia) Nanoparticles & Hydrogels Session Chair: Eugene Goldberg, University of Florida
0900 96. Wolfgang Parak; Characterization and biological application of colloidal nanoparticles 0925 97. Laurie Gower; Biomimetic processing methods for organic-inorganic particles 0950 98. Xinqiao Jia; Hyaluronan-based hydrogel microspheres for vocal fold regeneration 1005 Break 1025 99. Anna Musyanovych; Miniemulsions for biomedical applications
19 Technical Program
1040 100. Gershon Golomb; Targeting monocytes by nano-particulated formulations and vascular healing 1105 101. Ron Boch; Ocular drug delivery using lipid, polymer and suspension formulations 1130 102. Sandy Rosenthal; Imaging cell surface receptors with ligand conjugated nanocrystals 1155 103. Zhengmao Li; Oral insulin delivery with biodegradable and biocompatible nanocapsules 1210 End of Session
Session K1 (Monday, 0900-1230, Marathon-Cedar) Inhalation & Aerosols Session Chair: Hans Leuenberger, Institute of Pharmaceutical Technology, Basel
0900 104. Philippe Rogueda; Keynote - The nanoscale in inhalation delivery 0930 105. Uday Kompella; Particulate systems for pulmonary drug delivery: Safety, sustained release, and efficacy 0955 106. Amy M. Waligorski; An ingredient-specific method for particle size characterization of corticosteroid nasal sprays 1010 Break 1030 107. Mark Bumiller; A study of liquid properties and droplet size distributions of some common nasal decongestant spray formulations 1045 108. James R. Benson; New polymer enables zero-order release of drugs 1110 109. Jim Blanchard; Advances in liquid aerosol delivery systems 1135 110. Robert Cook; Embracing particle engineering for inhalation drug products 1200 111. Jimmy Yun; Design and production of ultrafine active pharmaceutical ingredients under high gravity environment 1230 End of Session
Session L1 (Monday, 0900-1200, Biscayne-Siesta) Cancer Targetting Session Chair: Afsaneh Lavasanifar, University of Alberta
0900 112. X.Y. (Shirley) Wu; Keynote - New polymer-lipid hybrid nanoparticles for enhanced anticancer drug delivery 0940 113. Kathleen F. Pirollo; A versatile tumor targeting nanoadelivery platform for cancer diagnosis and therapy 1010 Break 1030 114. Iris V. Enriquez; DNA mesospheres as drug delivery vehicles for localized cancer chemotherapy 1045 115. P. Low; Ligand-mediated targeting of nanoparticles and low molecular weight drugs to cancer cells and inflammatory immune cells 1110 116. Balaji Panchapakesan; Single wall carbon nanotube nanobomb agents for cancer therapeutics 1135 117. Bob (RJ) Lee, Folate receptor-targeted liposomes and nanoparticles for caner therapy 1200 End of Session
Session D3 (Monday, 0900-1230, Oleander A) Diagnostics
20 Technical Program
Session Chair: Wolfgang Fritzsche, Institute for Physical High Technology, Jena
0900 118. Brigitte von Rechenberg; Keynote - Superparamagnetic iron oxide particles as a drug delivery system for in vivo application in the joint 0930 119. Taeghwan Hyeon; Generalized and large-scale synthesis of monodisperse nanocrystals of ferrites and oxides and their applications as MRI contrast agents and in drug delivery 0955 120. Paul S. Russo ; Silica-polypeptide composite particles 1010 Break 1030 121. Mohammad S. Uddin; Surface functionalized nano-magnetic particles for adsorprtion/desorption of biomolecules 1045 122. Marc Porter, Strategies for the design and readout of ultrahigh density immunodiagnostic platforms 1110 123. Andreas Hütten; Magnetic nanoparticles: Aapplications beyond data storage 1135 124. Shy Chyi Wuang; Functionalization of magnetic nanoparticles with biomolecules 1150 125. Dave Thomas; Particle population classification by micro-flow imaging 1205 126. Nguyen TK Thanh; Development of magnetic nanoparticles for biomedical application 1230 End of Session
Session E2 (Monday, 0900-1200, Oleander B) …Somes Session Chair: Martin Caffrey, University of Limerick
0900 127. Gilbert Grant; Keynote - Liposomal bupivacaine: An ultra-long acting local anesthetic 0940 128. Indranil Nandi; Application of lipid based drug delivery system for water insoluble drugs 1010 Break 1030 129. Kenji Kono; Preparation of thermosensitive liposomes by use of amphiphilic block copolymers and their application to anticancer drug delivery 1100 130. Terrence Scott; PROMAXX microsphere drug delivery technology 1130 131. Laurence Navailles; Dislocation loop mediated smectic melting 1200 End of Session
Session M1 (Monday, 0900-1230, Jasmine) SCF Processing Session Chair: Paolo Caliceti, Universty of Padua
0900 132. Keith Johnston; Particle engineering and formulation for enhanced bioavailability of poorly water soluble drugs 0935 133. Steve Howdle; Particles for drug delivery and medical applications: A supercritical approach 1000 134. Jennifer A. Green; Investigation into particle size variations with process parameters using Nektar supercritical fluid technology 1015 Break 1035 135. Boris Shekunov; New particle technologies for drug delivery
21 Technical Program
1105 136. Ram B. Gupta; Formation of drug nanoparticles and microparticles using supercritical CO2 1135 137. Bob Sievers; Drug delivery applications of particle technology using CAN-BD processing 1205 138. Sung-Joo Hwang; Nanoparticles for enhanced solubility and bioavailability of poorly-water soluble drugs using supercritical fluid technology 1230 End of Session
Monday, May 15, 2006, Afternoon
Session A4 (Monday, 1400-1730, Magnolia) Nanoparticles & Hydrogels Chair: Renato Camata, University of Alabama at Birmingham
1400 139. Paul Braun; Double direct templating of periodically nanostructured ZnS hollow microspheres 1430 140. Takashi Miyata; Signal biomolecule-responsive gels 1455 141. Jonghwi Lee; Preparation of polymer/drug nano- and micro-particles by electrospraying 1510 Break 1530 142. Bruno G. De Geest; Polyelectrolyte microcapsules for biomedical applications 1545 143. James Talton; Oral nanoparticle deivery 1610 144. Eugene Goldberg; Drug-loaded protein and DNA nano-meso-microspheres for non-systemic intratumorial chemotherapy 1635 145. Chang Chun Wang; Preparation of magnetic polymeric microspheres for application in biomedical fields 1700 146. Aravind Chakrapani; Fabrication of biodegradable polymer microdevices for controlled drug delivery systems 1715 End of Session
Session B2 (Monday, 1400-1720, Biscayne-Siesta) Emulsions - Particles from Emulsions Session Chair: David Driscoll, Harvard University
1400 147. Ozgen Ozer; W/O/W multiple emulsions containing nitroimidazole derivatives for vaginal application 1425 148. Tommy Horozov; Mechanisms of emulsion stabilization by colloidal particles 1450 149. Katrin Ebert; Preparation of biodegradable nanoparticles by a membrane based process 1515 Break 1535 150. Hidekazu Yoshizawa; New functions of grafted-polymer layers of core/corona polymer nanospheres in bioseparation 1550 151. Dietmar Lerche; Rapid stability testing and shelf life determination of concentrated emulsions 1615 152. Christophe J. Barbé; Ceramispheres: Controlled release from sol-gel micro and nanoparticles 1630 153. Wei Lu; Double emulsion based synthesis of pegylated nanoparticles for brain delivery
22 Technical Program
1655 154. Yujie Dai ; Colloidal emulsion aphrons: Structure, principles, properties and applications 1720 End of Session
Session C2 (Monday, 1400-1730, Jasmine) Controlled Release Session Chair: Heike Bunjes, Friedrich Schiller University, Jena
1400 155. Michael Pishko; Keynote - Encapsulation of drug nanoparticles in self-assembled macromolecular nanoshells 1430 156. Nily Dan; Effect of drug characteristics on the degradation mechanism and drug release rate from biodegradable polymer matrices 1455 157. Sheng Qi; Investigation into the influence of fatty acid purity on the drug release behavior of taste-masking fatty acid microsphers 1510 Break 1530 158. Jacco Eversdijk; Functional microencapsulation using hybrid materials 1545 159. Hideki Ichikawa; Acrylate-based nanogels as microcapsular membrane components for peptide delivery 1610 160. Detlef Müller-Schulte; Thermosensitive magnetic nano- and microcarriers as contactless controllable drug carriers and tools 1635 161. Peter Markland; Microparticle formulation development - Considerations from bench top to clinical manufacturing 1700 162. Rick Rehrig; Vibrating nozzle processing in industrial microencapsulation 1715 163. Tinneke Van Thienen; On the synthesis and characterization of biodegradable dextran nanogels with tunable degradation properties 1730 End of Session
Session D4 (Monday, 1400-1720, Oleander A) Diagnostics Session Chair, Paul Russo, Louisiana State University
1400 164. Shawn P. Mulvaney; Fluidic force discrimination assays in complex media 1430 165. Geir Fonnum; Magnetic beads used in sample preparation for mass spectrometry 1500 166. Roel Wirix-Speetjens; Magneto-resistive immunosensors: Sensitivity enhancement by active guiding of superparamagnetic particles 1515 Break 1535 167. Wing Cheung Mak; Encapsulated microcrystalline particles as label systems for diagnostics 1550 168. Wolfgang Fritzsche; Chip-based molecular diagnostics using metal nanoparticles 1620 169. Dense F.S. Petri; Hybrid particles as substrates for biotargets and metallic ions 1635 170. Heinrich Haas; Assembly and in-vivo tracking of nanoparticulate carriers for neo-vascular targeting 1650 171. Swadeshmukul Santra; Tuning of luminescence intensity of manganese doped cadmium sulfide quantum dots by controlling surface passivation 1705 172. Matthias Frank; Single-particle bioaerosol mass spectrometry for medical and biochemical diagnostics and analysis 1720 End of Session
23 Technical Program
Session I2 (Monday, 1400-1730, Oleander B) Micelles, Dendrimers & Conjugates Session Chair: Sonke Svenson, Dendritic Nanotechnologies
1400 173. A.T. (Sandy) Florence; Keynote - Dendrimers, dendrons, dendrisomes and dendriplexes as carriers in drug and gene delivery 1440 174. Sonke Svenson; Dendrimers: A platform for custom-designed drug delivery 1510 Break 1530 175. Thor Nilsen; What constitutes a particle? Assembly of 30-500 nm dendritic (fractal) hyperpolymers via sequence recognition enabled polymers 1600 176. Victor Lin; Functionalized mesoporous silica nanoparticles for gene transfer, intracellular controlled release, and biosensor applications 1630 177. Joseph Backer; A standardized procedure for derivatization of nanoparticles with functionally active proteins 1700 178. Tao L. Lowe; Branched nanoparticles for drug delivery across the blood brain barrier 1730 End of Session
Session N1 (Monday, 1400-1700, Marathon-Cedar) Suspensions and Dispersions Session Chair: Mark Bumiller, Malvern Instruments
1400 179. Rainer H. Müller; Keynote - Nanosuspensions for the delivery of poorly soluble drugs - State of art and latest developments 1440 180. Ingrid Fischbach; Nanosuspensions of poorly soluble active ingredients 1510 Break 1530 181. Ingo Leubner; Size control of nanoparticles in continuous crystallizations 1545 182. Hemant Joshi ; Applications of nanotechnology in the pharma industry – An overview 1615 183. Christian Johansson; Electrostatic interacations between lysozyme and poly(acrylic acid) microgel particles 1630 184. Maria-Teresa Celis; Use of particles as emulsion and foam stabilizers 1700 End of Session
Monday, May 15, 2006, Evening
Poster Session P2 (Monday, 1800-1930, Citrus Ballroom) Session Chair: John Texter, Eastern Michigan University
General Papers
320. Shy Chyi Wuang; Surface functionalization of magnetic nanoparticles via surface-initiated atom-transfer radical polymerization (ATRP) 321. Krassimir P. Velikov; Colloidal delivery systems for functional foods 322. Alex I. Smirnov; Template-assisted self-assembly of polymerizable lipid nanotubes 323. Eik Leupold; The uptake mechanism of an APOE-derived peptide depends on its aggregational state
24 Technical Program
324. Daniel J. Burnett; Quantifying low levels of amorphous content using vapor sorption techniques 325. Daniel J. Burnett; Investigating the effects of protein concentration on the glass transition behavior of lyophilized protein-sugar formulations
Nanoparticles & Hydrogels
326. Meike Harms; Surface modified solid liIpid nanoparticles 327. Jessica Hain; Hybrid microgel particles with zinc sulfide inclusions 328. Larken E. Euliss; Exploiting emerging techniques in imprint lithobraphy to make highly uniform, shape-specific drugs, imaging agents, and nano-carriers 329. Nelly Fransén; Development and characterization of interactive mixtures with a fine particulate, mucoadhesive carrier for nasal drug delivery 330. Bruno G. De Geest; Monodisperse microgel synthesis inside microfluidic devices 331. Jerald W. Darlington; Modified nano-bentinite anit-viral activity against the herpes simples virus type 1, rhinovirus type 37, and rotovirus 332. José F. Almeida; Novel crosslinked chitosan and dextran gels as drug delivery systems 333. Christina A. Bauer; Silica nanoparticle formation in confined environments via biomimetic catalysis 334. Jean-Francois Berret; Nanoparticle-polymer complexation electrostatic self-assembly as a route to stable dispersions of hybrid nanocolloids 335. Etienne Duguet; One-step synthesis of spinel iron oxide-based core-corona nanoparticles for in vivo applications
Photodynamic & EM Therapy
336. Ji-Hun Yu; Fabrication and characterization of SiO2 and MgO coated Fe2O3 nanoparticles for hyperthermia applications 337. Sang-Eun Park; Preparation of gold nanocomposites with dielectric magnetic cores for targeted photothermal therapy
Diagnostics
338. Jaswinder Sharma; DNA-templated self-assembly of two dimensional and periodical gold nanoparticle arrays 339. Sofia Martins; Detection of hybridization reactions on microparticles by template dependent extension 340. Roberta Brayner; Formation of hybrid nanoparticle-alginate microcapsules for bio-detection applications 341. Angelika Müller; Magnetic nanoparticle-loaded erythrocytes as magnetic resonance imaging contrast agents 342. Beata Chertok; Magnetic nanoparticles for possible simultaneous MR imaging and drug therapy of brain tumors 343. Katarzyna Derwinska; Hydrogels and nanoparticles in microarrays for food control 344. Maciej Dybiec; Protein microarrays and spatially resolved photoluminescence in bio-conjugated quantum dots-probes for early cancer detection
25 Technical Program
345. Karsten Wegner; Production of biocompatible films by controlled gas-phase synthesis and deposition of nanoparticles 346. Sang Im Park; Synthesis of magnetic nanoparticles by thermal decomposition and biocompatibility
Gene Delivery
347. Tomohiro Asai, Gene silencing of mutant K-ras mediated by polycation liposome 348. Ravil A. Sitdikov, Amperometric immunosensor based on highly dipsersed graphite microparticles for influenza virus detection
Controlled Release
349. Divey Saini; Characterization of REP 2006 antiviral drug and drug delivery system for small animal models 350. Janet R. Wendorf; A practical approach to the use of nanoparticles for vaccine delivery 351. Hulda Shaidi Swai; Evaluation of nanoencapsulation performed by spray drying technique for the delivery of Tuberculosis drugs (ATD) 352. Edith C. Rojas; Double carrier systems as novel drug delivery vehicles 353. Sheng Qi; Investigation into using spray chilled Gelucire 50/13 microspheres as carriers for poorly soluble drugs 354. Sara Ponce; Long-term study of EPO-secreting myoblasts entrapped in microcapsules elaborated with five different commercial alginates 355. Jubo Liu; In vivo fate of the micelle forming block copolymer poly(ethylene glycol-block-caprolactone) in mice following intravenous administration 356. Yongwoo Lee; Trehalose bearing polymeric particles: Sorption induced hydrolytic degradation of pesticides and chemical agents 357. Bum Gil Kim; Biodegradable nanoparticles for vaccine delivery systems 358. Jae-Eun Jung; Effective stabilization of bioactive ingredients for long-terrm storage using W/O/W encapsulation and buffering layer 359. E Sócrates. T. Egito; Development of a new multiparticulate system for colon controlled drug delivery 360. E Sócrates. T. Egito; Sodium diclofenac incorporation into xylan microcapsules for colonic drug delivery 361. Masakazu Furuta; Drug release from nanoparticles made of radiation-crosslinked elastin model polypeptides
...Somes
362. Per Wessman; Melittin-lipid interactions: A comparison between liposomes and polymer-stabilized nanodiscs 363. Guifang Zhang; Nimodipine-nanoliposomes: Preparation, characterization and pharmacokinetics in rat 364. Maria R. Toler; Determining the dispersion behavior of formulated Bulk drug using light diffraction analysis
26 Technical Program
365. Jasna Urbanija; Giant phospholipid vesicles as a possible system for studying interactions of membranes with antibodies
Tuesday, May 16, 2006, Morning
Plenary 3 (Tuesday 0800-0850, Citrus Ballroom) Session Chair: Joe DeSimone, University of North Carolina
0800 185. Heinrich Hofmann; Superparamagnetic particles for diagnostic and Therapeutic applications
Session A5 (Tuesday, 0900-1230, Magnolia) Nanoparticles & Hydrogels Session Chair: Robert Pelton, McMaster University
0900 186. Joe DeSimone; Keynote - Exploiting emerging techniques in imprint lithography to make highly uniform, shape-specific drugs, imaging agents and nano-carriers 0930 187. Jim Lee; Molecular spring assemblies of nanoparticles and nanowires 0955 188. Renato P. Camata; Development of nanoengineered calcium phosphate-based particulates for gene delivery 1010 Break 1030 189. Joseph F. Bringley; Precision assembly of multicomponent nanoparticulate colloids 1045 190. Yiguang Jin; The Self-assembly behavior of the cholesteryl derivatives of acyclovir and he preparation of self-assembled nanoparticles 1100 191. Neal K. Vail ; Targeted nanoparticles for bone therapies 1125 192. Shannon Morrison; Iron nanoparticles as magnetic carriers 1150 193. Stan Farnsworth; Broad-band efficacy determination for discrete silver nanoparticles 1205 194. Cory Berkland; Nanoclusters as a unique drug delivery platform 1230 End of Session
Session G2 (Tuesday, 0900-1200, Jasmine) Particle Toxicology Session Chair: Janos Szebeni, Semmelweis University, Hungarian Academy of Sciences
0900 195. Vicki Stone; Keynote - The cellular and molecular toxicity of low solubility nanoparticles 0940 196. Robert DeLong; Formulation processes and characterization of DNA and siRNA nanoparticles 1010 Break 1030 197. Tridib K. Bhowmick; Physicochemical analysis and effects on yeast cells of a particulate traditional Indian medicine, jasada bhasma 1045 198. Gan-Moog Chow; Physical properties and cytotoxicity of inorganic nanoparticles for biomedical applications 1110 199. Robert Hurt; Green nanotechnology through collaborative research on mechanisms of nanomaterial toxicity 1135 200. James L. McGrath; Nanoparticle interactions with the cytoskeleton
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1200 End of Session
Session O1 (Tuesday, 0900-1145, Biscayne-Siesta) Vaccine Delivery Session Chair: Bill Turnell, Medivas
0900 201. Gary Fujii; Keynote - Nanoparticle based vaccine design: The VesiVax system 0940 202. Thomas Luby; Clinical development of a therapeutic microparticle formulation 1010 Break 1030 203. Ana M. Carmona-Ribeiro; Biomimetic particles for vaccine design 1045 204. Thierry Delair; Design of synthetic colloidal antigen carriers for vaccine purposes 1115 205. Balaji Narasimhan; Novel injectable biomaterials for protein and vaccine delivery 1145 End of Session
Session D5 (Tuesday, 0900-1230, Oleander A) Diagnostics Session Chair: J. Manuel Perez, University of Central Florida
0900 206. Orlin D. Velev; Keynote - Colloidal assembly extended into the biomaterials domain: Composite coatings and membranes from live cells 0930 207. Yi Lu; Genetic control of nanoparticle assembly and its application in colorimetric sensing 0955 208. Robert I. MacCuspie; Development of a multiplex peptide nanotube-based pathogen assay 1010 Break 1030 209. Frank van Veggel; Surface modified lanthanide doped nanoparticles as new robust biolabels 1045 210. Tania Q. Vu ; Nanoparticle quantum dots for targeted neuromodulation 1100 211. Yvon Durant; Targeted and functional nanoparticles for biomedical diagnostics 1125 212. Femke De Theije; Detection of drugs of abuse using a magnetic biosensor 1150 213. K. Bonroy; Sensing substrates modified with gold nanoparticle films 1205 214. Beth Goins; Radiolabeled lipid nanoparticles for imaging and radionuclide therapy 1230 End of Session
Session E3 (Tuesday, 0900-1200, Oleander B) …Somes Session Chair: Ijeoma F. Uchegbu, University of Strathclyde
0900 215. David Needham; Keynote - A new paradigm for local drug delivery: Temperature triggered release liposomes for anti-tumor applications 0940 216. David Sheff; Retrograde delivery of bacterial toxins passes through the recycling endosome of mammalian cells 1010 Break 1030 217. Ramin Darvari; Polyelectrolyte self-assembly to modify surface properties of PROMAXX insulin microspheres 1045 218. Dian R. Arifin; Polymersome encapsulated hemoglobin (PEH): A novel type of oxygen carrier 1100 219. Krzysztof J. Dziewiszek; Liposomal delivery of all-trans-retinoic acid: Challenges and results
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1130 220. Hiroyuki Nakamura; Transferrin-loaded nido-carborane liposomes: Synthesis and intracellular targeting to solid tumors for boron neutron capture therapy 1200 End of Session
Session J2 (Tuesday, 0900-1230, Marathon-Cedar) Gene Delivery Session Chair: Jon Preece, University of Birmingham
0900 221. Larry Brown; Keynote - Nucleic acid microspheres for Type 1 diabetes intervention 0940 222. Sean Sullivan; Development of nanotransfection complexes for systemic treatment of cancer 1010 Break 1030 223. Sarah W. Kamau; Enhancement of the efficiency of non-viral gene delivery by application of pulsed magnetic field 1045 224. David Thompson; Synthesis, characterization & triggered dethreading of cyclodextrin-polyethylene glycol polyrotaxanes bearing ceavable “click” endcaps 1115 225. Sang-Kyung Lee; Targeted neuronal delivery of a single antiviral siRNA protects mice against encephalitis caused by two distinct flaviviruses 1130 226. Chong-Su Cho; Receptor-mediated gene delivery using chitosan derivatives in vitro and in vivo 1200 227. Lou Zumstein; Nanoparticle mediated delivery of tumor suppressor genes 1230 End of Session
Tuesday, May 16, 2006, Morning
Session A6 (Tuesday, 1400-1730, Magnolia) Nanoparticles & Hydrogels Session Chair: Shannon Morrison, Virginia Commonwealth University
1400 228. Robert Pelton ; Glucose sensing microgels 1425 229. Peter Burkhard; Peptide nanoparticles for medical applications: Novel drug targeting, delivery and vaccination strategies 1450 230. James Worden; Preparation of monofunctional gold nanoparticle-dendrimer (pamam) conjugates by covalent approach 1505 Break 1525 231. Omid Veiseh; Chlorotoxin-bound superparamagnetic nanoparticles for brain tumor diagnosis and therapeutics 1550 232. L. Juillerat-Jeanneret; Functionalization of nanopaticles for disease detection and targeted drug delivery 1615 233. Hsien-Hsin Tung; Precipitation of an amorphous pharmaceutical solid 1630 234. Monique Smaïhi ; Semicarbazide functionalized silicate nanoparticles for peptide ligation 1645 235. Reynolds A. Frimpong; Integration of magnetic nanoparticles into temperature responsive hydrogel systems 1700 236. Brij Moudgil; Keynote - Engineered nanoparticulate systems for biomedical applications 1730 End of Session
29 Technical Program
Session K2 (Tuesday, 1400-1725, Marathon-Cedar) Inhalation & Aerosols Session Chair: Philippe Rogueda, AstraZeneca
1400 237. David L. Gardner; Keynote - Factors to consider in developing a dry powder inhaler 1430 238. Andrew Martin; Particle fabrication for aerosol drug delivery 1455 239. Philippe Rogueda; Particle aggregation in therapeutic aerosols 1510 Break 1530 240. Michael Lipp; An overview of Alkermes AIR® dry powder particle formulation technology for pulmonary delivery 1555 241. Matti Murtomaa; Electrostatic measurement methods in pharmaceutical aerosols 1620 242. Niklas Sandler; Powder surface image analysis in screening of functional bulk properties of pharmaceutical particles 1645 243. Jimmy Yun; Novel porous hollow silica nanoparticles for drug/pesticide release control 1700 244. Hans Leuenberger; FDA’S pharmaceutical CGMPS for the 21st century; Do we need a special research initiative in pharmaceutical powder particle technology to comply with the new requirements? 1725 End of Session
Session H2 (Tuesday, 1400-1705, Biscayne-Siesta) Photodynamic and EM Therapy Session Chair: Hubert van den Bergh, Swiss Federale Institute of Technology, Lausanne and Alfred Fahr, University of Jena
1400 245. Alfred Fahr; Liposomal formulations of a photosensizer: Green light for an old substance 1430 246. Gemunu Happawana; Use of a scattering element and direct deionized water cooling in high power semiconductor diode lasers to enhance the beam divergence and performance for use in photodynamic therapy (PDT) 1500 247. Dongling Ma; Developing multifunctional nano-architectures: Luminescent and superparamagnetic 1515 Break 1535 248. Gloria J. Kim; Cancer nanotechnology: Designing multifunctinal nanostructures for targetting tumor cells and vasculatures 1605 249. Sebastien Vasseur; La0.75Sr0.25MnO3-based magnetic nanoparticles as potential heat mediators in oncology 1620 250. Ross W. Boyle; Photosensitiser-macromolecule conjugates for targeted photodynamic therapy 1650 251. Jun Shao; Indocyanine green-loaded nanoparticulate delivery systems for superficial tumor diagnosis and treatment 1705 End of Session
Session D6 (Tuesday, 1400-1730, Oleander A) Diagnostics Session Chair: Kui Yu, Steacie Institute for Molecular Sciences, Ottawa
30 Technical Program
1400 252. J. Manuel Perez; Keynote - Magnetic biosensors in biodiagnostics 1430 253. Xiaohu Gao; Multifunctional quantum dots for molecular imaging and therapeutics 1455 254. Robert Nooney; Plasmon-enhanced fluorescence of dyes attached to polyelectrolyte coated metal nanoparticles 1510 Break 1530 255. Tao Liu; Nano-biosensor for detection of disease 1545 256. Hilde Jans; Biofunctionalization of water-dispersible gold nanoparticles 1600 257. Stephane Legastelois; A new particle-based immunoassay that depends on the quantitative detection of magnetic particles 1625 258. Jin Zhang; Cancer biomarker detection based on photoluminescence of semiconductor quantum dots and SERS of metal nanostructures 1650 259. Dave Thomas; Particle population classification by micro-flow imaging 1705 260. Kui Yu; Development of colloidal semiconductor nanocrystals as bio-imaging probes 1730 End of Session
Session I3 (Tuesday, 1400-1730, Oleander B) Micelles, Dendrimers & Conjugates Session Chair: Tao L. Lowe, Penn State University
1400 261. Ijeoma F. Uchegbu; Keynote - Nanomedicines for efficient drug and gene delivery 1440 262. Bill Turnell; Novel polymer technology for the delivery of biological macromolecules 1510 Break 1530 263. Robert W. Lee; Micellar nanoparticles 1600 264. Martin Woodle; Tissue targeted nanoparticles for systemic delivery of nucleic acid agents enables siRNA therapeutics 1630 265. Chun Li; Poly-L-glutamic acid as a building block for nanomedicine: From theapeutics to multimodality diagnostics 1700 266. Meike Harms; Formation of reverse micellar type versus surface modified solid lipid nanoparticles 1730 End of Session
Session M2 (Tuesday, 1400-1730, Jasmine) SCF Processing Session Chair: Ram B. Gupta, Auburn University
1400 267. Neil Foster; Keynote - Utilization of dense gas technologies for the fabrication and formulation of nano-biomaterials 1435 268. Matt Yates; Microencapsulation through hybrid aqueous/supercritical carbon dioxide processing of polymer colloids 1500 269. Ana Rita C. Duarte; Exploring supercritical fluid technology for the development of controlled drug delivery 1515 Break 1535 270. Katsuto Otake; Liposome formation with supercritical CO2 as an alternative solvent 1600 271. Gerhard Muhrer; Solid dispersions by compressed fluid antisolvent precipitation
31 Technical Program
1630 272. Geert Verreck, The use of compressed carbon dioxide to broaden the applicability of hot stage extrusion for drug delivery 1700 273. Paolo Caliceti, Fabrication of lipid nanoparticles for low molecular weight and biotech drug delivery by gas compressed techniques 1730 End of Session
32 Oral Abstracts
Abstracts of Oral Program
Plenary 1 TARGETTED BIORESPONSIVE NANOLIPID PARTICLES 1 Francis C. Szoka, Departments of Biopharmaceutical Sciences and Pharmaceutical Chemistry, University of California, San Francisco 94143-0446, USA; [email protected]
There is a growing expectation that targeted drug delivery will greatly improve anticancer therapy. This expectation is fueled by the increasing number of FDA approved passively targeted and actively targeted carriers of therapeutic agents. Doxil™, a sterically stabilized liposome that encapsulates doxorubicin, is an early example of a passively targeted nanosystem. Sequus, Inc. the company co-founded in 1981 by Demetri Papahadjopoulos and I developed Doxil™ a sterically stabilized liposome containing doxorubicin. But is there “room at the bottom” for more complex novel bioresponsive nanosystems? In this talk I will discuss various approaches our group have pursued to create and study pH and redox-responsive liposomes (nanolipoparticles), peptides and polymers. The possible advantages and barriers to the commercialization of systems studied by us will be discussed. As an example I’ll use polyamidoamine dendrimers, gene transfer reagents in 1993 and how the development of biodegradable dendrimers by the Frechet group now position them as outstanding candidates to make contributions in others aspects of medicine, from drug design and delivery to imaging and tissue repair. I will describe the special features of dendrimers, not enjoyed by liposomes---small diameter and monodispersity-and how dendrimer architecture and size influences their pharmacokinetic properties. Pharmacokinetics, bioresponsive drug release and biodegradation will be increasingly important as nanosized drug carriers approach clinical trials.
A1 – Invited Paper CARRIER FOR NANOPARTICLES - TARGETED DELIVERY OF 2 WATER-INSOLUBLE DRUGS Hans Bäumler, Steffen Bauer, Myriam Brähler, Andreas Lemke, Jan Möschwitzer, Verena Staedtke, Charité - Universitätsmedizin Berlin, D-10098 Berlin, Germany, [email protected]
Highly efficient drugs for the therapy of cancer or cerebral fungal infections are very often water insoluble. The design of nanoparticles of these drugs is very useful, but it is necessary to solve the problem of targeting as well as of drug availability at the target cells. We developed a method for loading erythrocytes with nanoparticles utilizing them as a potent carrier system characterized by excellent biocompatibility, biodegradability and non-immunogenic properties. As very poorly water soluble drug served Amphotericin B. The loading procedure leads to a final concentration of 4pg AmB per erythrocyte. It was proven that an amount of only 750 AmB-loaded erythrocytes per ml is sufficient to reach an antifungal effect, representing one millionth of the physiological erythrocyte
33 Oral Abstracts
concentration in human blood (4-6*109 per ml). Additionally loading the erythrocytes with magnetite nanoparticles and magnetically focussing them to the desired sites can realize targeting of organs or tissues. Loading erythrocytes simultaneously with both, AmB and magnetite, did neither reduce the AmB concentration per erythrocyte nor its bioactivity. Additionally, magnetite loaded erythrocytes can be visualized by MRI, offering the opportunity for diagnostic monitoring of the therapy. To achieve effectiveness in CNS fungal infections, we incorporated AmB loaded erythrocytes into RES cells. Due to damage of the blood brain barrier (BBB) in infections RES cells cross the BBB in a higher percentage than usual. In-vitro phagocytosis assays using AmB loaded erythrocytes show a significant inhibition of free fungal activity. This effect can be observed in viability assays using flowcytometric analysis and direct cell culture assay. The advantages of our erythrocyte-carrier-system are not limited to AmB. Any other water insoluble drug has potential to be loaded onto erythrocytes thereby exploiting the advantages of our carrier system. Due to their unique qualities erythrocytes can be used as therapeutically drug delivery systems, which cannot just deliver a high dosage of different drugs but also protects them from inactivating effects and minimizes side reactions. The additional diagnostic capacities as MRI contrast media and the possibility to focus on certain tissues make our delivery system one of the most versatile drug carriers.
A1 – Invited Paper DEVELOPING TUMOR TARGETED CONTROLLED RELEASE DRUG 3 VECTORS ON COLLOIDAL GOLD NANOPARTICLE Giulio F. Paciotti1, David Kingston2, Lonnie Myer1, and Lawrence Tamarkin1 1CytImmune Sciences, Inc. Rockville MD, 20850 and 2Department of Chemistry Virginia Polytechnic Institute and State University, Blacksburg, VA, 24061; [email protected] Colloidal gold nanoparticles are being used as a new drug delivery vector to deliver multifaceted drug therapies to solid tumors. We have developed two unique colloidal gold nanoparticle vectors designed to deliver both biologic and chemotherapeutic-based therapies to solid tumors. The first of these vectors, termed CYT-6091, is designed to target the delivery of tumor necrosis factor alpha (TNF) to solid tumors. Briefly CYT-6091 is comprised of a 26 nm colloidal gold nanoparticle that is covalently bound with TNF and a 20 kD form of thiolated PEG (PEG-THIOL). Each component in this multivalent vector performs a function: First the presence of the PEG-THIOL moiety prevents uptake and clearance of the vector by the reticuloendothelial system. Second the colloidal gold nanoparticle not only serves as the platform for assembling the nanoparticle vector but due to its size provides initial access to the tumor milieu by passive extravasation of the leaky tumor vasculature. Third, the particle bound TNF not only serves as the therapeutic but also acts as a tumor targeting ligand. By targeting the delivery of TNF to solid tumors, CYT-6091 improves both the safety and efficacy of TNF treatment. Nevertheless given the heterogeneity of tumor cells present in a given solid tumor and the heterogeneity of solid tumors themselves, it is unlikely that single agent therapies will, regardless of their ability to target the tumor, show efficacy over a broad spectrum of cancers. To address this potential limitation we have developed a second-generation vector, termed CYT-21001, that uses the tumor targeting and
34 Oral Abstracts therapeutic properties of TNF to target the delivery of a chemotherapeutic prodrug. In addition to the TNF and PEG-THIOL moieties, CYT-21001 contains a prodrug form paclitaxel, which can be triggered to form native paclitaxel at the tumor site to induce potent anti-tumor responses in solid tumors which are refractory to TNF. Thus, colloidal gold nanoparticles are the core of a new family of nanomedicines for the treatment of cancer.
A1 – Contributed Paper PRPEARATION AND CHARACTERIZATION OF 4 ELECTROSTATICALLY STABILIZED DISPERSIONS OF GOLD NANOPARTICLES FOR MEDICAL AND BIOLOGICAL APPLICATIONS Dan V. Goia and Daniel Andreescu, Center for Advanced Materials Processing, Clarkson University, Potsdam, NY 13699-5814, USA, [email protected]
The paper describes a convenient, rapid, and reproducible precipitation method to synthesize stable dispersions of uniform gold nanoparticles by reducing aqueous solutions of tetrachloroauric acid with iso-ascorbic acid at ambient temperature. The influence of the experimental conditions on the size of the gold particles and the stability of the final sols, investigated by dynamic light scattering and UV-VIZ spectrophotometry, will be discussed in detail. It was determined that the size of the resulting nanoparticles is affected by the concentration and the pH of gold solution, while the stability of the electrostatically stabilized final sols is strongly dependent on the excess of either reactant in the system, the ionic strength, and the temperature of the precipitation. Since the preparation process does not require the addition of dispersing agents to ensure the stability of the sols, the resulting gold nanoparticles have a clean surface and are, therefore, suitable for a wide range of surface modifications for applications in medicine and biology. It was also found that the addition of only trace amounts of organic molecules or inorganic ions to the sols triggers drastic changes in the position and the shape of the surface plasmon band of the gold particles, suggesting possible uses of these dispersions for sensing applications.
A1 – Contributed Paper LOW MOLECULAR WEIGHT HYDROGELATORS AS ORAL DRUG 5 DELIVERY SYSTEMS Arianna Friggeri, Kjeld J.C. van Bommel, Menno R. de Jong, Gieta van der Schaaf, Erna Bulten, Marijke Haas, Eduard Post, Anita Meter-Arkema, Biomade Technology Foundation, Nijenborgh 4, 9747 AG Groningen, The Netherlands; [email protected]
Low molecular weight gelators (LMWGs) are small organic molecules capable of self-assembling into extended three-dimensional networks by means of hydrogen bonding and other supramolecular interactions. Such networks very often consist of nano-sized fibers, with a well-defined molecular architecture, in which solvent molecules become trapped, leading to the formation of gels. LMWGs possess small and relatively simple
35 Oral Abstracts
structures. This means that they can be easily and uniformly functionalized. Furthermore, LMWG gels can show very rapid response times to stimuli such as temperature, pH, light, chemicals, or combinations of these. Biomade Technology has developed several classes of low molecular weight gelators that can be used for applications in pharmaceuticals formulation, as well as in cosmetics and personal care. These gelators can be used to thicken or gelate a wide variety of solutions, solvent mixtures, and emulsions. Oral administration is the most convenient way to treat patients, but preparing a suitable formulation for oral delivery is often hampered by the poor aqueous solubility of many active substances obtained via modern combinatorial synthesis procedures. Most of the approaches that have been used to overcome insolubility result in clinical problems such as poor and erratic bioavailability. BiOMaDe Technology has discovered that precipitation of poorly water-soluble drugs in the presence of the fibrous network formed by the gelator results in very finely dispersed drug particles that dissolve rapidly in the gastrointestinal tract. In vitro dissolution studies and in vivo bio-availability studies using lipophilic drugs, such as furosemide and itraconazole, formulated with low molecular weight hydrogelators for oral drug delivery will be presented.
A1 – Invited Paper CELL GROWTH AND CELL MANIPULATION BASED ON 2 AND 3 7 DIMENSIONAL ORGANIZATION OF NANOSIZED MATERIALS OF DIFFERENT MORPHOLOGY Michael Giersig, CAESAR Research Center, Ludwig-Erhard-Allee 2, 53175 Bonn, Germany; [email protected]
The fabrication of one-dimensional (1-D) inorganic nanomaterials (including nano-wires, tubes and nanoparticles) with a well-ordered structure has recently become of utmost importance for biomedical applications. A method based on conventional lithography for selective layer-by-layer material deposition is employed to fabricate stable networks having controlled geometry, topography, and composition. This procedure offers unconventional micro- and nanofabrication routes to yield complex, free-standing matrices, resulting in array designs with extremely high reproducibility. The array design consists of periodic compartments made up of intercrossed nanomaterials of different morphology. The creation of 2-D and 3-D scaffolds with regard to the size and morphology of growing cells will be discussed. In the second part, we will present the possibility of cell manipulation using nanomaterials. Our method allows us to deposit organic and inorganic nanomaterials in bacteria, cancer and yeast cells. The principles of this method and some examples of cell modification will also be presented.
36 Oral Abstracts
A1 – Invited Paper POROUS MICROPARTICLE TECHNOLOGY FOR IMAGING AND 8 DRUG DELIVERY Julie Straub, Don Chickering, Howard Bernstein, Acusphere, Inc., 500 Arsenal Street, Watertown MA 02472, USA; [email protected]
Porous particles in the submicron to micron size range have been produced using a patented porous microparticle technology. The technology uses a modified spray drying process that has been validated for aseptic operation. Particle size, porosity and morphology were engineered during the spray drying process using pore forming agents, such as volatile salts. These agents were added to the material to be spray dried and were removed during the drying process. The modified spray drying process was performed using a patented multi-chamber spray dryer. Three product candidates based on the porous microparticle technology have been studied in the clinic. The porous microparticle technology was first applied to the production of AI-700, an intravenously administered ultrasound contrast agent for detection of coronary artery disease. AI-700 consists of internally porous particles, about two microns in diameter, which contain perfluorobutane. The shell material is poly (lactide-co-glycolide). AI-700 is in Phase III clinical trials. The second product candidate studied in the clinic is AI-850, which is a readily dissolving porous microparticle formulation of paclitaxel for intravenous administration. AI-850 has been studied in a Phase I dose escalation clinical trial.The third product candidate studied in the clinic is AI-128, which is a sustained-release, dry powder formulation of a widely used asthma drug that is often administered multiple times daily. AI-128 consists of honeycomb-like microparticles of the asthma drug encapsulated in a slowing dissolving shell material. A Phase I clinical trial with AI-128 has been completed and demonstrated that the asthma drug was released from the microparticles in the lung over a 12 to 24-hour period. In summary, the spray drying process yields particles with average sizes in either the nanoparticle or microparticle size range which are appropriate for pulmonary, oral, and subcutanous administration.
A1 – Contributed Paper DEVELOPMENT OF MAGNETIC NANOCARRIER CONSISTING OF 9 GOLD AND IRON OXIDE FOR BIOMEDICAL APPLICATIONS Satoshi Seino, Takuya Kinoshita, Takashi Nakagawa, Takao A. Yamamoto, Grad. Sch. of Eng., Osaka Univ., 2-1, Yamadaoka, Suita, Osaka, Japan.; Takefumi Doi, Shinsaku Nakagawa, Grad. Sch. of Pharm. Sci., Osaka Univ., 1-6, Yamadaoka, Suita, Osaka, Japan.; Masakazu Furuta, Grad. Sch. of Sci., Osaka Pref. Univ., 1-2, Gakuencho, Sakai, Osaka, Japan.; and Noriyuki Ohnishi, Magnabeat Inc, 5-1, Goikaigan, Ichihara, Chiba, Japan.; [email protected]
We are developing a new technique for the preparation composite nanoparticles consisting of magnetic iron oxide and gold in an aqueous solution system using radiochemical or sonochemical process. Iron oxide nanoparticles were dispersed in an aqueous solution
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containing Au ions together with some alcohols and polymers. The solution was irradiated with gamma-ray, electron beam or ultrasonic wave to reduce Au ions. After the irradiation, small Au nanoparticles were immobilized on the surface of iron oxide nanoparticles. Some composite nanoparticles synthesized under appropriate conditions showed good dispersibility in an aqueous solution, and substantially no sedimentation occurs even after 50 days. The composite nanoparticle carries functions of the gold particle and maneuverability by a magnetic field. As the gold part firmly combines with molecules possessing mercapto groups, the composite nanoparticles can be easily modified with appropriate molecules for each purpose. We have already confirmed that the Au part in the composite nanoparticles can be combined with sulfur containing molecules such as amino acids (cystine and methionine), and thiol-modified oligonucleotide. We have demonstrated that a target oligonucleotide was easily and specifically picked up from an aqueous solution containing target and nontarget oligonucleotides by magnetically attracting these nanoparticles to which a probe oligonucleotide immobilized. The amount of separated target oligonucleotide depended on the size of Au grains in the composite nanoparticles. We found that 0.75 nmol oligonucleotide was picked up by 1-mg particles, and about 10 minute was enough for the hybridization process. The present results indicate that the composite nanoparticle is expected as a new magnetic nanocarrier for various biotechnological applications.
B1 – Keynote Paper OPTIMIZING DRUG DELIVERY – EFFICIENT FORMULATION 10 DEVELOPMENT AND SCALEABLE MANUFACTURING METHODOLOGY Irwin Gruverman, P.E., MFIC Corp.(Microfluidics Div.), 30 Ossipee Rd, Newton, MA 02464, USA; [email protected]
Formulation science enables delivery and performance of active pharmaceutical ingredients (API’s). In medicine, coatings, foods and other disciplines requiring normally unstable mixtures (dispersions, emulsions) formulation optimization is necessary. The quality of emulsions and dispersions is key to successful formulation, microemulsions, uniform nanosize structures are most stable, if few outlier sized structures exist to serve as nucleation sites for phase condensation. Surfactants and other additives are often needed. Minimum additive formulations are usually desired and enabled by appropriate high energy processing. Drug delivery by any modality requires control of particle or droplet size, to control penetration, retention and pharmacokinetics. Two major factors govern performance – formulation and process equipment to control energy input. Development is most efficient if composition and process trials are interactively developed. Thus, there is a need to perform many sequential and parallel studies as composition and process conditions are altered. Microfluidizer® Processors have enabled optimal formulation development for more than 20 years. The lab version is designed to clean rapidly and can perform dozens of trials in a day. Results obtained at 10 – 100 ml/min will scale linearly to 60 liters/min in production processors. Unique fixed geometry, constant shear conditions, and reproducible process pressure (up to 40000 psi) maximize and control energy deposition. Shear levels are an order of magnitude greater than in other process equipment,
38 Oral Abstracts such as conventional homogenizers and rotor/stator mixers. Fixed geometry operation assures minimal deviation in product characteristics. This talk will highlight 20 years of evolution of Microfluidizer processor systems to current equipment. It will address many application examples in biopharmaceutical formulation development and manufacturing, in pharmaceutical processing, and in protein harvesting from cell suspensions. Practical feedback from the more than 3000 processors in the field, as well as data developed in Microfluidics’ three applications laboratories (Boston, Los Angeles and Frankfurt, Germany) will be presented. Based on this considerable formulation applications database, we modestly conclude that modern formulation science is optimized by using Microfluidizer Processor technology and experience. Finally, a brief view of new technology based on Microfluidizer Processors, will be given. This is a new approach to continuous chemical reaction systems, which could revolutionize the manufacture of chemicals, including many API’s. It also allows easy solubilization by controlled recrystallization of insoluble refractory API’s which are potent drugs but which have resisted useful formulation development.
B1 – Invited Paper LIPID INJECTABLE EMULSIONS: PHARMACOPEIAL AND SAFETY 11 ISSUES David F. Driscoll, Harvard Medical School, Baker Building Suite 605, 185 Pilgrim Road, Boston, MA, USA; [email protected]
Lipid injectable emulsions intended for nutrition support of patients incapable of assimilating nutrients via the gastrointestinal tract have been administered for over 40 years. Despite this record of clinical use, there are no official pharmacopeial standards worldwide regarding its stability and subsequent safety in humans. Moreover, drugs have also been added to these emulsions such as the anesthetic agent, propofol. With a average droplet size of approximately 300 nm, these formulations are considered “mini-emulsions”, require energy to be formed and are thermodynamically unstable. Consequently, they have short shelf lives. Instability manifests as a growing population of large-diameter fat globules as a result of coalescence. As this population gets larger, the risk of embolic phenomena increases upon intravenous infusion. Embolism of the pulmonary, hepatic or cerebral microvasculature can prove to be fatal, especially in the face of critical illness. Recently, the United States Pharmacopeia has proposed Chapter <729> entitled “Globule Size Limits in Lipid Injectable Emulsions”. The not-to-exceed limits proposed universally apply to 10, 20 and 30%w/v oil-in-water nutritional emulsions, and include an intensity-weighted mean droplet diameter of < 500 nm, and a large-diameter fat globule limit expressed as a volume-weighted percentage of fat > 5-um or PFAT5 of < 0.05%. The rationale for these limits and associated methods of analysis, as well as bench and animal research in support of Chapter <729>, will be described.
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B1 – Contributed Paper DROPLET SIZE DISTRIBUTION OF LIQUID-LIQUID SYSTEMS FROM 12 UV-VIS SPECTRA Maria-Teresa Celis*, Luis H. Garcia-Rubio**, *Laboratory FIRP and Laboratory of Polymers and Colloids (POLYCOL) -Chemical Engineering Department, University of the Andes- Mérida Venezuela; **Chemical Engineering Department – University of South Florida- Tampa , FL, USA; [email protected]
The stirring of a surfactant/water/oil system result in the generation of droplet populations determined primarily by the nature and amount of emulsifier, mixing characteristics and emulsion preparation. The estimation of the droplet size and droplet size distribution is important data, not only because they are linked with the manufacturer process, but also they give valuable information on the properties of the dispersed phase. This research demonstrates the potential of spectroscopy as a tool for characterization of liquid –liquid systems in terms of droplet size distribution as a function of the oil concentration and of physicochemical variables. The opportunity of obtaining information from a single multiwalength measured makes UV-Vis spectroscopy a powerful tool for characterization of dispersed systems.
B1 – Invited Paper APPLICATIONS OF VITAMIN E BASED FORMULATIONS 13 Lynn Gold, and Alex Tustian, Sonus Pharmaceuticals Inc, 22026 20th Avenue SE, Bothell, WA 98021, USA; [email protected]
Vitamin E has been shown to facillate solubilization of various lipophilic drugs. Various parmeters have been optimized to produce drug emulsions which are suitable for evaluation as injectable drug products. In each case, nanodroplet (60-80 nanometer ) formulations have been achieved with 10 mg/mL drug loading. The goal of the nanodroplet is to localize in the tumor taking advantage of the leaky vasculature. These formulations are suitable for a variety of lipophilic drugs including taxanes and camptothecins. In the case of taxanes, the tolerability of an unmodified taxane has been improved, the drug loading has been increased, the volume injected has been reduced and the infusion time has been reduced with a vitamin E emulsion versus the marketed cremophor formulation. The final drug formulation is manufacturable under GMP specifications at a commercially viable scale. Early data on the campothecin emulsion shows similar benefits.
B1 – Invited Paper GENERATION OF MONODISPERSE DOUBLE EMULSIONS IN A 14 PLANAR MICROFLUIDIC NETWORK Takasi Nisisako, Toru Torii, Department of Precision Engineering, Graduate School of Engineering, the University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
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Tokyo, Japan; [email protected]
Double emulsions have attracted considerable attention because of their potential applications in foods, cosmetics, and pharmaceutics. Conventionally, two-stage vigorous mixing is employed in practical formulation of double emulsions, but resultant products have broad size distribution. Here, we present a planar microfluidic platform having hydrophobic and hydrophilic components for preparing monodisperse double emulsions. Monodisperse double emulsions can be formed by a two-step method of droplet breakup in a series of T-junctions. For a water-in-oil-in-water (W/O/W) emulsion, for example, to-be-enclosed aqueous drops of uniform sizes are periodically formed upstream at the hydrophobic T-junction; then, in a continuing series, monodisperse organic droplets, that encase the aqueous droplets, are formed downstream at the hydrophilic T-junction. Microchannels were fabricated on glass plates by conventional lithographic techniques. For aqueous droplet formation, channel surface was partially modified to be hydrophobic by using a water-soluble siliconizing agent. Surfactants were added to intermediating and external phases to stabilize the prepared multiple-phase dispersions. In the production of W/O/W emulsions, for example, each of the monodisperse aqueous drops (diameter = 52 μm, CV = 2.7%) could be encapsulated in an organic droplet (diameter = 83 μm, CV=2.8%). The size of the droplets and the number of internal droplets could be varied by changing the flow conditions. Both W/O/W and oil-in-water-oil (O/W/O) emulsions were prepared by reversing the order of hydrophobic and hydrophilic components. Also, double emulsions, including internal droplets of two different compositions were prepared using a cross-junction as the upstream component. Monodisperse polymeric microcapsules could also be engineered by curing intermediating organic phase in W/O/W droplets. We believe that the method we developed has many applications in various fields, such as, analysis of confined chemical reactions, biological screening, drug delivery systems and materials science, etc.
B1 – Invited Paper APPLYING NANOCOMPOSITE COATINGS ON MEDICAL 15 DEVICES USING ELECTRONANOSPRAY Robert A. Hoerr, Nanocopoeia, Inc., 1246 W. University Ave., St. Paul, MN 55104, USA; [email protected].
The drug-eluting coronary stent—first introduced in the United States in 2003—has transformed interventional cardiology by producing significantly improved clinical outcomes compared with earlier techniques that used balloon angioplasty or bare metal stents to restore blood flow through blocked coronary arteries. The market for drug-eluting stents is expected to exceed $6 billion in 2006, making this perhaps the fastest market introduction in medical device history. Despite this success, the current products represent only the first wave of development, and there is much opportunity remaining for both process and therapeutic advances. Nanocopoeia uses its novel, proprietary ElectroNanospray™ process to apply nanocomposite coatings onto coronary stents and other implantable medical devices. The ElectroNanospray™ process has several unique features and benefits. The cone-jet mode of spray operation produces highly charged,
41 Oral Abstracts uniform, monodisperse nanoparticles comprised of one or more components. Coating of the target is non-line-of-sight and directed by the established electrical field, which aids in the uniform coating of pieces with intricate architecture. The proprietary spray head enables the application of multiple agents in a nanocomposite matrix. The process accommodates a range of polymers, solvents, and drugs typical of those that are appropriate for coating stents and other implantable devices. Using these materials, coatings are applied as nanoparticles comprised of drug or drug and polymer in a solvent carrier. The resulting finishes of these coatings range from a highly preserved nanoparticulate architecture to a smooth surface. Characterization of these coatings by a variety of techniques, including cryomicrotomy, SEM and FTIR microscopy, demonstrates a highly uniform composite matrix that is stable as the stent is balloon-expanded.
B1 – Contributed Paper SILICONE BASED CONTROLLED RELEASE DRESSING FOR 16 ACCELERATED PROTEOLYTIC DEBRIDEMENT Kurt F. Bradstadt, 1 James W. Crissman, 1 Paal C. Klykken, 1 Csilla Kollar, 1 Thomas H. Lane, 1 Xavier Thomas, 1 Gregory S. Whitaker, 1 Richard Bott, 2 Grant Ganshaw, 2 Mark Gebert, 2 Isabelle Mazeaud, 2 Mae Saldajeno, 2 Lillian B. Nanney, 3 Jeffrey M. Davidson, 3,4 Dow Corning Corporation,1 Midland, MI; Genencor International,2 Palo Alto, CA; Vanderbilt School of Medicine 3 and VA TVHS Medical Center, 4 Nashville, TN; [email protected]
Unique solutions for rapid enzymatic debridement were developed by the combination of silicone materials that have demonstrated utility in wound care applications with the performance of a genetically engineered, broad spectrum protease. Silicone emulsion technology enables hydrophilic enzymes to be stabilized with hydrophobic silicone materials. The enzyme is a serine protease of the subtilisin family, which has not been tested previously as a debriding agent. The dried emulsion incorporates a protease in a dehydrated state that shows excellent performance in accelerated stability tests, with retention of ~90% of the initial activity for an equivalent of 18 months’ storage at room temperature. The combination was tested in a porcine burn model. The silicone delivery device acted synergistically with the enzyme to promote rapid debridement in the moist wound environment. Tissue fluids from the wound triggered reproducible and complete release of enzyme from the emulsion within eight hours. There was essentially complete removal of hardened eschar from full thickness burns after a single 24-hour treatment. Rapid debridement by the test material resulted in considerable exudate, which was easily managed with an absorbent dressing layer. There was a clear dose-response with various enzyme concentrations. Compared to controls and the benchmark material, debridement and progression to the healing phase were faster, and tissues unaffected by thermal injury appeared to remain normal. This technology, with the core silicone attributes of oxygen permeability, moisture retention, and friendly skin adherence properties, can serve as the basis for a number of drug delivery and wound care applications.
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C1 – Invited Paper SUPERCOOLED SMECTIC NANOPARTICLES - DEVELOPMENT AND 17 CHARACTERIZATION OF A NOVEL DRUG DELIVERY SYSTEM Heike Bunjes, Judith Kuntsche, Markus Drechsler, Friedrich Schiller University Jena, Institute of Pharmacy, Department of Pharmaceutical Technology, Lessingstr. 8, 07743 Jena, Germany, and Michel H.J. Koch, EMBL, Hamburg Outstation c/o DESY, Notkestr. 8, 22603 Hamburg, Germany; [email protected]
As an approach to improve the drug incorporation capacity of lipid nanoparticles with a nonliquid core, nanoparticles in the thermotropic liquid crystalline smectic state have been developed. These nanoparticles are based on cholesterol esters like, e.g., cholesteryl myristate. In the bulk, the materials of interest are crystalline at room temperature. When dispersed into the colloidal state, e.g., by melt-homogenization with physiologically acceptable emulsifiers the smectic state can, however, be retained for pharmaceutically relevant periods of time at room and body temperature in optimized systems. Using cholesteryl myristate as model matrix material, the stability against recrystallization of nanoparticles prepared with different surfactants was investigated. As a general trend, nanoparticles stabilized with surfactants containing fatty acid chains (e.g., phospholipids, fatty acids, sucrose ester) seem to be less stable against recrystallization than nanoparticles stabilized with typical polymeric surfactants (e.g., poloxamers, polyvinyl alcohol). This may result from different crystallization behaviors observed upon cooling in DSC experiments: In contrast to stabilization with polymeric surfactants, emulsifiers with fatty acid chains lead to multiple crystallization events. Phospholipid-containing dispersions display a usually bimodal crystallization event the course of which depends on the particle size distribution and seems to be correlated with ultrastructural parameters of the dispersion (ocurrence of cholesteryl myristate nanoparticles with different shapes). For these dispersions, a strong particle size dependence of the recrystallization tendency upon storage was observed. The stablity of the smectic state against recrystallization is improved when esters with a lower crystallization tendency like cholesteryl oleate or cholesteryl nonanoate are admixed to the cholesteryl myristate matrix. Cholesteryl nonanoate can also be used as the only matrix component to prepare stable smectic nanoparticles. First drug loading investigations on cholesteryl myristate nanoparticles revealed that the dispersions can stably incorporate model substances without negative influence on the matrix state and colloidal stability of the nanoparticles.
C1 – Invited Paper SUSTAINED AND ACTIVE PROTEIN DELIVERY FROM 18 PHOTO-CROSS-LINKED BIODEGRADABLE ELASTOMER MILLI-CYLINDERS Brian Amsden, Department of Chemical Engineering, Queen’s University, Kingston, Ontario, Canada K7L 3N6; [email protected]
Although the potential and advantages of implantable devices for localized, prolonged
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protein therapeutic delivery have long been recognized, there are currently very few formulations for this purpose currently in the clinic. This is a result of the difficulty in maintaining the activity of the protein within the delivery device prior to release. In an attempt to overcome this problem, we have investigated the potential of osmotic pressure induced rupturing of polymer systems for protein release. In these devices, the protein is distributed as solid particles within a rubbery polymer matrix. As it is desirable to have a biodegradable implant, a biodegradable elastomer was utilized that was cross-linked via irradiation with UV light. The cross-linking thus occurs at ambient temperature and does not denature the protein. To facilitate implantation, the device were prepared in milli-cylindrical form. The in vitro release kinetics of three therapeutically relevant proteins, interferon-gamma, interleukin-2, and vascular endothelial growth factor were measured and demonstrated to be constant and similar. Moreover, the bioactivity of the released proteins remained high during the release period. The release rate and duration was readily manipulated by varying formulation parameters such as the mechanical properties of the rubbery polymer used, and the amount and nature of excipients co-lyophilized with the proteins.
C1 – Invited Paper PHARMACEUTICAL APPLICATION OF CATANIONIC COMPLEXES – 19 SUSTAINED DRUG RELEASE FROM GELS Katarina Edsman, Uppsala University, Dept. of Pharmacy, Uppsala, Sweden; [email protected]
Gel formulations have many applications in different routes of administration, for example for ocular, cutaneous, nasal and oral use. Where an aqueous solution gives a short contact time to the mucosa a gel can prolong this period of time because of its rheological and mucoadhesive properties, making an enhanced absorption of the drug substance possible. However, a gel usually contains more than 90 % water, allowing the drug molecule to move almost as freely as through pure water. To fully benefit from the prolonged contact times the release of the drug must be sustained. One possibility of sustaining the release is to make the drug interact with or be part of a slower diffusing species. Recently the possibility of using catanionic aggregates with an active drug molecule was investigated for prolonging the release of the drug. Catanionic aggregates are formed spontaneously in aqueous mixtures of a large selection of cationic and anionic surfactants. At low concentrations of the catanionic components vesicles and micelles are formed, accompanied by two-phase regions and precipitates. The micelles formed in a catanionic mixture range from simple spherical ones, via rod-like micelles, to branched micellar aggregates. The properties of traditional catanionic mixtures are relatively well known. Lately we have shown that not only traditional surfactants form these mixtures, but also structurally more complex surface active drug compounds. We have investigated at what extent mixtures of drug substances and oppositely charged surfactants form catanionic aggregates and studied the release from gels using vesicle and micelle phases. The results show that catanionic aggregates seem to form frequently in these kinds of mixtures. The drug release studies show that catanionic drug surfactant mixtures is beneficial for obtaining prolonged release from gels, as the drug release using catanionic vesicles and
44 Oral Abstracts micelles was prolonged between 10 and 100 times compared to the release of pure drug substance from the gel. The mechanism suggested for this prolonged release is that the vesicles or micelles are larger than the pores of the gel, rendering them unable to move through the gel as large aggregates, whereas free molecules or smaller aggregates will diffuse through the gel.
C1 – Invited Paper MODIFIED POLYACRYLIC AND POLYSACCHARIDE 20 NANOPARTICLES FOR CONTROLLED EXTRACTION AND RELEASE P. Somasundaran and Somil Mehta, NSF Industry/University Cooperative Center in Novel Surfactants, Columbia University, New York, NY10025, USA; [email protected]
Polymeric nanoparticles are reservoirs which can encapsulate active substances in order to isolate them from the surrounding environment and then to release them as required. Nanoparticles can be used for preparation of formulations containing drugs, vitamins, steroids, proteins, enzymes, flavors and fragrances. Such nanohybrids are mainly based on crosslinked polymers. High pressure liquid chromatography (HPLC) and ultraviolet spectroscopy studies showed that the polymeric nanoparticles after modification with charged and hydrophobic groups show higher extraction of attributes such as drugs and fragrances. The release was observed to be pH dependent and thus offered a means of controlling the release of active substances in body. The potential of these nanoparticles for extraction of various active substances were investigated using Surface Plasmon Resonance (SPR) technique. The technique here is based on electromagnetic waves decaying evanescently at a substrate/solution interface. The evanescent behavior of the waves make this technique surface specific and sensitive to long and short changes in refractive index of the layer next to the substrate and hence to adsorption/desorption (extraction/release) of molecules in the surface layer. The release rates of drug and fragrance were thus established based on changes in the refractive index of tightly bound layers of nanohybrids. It was observed that the release can be controlled by varying properties such as cross linking density, temperature sensitivity and swelling/ shrinking capacity. The shape and size of the nanohybrids offer a good opportunity to produce a family of nanocarriers in the personal care industry.
C1 – Contributed Paper ENZYMATIC REACTIONS IN MICROCAPSULES 21 Dieter Trau1, Wing Cheung Mak2, Cheung Kwan Yee2, 1, 2 Division of Bioengineering and 1Department of Chemical & Biomolecular Engineering, National University of Singapore (NUS), Singapore; [email protected]
Here we report on the use of the Layer-by-Layer (LbL) technology to create microcapsules filled with different enzymes or mixture of enzymes and substrates and their use as a microreaction compartment to perform biochemical reactions. With our technology of “Matrix Assisted LbL-Encapsulation” we are able to encapsulate virtually any enzyme,
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mixtures of enzymes or complex mixtures of enzymes and substrates into LbL-capsules. Capsule permeability is controllable by the number of LbL-layers to control diffusion of substrates into the capsule and entrapment of polymeric products of enzymatic reactions. Encapsulation efficiency for proteins, enzymes and oligonucleotides was studied. Capsules with ~10 LbL-Layers (PAH/PSS) are extraordinary heat stable and successful PCR reactions within microcapsules could be achieved. The use of microcapsules as reaction compartment has several advantages: Large number of individual reactions in parallel; small reaction volumes; small reagent/substrate consumption; high throughput. Our novel encapsulation technology, encapsulation efficiency, stability, PCR reactions in microcapsules and various applications will be presented.
C1 – Contributed Paper BIO GOES NANO OR NANO GOES BIO 22 Sabine Fischer, Lawrence Batenburg, Renz van Ee, Jacco Eversdijk, Klaas Timmer, TNO Science and Industry, Innovative Materials, Eindhoven, The Netherlands; [email protected]
TNO as an institute for applied science is working for more than 10 years in the field of nanotechnology, especially in the preparation of hybrid nano-composites. Our group has developed a new technology – the so-called Planomers® Technology - that enables the incorporation of inorganic nanoparticles into the most existing polymer matrices on a real nano-scale. By applying this technology to commodity and engineering plastic an enormous improvement of properties like mechanics, barrier or flame retardancy can be achieved. During the last years our research activities became more and more focused on the application of these nanotechnological principles to natural polymers (biopolymers), like polysaccharides and proteins to develop new biodegradable packaging materials and controlled release systems for agriculture and building industry. On the other hand, our knowledge on nano-composites in general has lead to new applications in the field of food, pharmacy and biomedical materials. The TNO Planomers® Technology is not only improving the properties of plastics but can also lead to healthier food by incorporating inorganic (nano)minerals, to improved drugs by using nanocomposite materials as drug carriers or to the 3D-processing (Rapid manufacturing) of nanocomposite materials for biodegradable and biocompatible scaffolds for tissue engineering. The research results of the nanotechnology for hybrid biological system and its practical application in food, pharmacy and medical industries will be presented and discussed in detail.
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C1 – Invited Paper DEVELOPMENT OF FORMULATIONS AND EQUIPMENT FOR THE 23 SPRAY DRYING OF HIGH VALUE ACTIVE INGREDIENTS FOR THE PHARMACEUTICAL INDUSTRY Irwin C. Jacobs, Robert E. Sparks, John Cooper; Particle and Coating Technologies, Inc. 5445 Highland Park Drive, St. Louis, MO, 63110, USA; [email protected]
An apparatus was assembled which was designed to spray dry gram to sub-gram quantities of active pharmaceutical ingredients in solution for formulation development. The idea was that existing commercial equipment produced product at very low yields and added thermal stress with severe limitations on achievable particle size. With such small quantities atomized, some consideration had to be given to simplicity and thoroughness of cleaning without excessive effort. Lastly, the equipment should be capable of atomization of molten feeds (spray chilling) and atomization to achieve a fairly uniform particle size distribution to reduce heat stressing normally required in drying the larger particles in an atomized distribution. The apparatus was assembled with necessary controls and temperature monitoring. It has been found to produce yields of approximately 85 to 90% based on collected product vs. atomized solids and at a size distribution consistently of about d90/d10 of 2.2. The atomization of feed parameters can be adjusted to produce particles ranging from approximately 5 microns to 100 microns. It is capable of spray chilling. The equipment has been used for the spray drying of active ingredients in solution with various polymers to prepare amorphous matrices for preparation of enhanced bioavailability formulations. Also, atomization conditions can be varied to produce smooth or porous surface morphologies. Solvents including water, acetone, methylene chloride and even glacial acetic acid have been used. Proteins and live bacteria have been spray dried with good retention of activity or viability. Hard to dry sugars have been dried successfully and free-flowing product achieved. The technique has also been used more recently to achieve high dilutions of extremely active pharmaceutical ingredients to assure one of homogeneous dilution of the formulation. Lastly, the ultrasonic atomizer has been adapted with a concentric tube for production of true core/shell morphologies by this spray drying technique.
C1 – Contributed Paper ALL-DRY ENCAPSULATION OF FINE PHARMACEUTICS FOR 24 CONTROLLED DRUG RELEASE Kenneth K. S. Lau and Karen K. Gleason, Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA; [email protected]
Current methods for encapsulating pharmaceutical powders are prone to particle agglomeration as a result of strong binding forces from liquid bridges which form during drying of the polymer solution that is applied to the particles. This condition becomes increasingly severe as particle dimensions go below 100 µm. To circumvent this limitation,
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we present an all-dry encapsulation technology, initiated chemical vapor deposition (iCVD), that enables fine pharmaceutical powders well below 100 µm in size to be coated with controlled release polymer formulations. iCVD enables polymers with stoichiometric compositions to be made in a CVD environment, unlike plasma CVD, which tends to lead to undesired crosslinking and loss of chemical functionality. iCVD combines polymerization and coating in a single efficient step, achieving individual particle layering. We present results on methacrylic acid copolymer systems for modifying the release of ibuprofen microcrystals down to 25 µm in size. FTIR and XPS reveal the composition of these polymers, demonstrating the tunability of copolymer ratios. SEM show the morphology and conformality of the coatings, demonstrating coating without particle agglomeration. Controlled release studies evidence delayed release based on pH conditions, demonstrating enteric release of the core material. iCVD kinetic modeling reveals a surface-driven polymerization that is similar to bulk phase free radical polymerization. The iCVD process is believed to involve the delivery of monomer and initiator vapors into the coating reactor; thermal activation of the initiator to create primary radicals; diffusion and adsorption of the primary radicals and monomer onto the surface; and finally surface polymerization via initiation, propagation and termination to yield a polymer encapsulation layer on the surface.
C1 – Keynote Paper DRUG, PROTEIN, AND DNA DELIVERY WITH 25 GLUTATHIONE-MEDIATED RELEASE USING GOLD NANOPARTICLES Vincent M. Rotello, Department of Chemistry, University of Massachuetts, Amherst, MA 01003 USA; [email protected]
Monolayer-protected gold nanoparticles provide effective vectors for the delivery of drugs and biomolecules. The ability to attach targeting functionality presents a means of delivering these carriers to target organs and tissues. Additionally, the capability of tuning surface properties provides systems that are rapidly internalized by cells, with intracellular targeting possible through the use of appropriate functionality. In addition to their ability to be functionalized in modular fashion, gold nanoparticles provide a tunable method for payload release. Glutathione (GSH) is present in low micromolar concentrations extracellularly. Intracellular GSH concentrations, however, range from 1-10 millimolar. This dramatic increase in GSH concentration within the cell provides a novel means for release: GSH can displace thiol functionality from the particle surface. This displacement process can be used directly to release drugs and prodrugs. Moreover, the addition of anionic GSH can be used to change the surface potential of cationic particles, providing an effective means for release of electrostatically-bound DNA and proteins. All of these release processes are tunable via control of the monolayer structure, with release rate correlating with monolayer length..
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D1 – Keynote Paper SYNTHESIS AND CHARACTERIZATION OF NANO AND 26 MICRON-SIZED PARTICLES OF NARROW SIZE DISTRIBUTION FOR MEDICAL IMAGING APPLICATIONS S. Margel, T. Lublin-ennenbaum, A. Galperin, B. Perlstein, O. Ziv and M. Tsubery, Bar-Ilan University, Ramat-Gan 52,900, Israel; [email protected]
Polystyrene (PS) microspheres of narrow size distribution were prepared by dispersion polymerization. These microspheres were then coated with silica and/or magnetite/silica thin films. Uniforms hollow silica and hollow silica/magnetite micron-sized particles were prepared by burning off the silica/PS and silica/magnetite/PS composite particles, respectively. The potential use of these air-filled hollow particles for ultrasound imaging have been demonstrated. Maghemite nanoparticles (10-100 nm) of narrow size distribution were formed by nucleation and the growth of thin maghemite films onto desired nuclei. The surface of the maghemite nanoparticles was then coated with dextran, followed by crosslinking with divinyl sulfon. The residual double bonds of the divinyl sulfon have been used for covalent binding of various bioactive reagents. The potential in vitro and in vivo use of the non-conjugated and conjugated maghemite nanoparticles for MRI imaging have been demonstrated. Polymeric nano and micron-sized particles containing high loading of iodine were formed by emulsion and dispersion polymerization, respectively of acrylate monomers containing iodine. The potential in vitro and in vivo use of these particles for x-ray imaging has also been demonstrated.
D1 – Invited Paper DYNAMIC NMR CHARACTERIZATION OF NANOPARTICLES FOR 27 MEDICAL IMAGING APPLICATIONS Yuzhuo Li and Fadwa Odeh, Center for advanced Materials Processing, Department of Chemistry, Clarkson University, Potsdam, NY 13699, USA; [email protected]
Particle size is crucially important in determining the usefulness of a nanoparticle system for medical imaging applications. Until now, the particle size of a liposome or lipid nanoparticle systemis typically determined using a light scattering method. The method often suffers from interferences from air bubbles, dusts, and unrelated particles. Although the NMR pulsed field gradient (PFG) experiments are known to yield self-diffusion coefficient for micelles and other particulate matters, the application of this technique to vesicle and lipid systems has been limited due to the rigid nature of the lipid bilayer which usually does not give well-defined NMR signals for PFG experiments. This study shows that the polyethylene oxide chains on a pegylated lipid could serve as an excellent tracer to measure the self-diffusion coefficient via PFG method. In addition, liposomes or lipid nanoparticles containing a hydrotrope also give adequate signal from the lipid for PFG experiments due to the increased flexibility of the lipid molecules. In addition to particle size, PFG-NMR is also used to determine the extent of association of a hydrotrope with the liposome bilayer.
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D1 – Invited Paper PERFLUOROCARBON NANOBUBBLES AS DIAGNOSTIC AND 28 THERAPEUTIC AGENTS Terry Matsunaga, Evan Unger, ImaRx Therapeutics, Inc., 1635 East 18th St., Tucson, Arizona 85719, USA; [email protected]
Perfluorocarbon gas-water interfaces are exquisitely efficient at reflecting ultrasound waves. As such, they can act as excellent vascular contrast agents. Currently, there are two bubble products that are commercially available as echocardiographic contrast agents. Moreover, in the presence of clinical ultrasound frequencies on the order of 1 – 10 MHz, gas bubbles ranging in size 5 μm or less, are subject to oscillations and cavitation. This cavitational effect can be used as a therapeutic modality to aid in the dispersion of vascular thrombi. We refer to this as SonoLysis® Therapy. We have combined diagnostic and low mechanical index therapeutic ultrasound to both detect regions of thrombotic occlusion and to affect recanalization of those vessels. Work has been performed to demonstrate the physical phenomenom of bubble cavitation as well as preclinical studies to confirm thrombus dispersion. Pre-clinical studies have focused upon the efficacy of Sonolysis in a series of applications. In addition, we will provide insight into the potential of SonoLysis therapy for the treatment of vascular occlusions.
D1 – Contributed Paper MOLECULARLY IMPRINTED NANPARTICLECLES FOR 29 ANALYTICAL AND PREPARATIVE SEPARATION OF AMINO ACID DERIVATIVES Marc Herold, C. Gruber, M. Herold, A. Weber, M. Dettling, S. Sezgin, Günter E. M. Tovar, H. Brunner, Institute for Interfacial Engineering (IGVT), University of Stuttgart & Fraunhofer Institute for Interfacial Engineering and Biotechnology (IGB), Nobelstr. 12, D-70569 Stuttgart, Germany; [email protected]
Molecular imprinting is a template polymerization which produces artificial binding sites in polymers. Their specific cavities can be used for molecular recognition reactions mimicking the antibody-antigen interaction, but now with a completely synthetic system. Nowadays particulate MIP’s with a defined morphology and of colloidal size open new applications for these artificial receptor material. In a new approach, we synthesized MIP’s as nanoscopic spheres. A variety of highly crosslinked copolymer networks, e.g. poly(methacrylic acid)-co-(ethylene glycol dimethacrylate) nanoparticles were prepared by miniemulsion polymerization in presence of a molecular template (e.g. amino acid derivatives L- or D-boc-phenylalaninanilid) with a yield of (98 ± 2) %. Coagulate-free and stable latexes were obtained. The prepared polymer colloids are in the size range of 50 – 300 nm. The efficiency of the imprinting process was quantified by ligand binding experiments using UV/Vis and HPLC. The nanoscopic molecularly imprinted polymers (MIP’s) allowed for the first time for an examination of the non-covalent interaction
50 Oral Abstracts between the synthetic receptor and various ligands by microcalorimetry. The interfacial preparation method introduced here now opens the way to imprinting more complex templates such as peptides or proteins. The preparation and the use of the colloidal MIP’s, e.g. as the surface coating of an optical waveguide sensor, and as a selective layer in a composite membrane are presented.
D1 – Invited Paper NANOMAGNETIC PARTICLES IN RAPID DIAGNOSTICS 30 Yousef Haik, Department of Mechanical Engineering, United Arab Emirates University, Al Ain, UAE and Center for Nanomagnetics and Biotechnology, Florida State University, Tallahassee, FL 32310, [email protected], Reyad Sawafta, Quartek Corporation, 4180 Piedmont Parkway, Greensboro, NC, 27410, USA; [email protected]
Today, with increase emphasis in cost-effective decision-making at the point of care, first responders are in need for rapid and effiecient diagnsotic devices. The use of magnetic immunoassays has grown considerably in recent years. This study identifies a simple process that uses nanomagnetic particles to quickly condcut an anlysis, under field conditions, for detection of analytes. The process has been utilzed for rapid diagnostics of acute mycorial infarction (AMI) at the point of care and for diagnosis of food born pathogens. The magnetic immunoassya takes advantgae of multiple epitopes that are found on the target. On ne epitope a magnetic particle is attached. On another epitope a colorimetric marker is attached. Magnetic nanoparticles of different classes of magnetic compoistions were prepared. The compositions include, Fe-Zn-Fe, Fe-Gd-Zn, Fe-Nd-B, and Ni-Cu. Magnetic moment measurments showed a substantial increase when compared with the iron oxide particles. These particles were then coated with bovine serum albumin. The surface of the coated particles was then modified to couple with the target. Site speific antibodies were used to isolate cardiac protiens (Troponin, Myoglobin, FABP) for the AMI daignosis and to isolate food born pathogens (E. coli and S. enteritidis). A secondary antibody with a colorimetric marker (AP or HRP) was attached to the analyte. Different serial concentration of cardiac markers (0-700 μg/L) were used in forming the conjugate. The resulted color intensities were corrolated to the amount of the cardiac marker used in the conjugate. The experimental results also showed that HRP perfromed better than that of AP particularly at high concentrations of cardiac markers. Serial dilutions of E. coli O157:H7 concentrations in buffer samples were used. Both HRP and AP conjugates were used. A higher sensitivity of the assay was observed when using the HRP compared to AP labeled antibodies. The specificity of the assay for selecting E. Coli in the presence of other pathogen (S. enteriditis) was also tested. The assay specifically identified E. Coli and it yielded negative results for the sample containing S. enteriditis as well as for the blank sample. The nanomagnetic immunoassay has showed a senstivity of detecting the presence of a single bacterium in a meat spiked with E. coli and S. enteritidis. E. coli O157:H7 and S. enteritidis concentrations in buffer and meat samples were confirmed by plating 0.1 ml sample onto plate count agar. For the E. coli O157:H7 samples, the highest dilution contained approximately 6 cells/ml of buffer and 1 cell/g of meat, respectively. For the S. enteritidis samples, the highest dilution contained approximately 9 cells/ml of buffer
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and 1-2 cell/ g of meat, respectively. The process of detection for both the cardiac markers and pathogens was performed in few minutes.
D1 – Invited Paper REAL-TIME VISUALIZATION OF NANO-LIPOSOMAL DELIVERY 31 INTO PRIMATE BRAIN BY MAGNETIC RESONANCE IMAGING Krystof S. Bankiewicz, Michal T. Krauze, John R. Forsayeth, Department of Neurological Surgery, Brain Tumor Research Center, University of California San Francisco, CA; Charles O. Noble, Daryl C. Drummond, Dimitri B. Kirpotin and John W. Park, Division of Hematology-Oncology, University of California San Francisco, CA; Peter J. Dickinson, Robert Higgins, Rick LeCouteur, Department of Surgical and Radiological Sciences, University of California, Davis, CA; [email protected]
Liposomes loaded with Gadoteridol (GDL), in combination with convection-enhanced delivery (CED), offer an excellent option to monitor CNS delivery of therapeutic compounds with MRI. In our studies, we investigated possible clinical applications of liposomes to the treatment of brain tumors. In our study, up to 700 μl of Gadoteridol/rhodamine-loaded liposomes were distributed in putamen, corona radiata and brainstem of non-human primates. Distribution was monitored by real-time MRI throughout infusion procedures, which allowed accurate calculation of volume of distribution within anatomical structures. We found that different regions of the brain gave various volumes of distribution when infused with the same volume of liposome. Furthermore, we investigated multiple real-time infusions in the same regions for reproducibility, and possible long-term changes in primate brain due to liposome convection. In parallel with the primate experiments, we performed CED of nano-liposomal therapeutics (Ls-CPT-11, Doxorubicin) into rodent brain tumor xenografts. Therapeutic liposomes mediated metronomic chemotherapy to brain tumors as controlled release could be detected up to 40 days after single delivery. These experiments also showed tremendously increased survival, and a very broad safety profile. Based on these findings, real-time delivery of therapeutic liposomes was initiated in canines with spontaneous brain tumors. Initial findings suggest that spontaneous tumors are amenable for CED of liposomes. MRI is highly accurate in monitoring distribution, thereby avoiding unwanted distribution into CSF. This work underlines the importance of monitoring drug delivery to CNS, and enables accurate delivery of drug-loaded liposomes to specific brain regions with a standard MRI procedure. Real-time CED of liposomes is a promising drug delivery technique for brain pathologies in which localized delivery of therapeutic agents is indicated.
D1 – Contributed Paper CONTROLLED CLUSTERING OF SUPERPARAMAGNETIC 32 NANOPARTICLES USING BLOCK COPOLYMERS : DESIGN OF NEW CONTRAST AGENTS FOR MAGNETIC RESONANCE IMAGING Jean-François Berret, Matière et Systèmes Complexes, Université Denis
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Diderot, Paris-VII, 140 rue de Lourmel, F-75015 Paris, France, and Olivier Sandre, Laboratoire Liquides Ioniques et Interfaces Chargées, Université Pierre et Marie Curie, Paris-VI, 4 place Jussieu, F-75252 Paris, France; [email protected]
During the past years we have investigated the complexation between nanocolloids and oppositely charged polymers. The nanocolloids examined were ionic surfactant micelles and inorganic oxide nanoparticles. For the polymers, we used homopolyelectrolytes and block copolymers with linear and comb architectures. In general, the attractive interactions between oppositely charged species are strong and as such, the simple mixing of solutions containing dispersed constituents yield to a precipitation, or to a phase separation. We have developed means to control the electrostatically-driven attractions and to preserve the stability of the mixed solution. With these approaches, we designed novel core-shell nanostructures, e.g. as those obtained with polymers and iron oxide superparamagnetic nanoparticles (Berret et al., JACS (2006)). In this presentation, we show that electrostatic complexation can be used to tailor new functionnalized nanoparticles and we provide examples related to biomedical applications in the domain of contrast agents for Magnetic Resonance Imaging.
D1 – Invited Paper MULTILAYERED NANOPARTICLES: CHARACTERIZATION AND 33 BIOANALYTICAL USE Karin D. Caldwell, Department of physical and analytical chemistry, Box 577, Uppsala University, SE-75123 Uppsala, Sweden; [email protected]
The attachment of biologically active proteins to solid surfaces requires utmost care to the manner in which the linking of these often fragile macromolecules is carried out. Issues of concern relate to structural protection, but also to the presentation of reactive features to the environment. One attractive way of addressing these issues is to attach the molecules to solid particles in the 100-250 nm size range through the intermediary of a system of surface linked tethers. These structures protect the protein from direct contact with the substrate while allowing it substantial freedom of motion. Particularly in the design of biosensor systems there is a frequent need to attach not only single proteins but also more complex combinations of molecules which, in a cascade fashion, help to amplify a desired signal. In order to maintain compositional control over such composite systems it is desirable to exactly manage the concentrations and activities of all features bound to the surface. It is in this respect that immobilization via a cocktail of differentially activated tethers becomes particularly attractive. The utilization of this strategy will be exemplified by way of a bioluminescence based sensor chip, containing localized islands of immobilized nanoparticles decorated both with capture antibodies and with the signal generating enzyme firefly luciferase. As the analyte binds the antibody it is made to produce ATP that rapidly diffuses the short distance to the neighbouring enzyme, which it binds and assists in generating a light pulse. The exact surface concentrations of tethers as well as active biomolecules are determined by sedimentation FFF, in a series of analyses that reveal the detailed construction of the surface coating.
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E1 – Keynote Paper COMPARATIVE PERFORMANCE OF TRANSFECTION COMPLEXES 34 DERIVED FROM A FAMILY OF BISVINYL ETHER CATIONIC LIPIDS David H. Thompson, Jeroen Van den Bossche, Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, IN 47907, USA; [email protected]
The development of synthetic vehicles for gene delivery that can rival the efficiency of viral vectors is an unmet challenge in non-viral vector design. Of the many known barriers to efficient transfection, control over intracellular trafficking and release from acidic endosomal compartments are among the most poorly understood steps in this multistep process. We report the synthesis and performance of a series of structurally-related acid-labile cationic lipid transfection agents that have been designed to degrade within acidic endosomes and promote endosomal escape by permeabilizing the endosomal membrane via detergent action of the lipid hydrolysis products. These cationic lipid derivatives contain vinyl ether-linked lipid chains and diethylenetriamine headgroups that were designed to be more responsive than the prototype BCAT lipid that was previously reported by this laboratory. The synthesis, complexation behavior, intracellular uptake, and expression levels of these materials will be described.
E1 – Invited Paper LIPIDIC CUPIC PHASES: APPLICATIONS IN MEMBRANE PROTEIN 35 CRYSTALLOGENESIS AND IN UPTAKE AND DELIVERY Martin Caffrey, Department of Chemical and Environmental Sciences, Plassey Park, University of Limerick, Limerick, Ireland; [email protected]
One of the primary impasses on the route that eventually leads to membrane protein structure through to activity and function is found at the crystal production stage. Diffraction quality crystals, with which an atomic resolution structure is determined, are particularly difficult to prepare currently when a membrane source is used. The reason for this lies partly in our limited ability to manipulate proteins with hydrophobic/amphipathic surfaces that are usually enveloped with membrane lipid. More often than not, the protein gets trapped as an intractable aggregate in its watery course from membrane to crystal. As a result, access to the structure and thus function of tens of thousands of membrane proteins is limited. The health consequences of this are great given the role membrane proteins play in disease; blindness and cystic fibrosis are examples. In contrast, a veritable cornucopia of soluble proteins have offered up their structure and valuable insight into function, reflecting the relative ease with which they are crystallized. There exists therefore a pressing need for new ways of producing crystals of membrane proteins. A novel approach that holds promise makes use of the lipidic cubic phase. In this presentation I will describe the method and our progress in understanding how it works at a molecular level. The practicalities of implementing the method in low- and high-throughput modes will be examined. Transport properties of the cubic phase are critical to the crystallization process and have been explored from this perspective but also
54 Oral Abstracts with regard to their possible use in updake and delivery applications. Supported in part by Science Foundation Ireland (02-IN1-B266), the National Institutes of Health (GM61070, GM075915), and the National Science Foundation (DBI9981990).
E1 – Invited Paper SPATIALLY ADDRESSABLE PROTEIN ARRAYS BASED ON 36 PROTEOLIPOSOME SELF-ASSEMBLY INTO MICROWELLS Alexander Couzis, Charles Maldarelli, Lane Gilchrist, Nikhil Kalyankar, Shyam Vaidya, Gerson Aguirre, Dept. of Chemical Engineering, The City College of New York, NewYork, NY 10031;[email protected]
Protein microarrays - high throughput diagnostic methods in which proteins are arrayed in spots on a surface and screened in one step against a solution of potential binding partners – has emerged as the primary tool in mapping the protein binding interactions of the proteome. This paper outlines a research program focused of the development of a new platform for a protein array, which, extends current technologies and can represent the next generation in protein microarray technology. The array platform addresses two issues in current protein array technology. The first is further miniaturization: Current technologies use fluid spotting to array spots 102 microns in diameter on surfaces with active areas of the order of millimeters by millimeters to generate up to thousands of spots per array. The second is the inability to display functioning cell membrane surface receptors; while antibodies and small molecule ligands can easily be spotted onto a surface, cell membrane proteins require a lipid environment to maintain functionality. The inclusion of membrane proteins is particularly significant because these proteins control cell signaling pathways. Our array platform uses 500 nm – 1000nm in diameter liposomes to sequester and display cell membrane proteins (proteoliposomes) and quantum dot (QDs) encoded beads to label the liposomes with a fluorescent label and provide for spatial addressability. Sequestering the proteins in the liposome bilayer retains their biological activity and the barcoding in beads allows assaying on the nanoscale. Mixtures of the proteoliposomes, each with a different protein and corresponding barcode, are then arrayed on a surface in wells separated by 500-3000 nm. For an active area of 500 μm x 500 μm, upwards of 106 registries can be assembled. In this formats, GPCR membrane proteins and glycolipids will be employed to demonstrate the viability of the arrays, using fluorescence detection with imaging microscopy.
E1 – Invited Paper USE OF NOVEL INTRALIPOSOMAL STABILIZATION STRATEGIES 37 FOR FORMING HIGHLY STABLE AND LONG CIRCULATING NANOLIPOSOME CONSTRUCTS FOR CAMPTOTHECINS AND VINCA ALKALOIDS Daryl C. Drummond, Mark E. Hayes, Charles O. Noble, and Dmitri B. Kirpotin, Hermes Biosciences, 61 Airport Blvd, South San Francisco, CA; and John W. Park, University of California at San Francisco, San Francisco, CA; [email protected]
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Controlling the rate of drug release from liposomal carriers is essential for optimum drug delivery. We have developed a novel nanoliposome construct encapsulating either CPT-11 (irinotecan) or vinorelbine (VRL) with a high degree of drug loading efficiency and in vivo retention. Using a modified gradient loading method featuring a sterically hindered amine with highly charged, multivalent anionic trapping agents, either polymeric (polyphosphate) or non-polymeric (sucrose octasulfate), liposomes were capable of entrapping CPT-11 at extremely high drug-to-lipid ratios (> 800 g CPT-11/mol phospholipid) and retaining encapsulated drug in vivo with a half-life of drug release in the circulation of 56.8 h. The release rate (t1/2 = 1.8-27.2 h) of vinorelbine from the liposome could be improved by either modifying the chemical nature of the trapping agent or by altering the concentration of drug loaded into the liposomes, with liposomes prepared using sucroseoctasulfate displaying the longest half-life in the circulation (9.3 h) and in vivo retention in the nanoparticle (t1/2=27.2 h). CPT-11 was also protected from hydrolysis to the inactive carboxylate form and from metabolic conversion to SN-38 while circulating. The maximum tolerated dose in normal mice was determined to be 80 mg/kg for free CPT-11 and >320 mg/kg for nanoliposomal CPT-11. The acute toxicity in normal mice was not increased upon encapsulation of vinorelbine in liposomes, with an MTD of 17.5 mg VRL/kg for free vinorelbine and 23.8 mg VRL/kg for nanoliposomal vinorelbine. Nanoliposomal CPT-11 demonstrated markedly superior efficacy when compared to free CPT-11 in human breast (BT474) and colon (HT29) cancer xenograft models, while nanoliposomal vinorelbine displayed improved activity in a wide range of lung, colon, and breast cancer xenograft models. These studies shows that intraliposomal stabilization of CPT-11 or VRL using a polymeric or highly charged, non-polymeric polyanionic trapping agent results in strikingly active antitumor agents with low toxicity.
E1 – Contributed Paper COMPARATIVE PERFORMANCE OF TRANSFECTION COMPLEXES 38 DERIVED FROM A FAMILY OF BISVINYL ETHER CATIONIC LIPIDS David H. Thompson, Jeroen Van den Bossche, Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, IN 47907, USA; [email protected]
Microvascular endothelial cells are relevant targets for targeted liposome mediated drug delivery, because of their involvement in pathophysiological processes such as chronic inflammatory disease. Furthermore endothelial cells are direct accessible for substances transported by the blood. The heterogeneity of the endothelium with respect to appearance and function allows for disease specific drug delivery approaches. E.g., activated endothelial cells present in areas of inflammation selectively (over)express various adhesion molecules. Among these, E-selectin is solely expressed by activated endothelial cells while it is absent in non-diseased organs. Therefore E-selectin is an ideal target to deliver anti-inflammatory drugs into activated endothelial cells. The aim of the study was to develop immunoliposomes targeted to E-selectin on activated endothelial cells for intracellular delivery of drugs for the treatment of inflammatory diseases. We prepared immunoliposomes containing dexamethasone, using anti-E-selectin IgG as a homing
56 Oral Abstracts device. In TNFα activated human umbilical vein endothelial cells, association of anti-E-selectin immunoliposomes was 20 fold higher than in non-activated endothelial cells. This association could be blocked by excess of free anti-E-selectin but not by control IgG, demonstrating specificity of the interaction. Confocal laser scanning microscopy demonstrated that the immunoliposomes were endocytosed via a clathrin mediated pathway. In vivo, in a mouse model of anti-glomerular basement membrane induced glomerulonephritis, injection of dexamethasone containing anti-E-selectin immunoliposomes revealed specific uptake in the kidneys as compared to control liposomes. Within the kidney the anti-E-selectin immunoliposomes were located in the diseased glomeruli, corresponding to the glomerular expression of E-selectin in this model. The immunoliposomal delivery of dexamethasone into the diseased glomeruli resulted locally in a significant downregulation of inflammation related genes. Systemic hyperglycemia induction seen with free dexamethasone treatment was absent in the dexa-immunoliposome treated group. These data demonstrate the feasibility of selective delivery of anti-inflammatory drugs by immunoliposomes into disease-activated endothelium to exert local pharmacological effects.
E1 – Invited Paper TRM-484, STEROID INCLUDED IN LONG CIRCULATING CATIONIC 39 LIPOSOMES ACTIVELY TARGETING INFLAMMATORY TISSUES Junji Kimura, Katsumi Morimoto, Shinichi Kaneda, Masaharu Miyajima, Hiroaki Kasukawa, TERUMO Corporation R&D Center, 1500 Inokuchi, Nakai-machi, Ashigarakamigun, Kanagawa, 259-0151 Japan; [email protected]
We have been developing a novel liposome actively targetable to diseased cells without losing a long-circulating ability in the blood. Our novel active targeting factor, TRX-20 (3,5-dipentadecyloxybenzamidine hydrochloride), has been reported to interact with some kind of chondrotin sulfate proteoglycans (CSPGs) on the subendothelial cell surfaces under certain diseases such as inflammation, but not with heparan sulfate proteoglycans. However, to make satisfy both long-circulating and selective targeting, an optimal composition ratio of polyethylene glycol and TRX-20 on the liposomal surface was confirmed to be important. We are developing a prednisolone-encapsulated liposome (TRM-484) using this technology mentioned above. In this presentation, we would show the mechanism of action and its pharmacological advantages with some in vitro and in vivo studies. In summary, TRM-484 bound to cultured synovial cells derived from RA patients via mediating CSPGs on these cell surfaces, introduced into the cell inside and then released prednisolone. Also in the rat arthritic model TRM-484 localized in the synovial lining cells, but not in the normal joint tissues. Effective dose of prednisolone with this liposomal formulation could be reduced by 1/10 of oral dose. Such local effect and dose reduction are expected to decrease side effects due to lower systemic exposure of prednisolone. Currently TRM-484 is in a phase 1 study.
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E1 – Invited Paper DNA BASED HYDROGELS, LIPOSOMES AND NANOBARCODES 40 Dan Luo, Soong Ho Um, Jung Bum Lee, Nokyoung Park, Sang Kwon Dept. of Biological and Environmental Engineering Cornell University, Ithaca, NY 14853, USA; [email protected]
DNA can be engineered as a generic instead of a genetic material -- the genetic information-coding property of DNA can be completely and purposely ignored. Instead, by taking advantage of nanoscale, molecular biology tools (including enzymes) we have employed DNA as a true and novel polymer. Branched, networked, and dendritic DNA has been fabricated as additional building blocks for novel materials. In particular, we have made DNA based hydrogels in a variety shapes and sizes, from centimeter to nanometer, and from a complicated pattern (“Cornell”) to spherical, microdroplets. These DNA gels are water soluble, biocompatible, biodegradable and most importantly can encapsulate drugs and even live cells in situ, eliminating the drug loading step and also avoiding denaturing conditions that are typical in most hydrogel-based, controlled-release systems. The efficiency of encapsulation of the DNA gels has reached close to 100%. DNA hydrogels will have great potential for controlled drug delivery, tissue engineering, 3D cell culture, cell transplant therapy, and other biomedical applications. We have also created DNA-based, anisotropic dendrimers that can be tagged with a variety of chemical moieties. The process is robust and totally controlled. The anisotropic structures were also very stable. Although we demonstrated anisotropic DNA dendrimers with only fluorescent tags (which were used as DNA based nanobarcodes), the procedures can be easily extend to almost any chemical moieties as long as they can be conjugated to DNA strand. Thus it is possible to use DNA as framework to create anisotropic materials for a variety of applications such as a barcode system for molecular detection and a heterogeneous drug delivery system with different drugs along with specific targeting molecules. In addition, we have also created DNA-liposomes from DNA dendrimers, The dendritic DNA components embedded in these amphiphiles offered not only greater control over general morphology, but also a unique method for tracking the liposomes’ core-shell nanostructures via a simple labeling with DNA and lipid specific fluorescent dyes. It is expected that DL-DNA-lipid amphiphiles and DNA-liposomes will become new tools for designing and synthesizing novel, drug delivery materials and for tracking self-assembling processes.
F1 – Invited Paper CHARACTERIZATION OF MAGNETIC NANOPARTICLES USING 41 QUADRUPOLE MAGNETIC FIELD-FLOW FRACTIONATION P. Stephen Williams, Francesca Carpino, and Maciej Zborowski, Cleveland Clinic, Lerner Research Institute, Department of Biomedical Engineering, 9500 Euclid Avenue, Cleveland, OH 44195, USA; [email protected]
Field-Flow Fractionation (FFF) is an analytical scale separation and characterization technique for macromolecules and particles (from ~0.01 to 100 μm in diameter). The
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technique was conceived by Cal Giddings in 1966. It is a separative elution technique, similar to chromatography, in which different components of a small sample elute from a separation channel at different times. Whereas chromatography exploits differences in partition between the mobile and stationary phases to separate sample components as they are carried along a column, FFF separation is achieved within the mobile phase alone. The separation device takes the form of a thin, parallel-walled channel, across the thickness of which is applied a field of some type. Particles that interact strongly with the field are confined to thin zones adjacent to the wall where fluid velocity is low, and they elute more slowly than particles which interact less strongly with the field. Furthermore, the quantitative theoretical foundation of FFF allows the determination of the strength of interaction of particles with the field as a function of their elution time. The nature of the applied field determines the sample property that is characterized. Our approach to magnetic FFF (MgFFF) uses the radially symmetric magnetic field generated by a quadrupole electromagnet. A thin helical channel exploits the quadrupole field efficiently. The field strength is gradually reduced during sample elution in order to elute very polydisperse samples. MgFFF has been used to characterize magnetic nanospheres used for immunospecific labeling of biological cells for magnetic separation. Samples of different production lots were kindly supplied by BD Biosciences Pharmingen. The samples were eluted under a range of different mobile phase flow rates and programmed field decay conditions. Distributions in magnetite content were determined for the sample lots supplied. The method appears to be a useful technique for quality control purposes.
F1 – Invited Paper COLLOIDAL STABILITY OF EMULSIONS AND NANOPARTICLES IN 42 PHARMACEUTICS Hélène Dihang, Pascal Bru, Gérard Meunier, Formulaction, 10 impasse Borde, Basse 31240 L’Union, France; and Patrick Snabre C.R.P.P., avenue Albert Schweitzer, 33600 Pessa, France; [email protected]
The world of drug delivery is getting every day more efficient with new product forms, which are directly targeting the deficient organs. Colloidal systems such as emulsions (simple or multiple) or nanoparticle suspensions are now widely used as drug carriers or nutritional solutions in this respect. These new product forms allow better targeting and assimilation for the patient, and give rise to new solutions for the drug designers. However, the stability of these inherently unstable colloidal systems makes them complex to formulate and study. Despite the existence of models and increasing knowledge on physical chemistry to help the formulator to tend towards more stable formulations, he cannot take the risk to select an industrial formula for its sole end-use properties without testing the stability experimentally, when an unstable formulation can risk the life of the patient he is trying to cure. Therefore, the companies developing instruments have worked in collaboration with the physical chemists, from industry and research, to develop new techniques of analysis for either predicting the stability of these mixtures, or reducing times of ageing tests. These techniques, although very powerful, require a good knowledge of measurement to interpret the results. This presentation aims to review the main techniques available to monitor the physical stability of colloidal systems for pharmaceutical applications.
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F1 – Contributed Paper PARTICLE SIZE ANALYSIS: COMPARTIVE RESULTS FROM 43 SEVERAL TECHNIQUES. Frank M. Etzler and Richard Deanne. Boehringer-Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Road, Ridgefield, CT 06877, USA; [email protected]
Particle size analysis is important to a number of industrial processes and influences the performance of many products. In the context of the pharmaceutical industry particle size affects the performance, for instance, of inhalable formulations and the dissolution rate of poorly soluable compounds. Processes relying on powder flow are also affected by particle size and partticle size distribution. Particle size analysis is difficult for a number of experimental and philosophical reasons. Particle size unlike many often experimentaly measured quantities is ill defined in many instances. The size of spherical particles can be expressed as a single number – the radius or diameter. In contrast, particles of complex geometry particularly disks, flakes and needles cannot be completely described using a single number. Many particles are also significantly cohesive and may not disperse into parimary particles on a least some kinds of particle sizing equipment. The dispersion charateristics of commercially available equipment is not hydrodynamically defined. Laser diffraction instruments have found almost Procrustean use in the pharmaceutical industry principally because they are relatively easy to use. The theoretical basis for the construction of such instruments is complex and in many senses incompletely implemented on commercially avaliable for a number of technical reasons. One consequence is that in many instances the response to changes in subpopulations particles may not be reflected by the instrument in a linear manner resulting either in an underestimate or overestimate of the change in the subpopulation. In this work compartive performance of serveral instruments is investigated. The influence of powder properties and instrument design is ilustrated. Recommendations for future work are also made.
F1 – Invited Paper PARTICLE SIZE AND SHAPE CHARACTERIZATION OF NANO- AND 44 SUBMICRON SUSPENSIONS Arjen Tinke, Johnson & Johnson Pharmaceutical R&D, Turnhoutseweg 30, B-2340 Beerse, Belgium; [email protected]
Nanonization and micronization of highly (water) insolible compounds by means of wet milling is generally applied in function of a modication of the in-vivo release profile of active pharmaceutical ingredients (API). More precisely, the technology is used as basis for the pharmaceutical development of various types of dosage forms, such as for example sustained release and depot formulations. Since the dissolution of the particles is a dominant factor in the release mechanism of these drugs, it is an absolute necessity to characterize the particles in terms of their morphology (or shape) and particle size distribution (PSD). An adequate characterization of the shape and size distribution will enable the evaluation of the dosage form in terms of release profile, burst effect, Ostwald
60 Oral Abstracts ripening, etc. In practise, the size distribution characterization of nano- and submicron particulate systems is often far from straightforward, and the different analytical techniques easily lead to different results. Based on experimental data as obtained for the development of one of our formulations having a rather broad PSD, the capabilities of laser diffraction (LD) and centrifugal sedimentation (CS) are demonstrated for the size distribution characterization of submicron suspensions. In addition to this, both qualitative and quantitative environmental scanning electron microscopy (ESEM) data are shown that demonstrate the capabilities of this rather new approach in the evaluation of both the morphology and the PSD of the product. Though photon correlation spectroscopy (PCS) has shown to have limited capabilities in the PSD characterization of submicron suspensions, with PCS related technologies the stability of the product could be assessed by measuring the zeta potential of the drug particles. Finally, the interpretation of the PSD data in function of the dissolution behavior of the product is illustrated by means of correlation of the in-vitro release and PSD data. Below ca. 100 nm things get much more complicated since in this area LD and CS have limited capabilities. The lack of reliable and robust PSD technologies for the analysis of nano suspensions (i.e. < 100 nm) stresses the need for other techniques in the accurate PSD characterization of these products. For this reason, the latest developments on this area are discussed in order to help the applicant in the selection of the right preferred analytical technologies.
F1 – Contributed Paper 45 AFM CAPABILITIES IN CHARACTERIZATION OF PARTICULATE MATTER: FROM ANGSTROMS TO MICRONS Paul West, Natasha Starostina, PacificNanotechnology, Inc., 17984 Sky Park Circle, Suite J, Irvine, CA 92614, USA; [email protected]
Scanning Probe Microscopy has been successfully employed as surface characterization technique for more than 20 years. Atomic Force Microscopy is the most widely used subset of SPM, which can be used in ambient conditions with minimum sample preparation. AFM is capable to deliver unique three-dimensional topography information from angstrom level to micron scale with unprecedented resolution. AFM is well suited for individual particle characterization. Standard set of measured parameters include: volume, height, size, shape, aspect ratio and particle surface morphology. With single-particle technique, physical parameters for each particle in a set can be recorded and the data set can be processed to generate a statistical distribution ( i.e. ensemble-like information) for entire set of particles. Speeding up the process of obtaining data is critical for many reasons and definitely make AFM more attractive for individual particle imaging. In general AFM individual particle characterizations is both cost and time effective compare to electron microscopy. The resolution of AFM is greater or comparable to SEM/TEM. The main advantage of AFM for particle characterization is unambiguous morphology determination along with direct measurements of height, volume and 3D display.
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F1 – Contributed Paper FEMTOSECOND PULSE VACUUM-ULTRAVIOLET-FREE ELECTRON 46 LASER DIFFRACTION SCATTERING OF POLYSTYRENE NANOPARTICLES Michael Bogan, Anton Barty, Stefano Marchesini, Henry Benner, Bruce Woods, Matthias Frank, Stefan Hau-Riege, Henry Chapman, Lawrence Livermore National Laboratory, 7000 East Ave, Livermore, CA, 94568, USA; Sebastien Boutet and Janos Hadju , Uppsala University, Uppsala, Uppsala Biomedicinska Centrum BMC, Husarg. 3, Box 596, 751 24 Uppsala, Sweden; [email protected]
A free electron laser for wavelengths down to 6 nm in the vacuum-ultraviolet and soft X-ray regime (VUV-FEL) is under construction at the TESLA Test Facility at DESY. It is operated in the self-amplified spontaneous emission mode and delivers sub-picosecond radiation pulses, with gigawatt peak powers. User experiments using this unique radiation started in August 2005 and we performed some of the first experiments in January/February 2006 using a wavelength of 32 nm. At this wavelength, the FEL pulse intensities from vary typically from a few µJ up to more than 100 µJ have been obtained with pulse lengths between 20 and 50 fs. Here, I will present femtosecond pulse diffraction scattering data from experiments using 32 nm radiation with a target of size-monodisperse nanoparticles. Polystyrene particles of sizes ranging from 65-145 nm were prepared for analysis by aerosolizing them via charge-reduced electrospray, size-selecting them through a differential mobility analyzer, and capturing them onto 20 nm thick silicon nitride windows microfabricated in silicon wafers. Scanning electron microscopy was used to calculate the resulting number densities on the windows (<1 to ~10 particles/μm2). Correlation with particle number density in the aerosol, as sampled using a condensation particle counter, enabled estimation of the number density on the window in real-time. I will also discuss recent data collected at the Lawrence Berkeley National Lab Advanced Light Source using 1.65 nm radiation that validated size-monodispersities of <3.5% (for ~100nm particles) after differential mobility filtering. This work was performed under the auspices of the U.S. Department of Energy by University of California, Lawrence Livermore National Laboratory (LLNL) under contract No. W-7405-ENG-48 and supported by LLNL through laboratory directed research and development grants no. 02-ERD-047 and 05-SI-003.
F1 – Invited Paper UNDERSTANDING THE EFFLORESCENCE OF SUPERSATURATED 47 AEROSOLS USING FLUORESCENCE SPECTROSCOPY Man Yee Choi and Chak K. Chan, Department of Chemical Engineering, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong; [email protected]
The knowledge of the state of water molecules, particularly the amounts of solvated water and free water in aqueous droplets, is valuable in understanding the hydration properties
62 Oral Abstracts and crystallization properties of supersaturated solutions or suspended droplets. A novel technique combining the use of an electrodynamic balance (EDB) and a fluorescence dye, 8-hydroxyl-1,3,6-pyrenetrisulfonate (pyranine), was used to study the state of the water molecules in single levitated aqueous droplets from subsaturation to supersaturation concentrations. The steady state fluorescence spectra of NaCl, (NH4)2SO4, Na2SO4, MgSO4, Mg(NO3)2 and mixed NaCl and Na2SO4 (in 1:1 mole ratio) solutions doped with 100 ppm pyranine were measured. The fluorescence emission of pyranine is sensitive to the proton transfer capacity of its microenvironment. When excited by radiation at around 345nm, pyranine fluoresces and the spectrum consists of two peaks, one at about 440nm and the other at about 510nm, which correspond to the presence of solvated and free water, respectively. The fluorescence peak intensity ratios of the 440nm peak to the 510nm peak and the hygroscopic measurements were used to calculate the amounts of solvated and free water in the droplets as a function of relative humidity (Choi et al., 2004). We found that the equality of the amounts of solvated and free water is a necessary but not sufficient condition for efflorescence. For efflorescing compounds such as NaCl, Na2SO4, (NH4)2SO4, and a mixture of NaCl and Na2SO4, the amount of free water decreases, while that of solvated water is roughly constant in bulk measurements and decreases less dramatically than that of free water in single-particle measurements as the relative humidity (RH) decreases. Efflorescence of the supersaturated droplets of these solutions occurs when the amounts of free and solvated water are equal (Choi and Chan, 2005). This study demonstrates that fluorescence spectroscopy is a unique tool in understanding the hydration properties, the efflorescence and the structural heterogeneity of aqueous droplets.
F1 – Contributed Paper THREE DIMENSIONAL CHARACTERIZATION OF 48 PHARMACEUTICAL POWDERS BY SCANNING WHITE LIGHT INTERFERENCE MICROSCOPY Tim B. Vander Wood, Rachel Rose and Mary A. Miller; MVA Scientific Consultants, 3300 Breckinridge Blvd. #400, Duluth, GA 30096; [email protected]
Particle dissolution properties depend critically on the three dimensional characteristics of the particle, specifically on the surface area to volume ratio. Traditional methods of characterizing pharmaceutical particles and powders in three dimensions typically rely on estimates derived from measurements made in only one dimension (e.g. light scattering estimates of particle diameter) or, less frequently, in two dimensions (e.g. particle feret diameters measured from micrographs of the particles in question). Rarely, three dimensional measurements are made using stereographic properties of pairs of two dimensional micrographs or using images of particles tilted through a known angle. These methods are particularly suited to regular particles with a square prism habit, and are labor intensive. We present here a method of directly characterizing particles in powder samples using scanning white light interference microscopy. This method combines data taken from the two dimensional (x,y) projection of each particle (as in a typical micrograph) with direct measurement of the height (z) of each particle at every pixel encompased by the
63 Oral Abstracts projection of each particle. This x,y,z data array is then used to directly calculate the volume and surface area of each particle. Alternatively, the data may be used to infer an “ideal” particle shape, and volume and surface area calculations performed for the ideal shape. We have established the reliability of this technique for x,y dimensions of greater than or equal to approximately 3 µm and z of greater than or equal to approximately 100 nm. We are working to extend this concept to submicrometer particles utilizing atomic force microscopy.
F1 – Contributed Paper POTENTIAL OF MICROCAPSULES MADE FROM PLANT POLLEN FOR 49 APPLICATION IN CHROMATOGRAPHY Holger Woehlecke, Kristina Lerche, Dietmar Lerche, Dr. Lerche KG, Rudower Chaussee 29, D-12489 Berlin, Germany; and Rudolf Ehwald, Humboldt-University, Institute of Biology, Invalidenstr. 42, D-10115 Berlin, Germany; [email protected]
The exine, outer wall of pollen cells, consists of sporopollenin, a polymer material which has unique chemical and physical properties. We used mild purification methods to obtain chemically almost unmodified sporopollenineous microcapsules from various pollen species. Outstanding physical and chemical properties of these microcapsules encourage their application in separation technology. They have a suitable size for chromatography and a narrow and uniform size distribution. They are resistant to heat, organic solvents, strong acids and alkaline solutions and they show excellent hydraulic and mechanic properties. Using microcapsules in chromatography one has to consider the large free liquid phase, which is enclosed by the capsule wall within the capsules. This liquid acts as stationary phase and has to be accessible for molecules to be separated. As we have found a highly permeable exine structure in the pine pollen air bladders, which represent capable microcapsules, the enclosed stationary liquid phase can be used as partition space for permeable molecules. Because of the high permeability of this special microcapsule walls extremely large unpermeable hydrocolloids or even microparticles can be isolated from other polymers of rather large size, e.g. most proteins. The diffusional exchange of this large permeable polymers with the enclosed stationary eluent is very fast and not membrane controlled. This allows for an efficient separation by size exclusion chromatography. We present exemplary and practical applications in that field including rapid and fast separation of giant polymer molecules as genomic DNA or high-molecular-weight fractions of dextran, polyethylene glycol and hyaluronic acid from permeable proteins and other polymer molecules with a Stokes' radius of up to 50 nm on short columns (3-4 cm).
A2 – Invited Paper NOVEL METHODS OF PRODUCING NANO- AND MICROSIZED 50 PARTICLES COMPOSED OF NATURAL POLYELECTROLYTES Satoshi Iwamoto, Gifu University, Faculty of Applied Biological Sciences, 1-1,Yanagido, Gifu 501-1193, Japan; [email protected]
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Two kinds of natural polyelectrolyte complexes such as chitosan and carboxymethyl cellulose, or gelatin and acacia were used in order to prepare nano- and microsized particles. Two types of nanoparticles with an average diameter of around 200 nm were formed only by mixing hydrolysates of chitosan and carboxymethyl cellulose. Nanoparticle A was produced from chitosanase hydrolysate of chitosan and cellulase hydrolysate of carboxymethyl cellulose, and nanoparticle B was produced from lysozyme hydrolysate of chitosan and the carboxymethyl cellulose hydrolysate. Negatively charged compounds with high molecular weights were maintained in the particles even at the higher pH levels than the pKa of the amino groups of chitosan. Entrapped compounds were gradually released from nanoparticle A by lysozyme treatment. In contrast, there was no release from nanoparticle B. These results indicate that nanoparticle A can be applied to controlled-release drug delivery systems, and that nanoparticle B is stably retained in the body without releasing the entrapped compounds. Another kind of complex composed of gelatin/acacia coacervate was used to create monodisperse microparticles. In order to product monodisperse microparticles, emulsion droplets with a narrow size distribution were required, so microchannel (MC) emulsification, a novel method for preparing monodisperse O/W and W/O emulsions, was applied. Generally, surfactants are necessary for MC emulsification, but they can also inhibit the coacervation process. In this study, surfactant-free systems were examined. The results demonstrated the potential use of gelatin for MC emulsification. Moreover, low bloom gelatin was found to be suitable for obtaining monodisperse emulsions. Finally, surfactant-free monodisperse droplets prepared by MC emulsification were microencapsulated with gelatin/acacia complex coacervate. The microsized particles produced by this technique were observed with a confocal laser scanning microscope. Average diameters of the inner cores and outer shells were 37.8 and 51.5μm; their relative standard deviations were 4.9 and 8.4%.
A2 – Invited Paper PEPTIDE AND POLYMER ASSEMBLY-CONTROLLED 51 NANOPARTICLE FORMATION Andreas Taubert and Wolfgang Meier, Department of Chemistry, University of Basel, Klingelbergstr. 80, CH-4056 Basel, Switzerland; [email protected]; [email protected]
Many organisms are able to crystallize inorganic minerals like calcium phosphate in their cells at room temperature and ambient pressure in aqueous media. Birds, bacteria, fish, humans and many others not only crystallize calcium phosphate but also iron oxide and many other minerals. These biogenic hybrid materials have advantageous properties like high abrasion resistance or high mechanical strength. These properties often arise from a sophisticated interplay between inorganic and organic components, which is fine-tuned by a given organism to meet a specific requirement. One of the main questions in current materials science is how one can in the laboratory generate materials with properties similar to natural materials. Ideally one is able to generate a “toolbox” with which one is able to change or adapt materials properties by design rather than by accident. We will present an approach towards new calcium phosphate (CaP)/organic hybrid materials, that
65 Oral Abstracts shows great promise of becoming such a toolbox. Specifically, we use synthetic water-soluble polymers, which, in aqueous solution self-assemble into various structures such as micelles, vesicles, polymer tubes, or lyotropic mesophases. These self-assembled entities strongly affect the growth of CaP from aqueous solution. They can also be modified with either short peptides or proteins to modify the CaP growth.
A2 – Contributed Paper DEVELOPMENT OF NANOENGINEERED PLATELETS 52 Kent Coulter, John Cernosek, and Jim Arps, Mechanical & Materials Engineering Division, Southwest Research Institute, 6220 Culebra Rd, San Antonio TX, 78238-5166, USA; [email protected]
Iron nanoparticles that are superparamagnetic are being utilized in magnetic resonance contrast enhancement, cell and DNA separation, drug delivery, and gene cloning but while these spherical nanoparticles exhibit unique properties, the material handling, utilization in application, and potential health and environmental issues have lead to the examination of engineered platelet technologies that can be macroscopically manipulated and maintain the unique nanoparticle functionality. Multilayer structures composed of alternating layers of Fe and non-magnetic layers exhibit a range of novel magnetic properties that can be manipulated by varying the layer thickness, topography of the interfaces and the layer crystalline structure. Vacuum deposition of a thin film onto a substrate and the subsequent comminuting of the film into a flake/ platelet results in unique attributes of layer thickness control, multilayer construction, and planar geometry that are advantageous for multilayer magnetic platelets used in biomedical applications. The development of a multilayer iron/alumina platelet that is capped with a layer of gold provides the opportunity to take advantage of the enhanced magnetic properties of these multilayer platelets in a biocompatible structure. Characterization of the layered structures and resulting particles using optical microscopy, XRD, SEM, and magnetic techniques provides information on the platelet size and shape, particle size distribution, surface chemistry and magnetic response. The design considerations, fabrication process, and film and platelet properties will be discussed.
A2 – Invited Paper GAS-PHASE SYNTHESIS AND COATING OF MULTIFUNCTIONAL 53 NANO- AND MICROPARTICLES FOR DRUG DELIVERY Esko I. Kauppinen, Janne Raula, David P. Brown, Anna Lähde, NanoMaterials Group, Center for New Materials and Laboratory of Physics, Helsinki University of Technology, P.O. Box 1000, FIN-02044 VTT, Finland, Hua Jiang, Juha A. Kurkela, VTT, P.O. Box 1000, FIN-02044 VTT, Finland; [email protected]
Good flowability of pharmaceutical powders is an important feature for the handling, processing and dispersion of dry powders. To improve flowability, the optimization of the powder characteristics such as surface morphology and hardness is needed. It is known that
66 Oral Abstracts a rough and hard surface minimizes the area between contacting objects and accordingly the forces between particles, and thereby, improves detachment and flowability. This work presents a novel aerosol flow reactor i.e. gas-phase method for in-situ synthesis and surface-modificiation of nano- and microparticles by a volatile peptide i.e. L-leucine. In the method, the starting solution containing drug molecules and L-leucine are atomized and fed into a heated laminar flow reactor with the aid of carrier gas. Particles dry up followed by evaporation of leucine. Coating on the surface of drug particles is formed by physical deposition of L-leucine vapor. The thickness and the morphology of L-leucine layer on the spherical salbutamol sulphate and sodium chloride core particles is controlled by saturation conditions and residence time of aerosols in the reactor. Downstream of the reactor, particle size distributions were determined with aerosol size spectrometers and particle morphology was characterized with SEM and TEM. During condensation, L-leucine formed small crystallites (nanoflakes) on the surfaces of drug particles with their length being determined mainly by the concentration of L-leucine vapor. These nanoflakes increased the distance and reduced the area of contact between particles. The coated micropowders showed excellent flowability as well as dispersibility when tested with a novel deagglomeration apparatus. The coated powders could be fed at stable rate without the use of the lactose carrier. Coated salbutamol sulphate micropowders were fed to study their dispersion with and without lactose carrier particles. Dispersion improved with increased dispersion air flow rate and the degree of L-leucine coating.
A2 – Invited Paper DNA CONDENSATION MECHANISM REVEALED UNDER 54 FLUORESCENCE VIDEO-MICROSCOPE Yuichi Yamasaki, University of Tokyo, School of Eng., Dept. of Material Eng., 7-3-1 Hongo, Bunkyo, Tokyo 113-8656, Japan; [email protected]
DNA condensation has been treated as a key phenomenon to clarify DNA packaging into a viral capsid head or a cell nucleus, and recently attracted much attention in relation to the non-viral gene therapy. In addition, this phenomenon is closely related to the traditional problem of polymer physics, i.e., coil-globule transition. It has so far been believed that DNA conformation tends to gradually shrink to a condensed state via q-state. Recently, however, a fluorescence microscopic observation has clarified that a single DNA chain changes its conformation from the elongated to the compact collapsed state in a discrete manner. During the transition region, both elongated and collapse DNA chains coexist without the presence of an intermediate conformation corresponding to the q-state, indicating the bimodality of DNA conformation. The conformational change of DNA seems to be related to the electrostatic property of DNA complexed with condensing agents. Thus, the free solution DNA electrophoresis under the fluorescence microscope was performed to obtain the relationship between DNA conformation and its residual charges at the coexistence region of the DNA condensation transition. To evaluate both DNA conformation and its residual charges simultaneously, an electrophoretic chamber attachable to a microscope stage was designed. When the DC electric field (6 V/cm) was applied, the collapsed DNA behaves as a Brownian particle, while the elongated DNA
67 Oral Abstracts migrated toward an anode electrode with a constant mobility 1.8×10-8 m2V-1s-1. This result indicates that the collapsed DNA was completely neutralized, while the elongated chains still had the residual charges. At the coexistence region of the DNA collapse transition, bimodalities not only in DNA conformation but also in its residual charges are demonstrated using direct observation of free solution DNA electrophoresis. It is suggested that the DNA conformational change closely correlates with the change in its residual charges.
A2 – Contributed Paper NOVEL NANOPARTICLE TECHNOLOGIES FOR THE 55 COST-EFFECTIVE, TARGETED, NON-SYSTEMIC DELIVERY OF THERAPEUTIC AGENTS Andrew Loxley, Christy Law, David Fairhurst, Particle Sciences Inc., 3894 Courtney St. Suite 180, Bethlehem, Pennsylvania, 18017, USA; [email protected]
Nanoparticles are used to carry therapeutic agents, either attached to their surfaces, or encapsulated within the particle matrix. Early work with nanoparticle carriers focused on injectable formulations for systemic delivery of therapeutics. We describe here a novel technique for preparing nanoparticles for non-systemic (topically administered) targeted delivery of therapeutic agents. Nanoparticles were prepared from a melt-emulsification-chill process and surface-engineered to enable targeting of the particles to specific biological surfaces including hair follicles, buccal- and vaginal- mucosal surfaces and the cell surfaces of pathogens. Particles were prepared with varying surface charge (sign and density) enabling electrostatic attachment to oppositely charged biological surfaces, and are amenable to coating with, for example, ligands for specific cell surface receptors. The production process uses low-cost, readily available, biocompatible/biodegradeable materials, and low-cost processing. In this respect the technology is ideally suited to developing world applications. A first step in the design of a targeted therapy often involves the characterization of the targeted surface, and a novel approach to characterizing the properties of host- or pathogen- cell surfaces, "electrophoretic fingerprinting" is introduced that provides useful information for use in the rational design of targeted delivery vectors, as demonstrated by the mapping of surface properties of human T-cells.
A2 – Invited Paper SILK MICROSPHERES PREPARED BY SPRAY-DRYING OF AN 56 AQUEOUS SYSTEM Tomoaki Hino, The College of Pharmacy, Kinjo Gakuin University, 2-1723 Ohmori, Moriyama, Nagoya 463-8521, Japan; [email protected]
Scoured silk fiber was dissolved in an aqueous solution of calcium chloride mixed with ethanol. After dialysis against purified water, theophylline as a model drug was added in the solution. Silk microspheres were obtained by spray-drying. The resultant silk
68 Oral Abstracts microsphere was round but somewhat wrinkled, and its diameter was about 5 μm. In this way, we developed a new drug carrier without using poisonous organic solvents nor glutaraldehyde. Silk microspheres prepared by spray-drying were exposed to humid atmosphere (89 % relative humidity, RH). Change in the secondary structure of silk fibroin during preparation of silk microspheres and exposure to high humidity was studied. Silk fibroin and theophylline were found to be amorphous in the microsphere by means of powder X-ray diffractometry. Fibroin formed β-sheet conformation in the scoured silk fiber while it is composed of irregular structure in the microsphere according to FT-IR and Raman spectra. Fibroin recrystallized and its secondary structure changed to β-sheet conformation by exposing the microspheres to an atmosphere of 89 % RH.
A2 – Contributed Paper SYNTHESIS AND APPLICATIONS OF ARRAYS OF SILICA 57 NANOTUBES Igor Sokolov, Yaroslav Kievsky, Dept. of Physics and Dept. of Chemistry, Clarkson University, Potsdam, NY 13699, USA; [email protected]
The problem of how to “package” nanodots, carbon nanotubes, fullerenes, and other popular nano objects into larger scale devices is one of the most important problems of modern nanotechnology. The self-assembly of nano objects into larger functional shapes, devices is a very attractive way of addressing this problem because of its intrinsic simplicity and low-cost. An ultimate example of such hierarchical self-assembly exists already in the world of biological objects. While rational design of bio objects is definitely not a task of nowadays, an example of such hierarchical self-assembly is already well-known. It is the coassembly of organic liquid crystals with inorganic precursor of some metal oxides, in particular, silica. In some specific conditions such synthesis can result in assembly of complex shapes, which resemble the shapes typically found in the biological world. From a structural point of view, these shapes are the arrays of silica nanotubes (ASNT). These materials are in a prime position to be used in a broad variety of applications. The major obstacle here is a broad polydispersity of the synthesized arrays-shapes. Here we demonstrate that this problem can be solved for at least one specific synthesis of ASNT with high uniformity of shapes for the case of fibers, hexagonal cylindrical bundles of straight silica nanotubes. Each such fiber contains about quarter of million of parallel silica nanotubes, 3nm each. This is the first indication that hierarchical self-assembly can be used for controllable “mass-manufacturing” of larger nanostructured objects. This will contribute toward a new way of thinking about templated synthesis of materials over length scales of tens of microns that is typically outside of the range of solid state chemistry. Immediate applications of ASNT are drug delivery, new self-healing materials, assembly of templates for dye microlasers, photonic microsensors, photonic chips, etc. We will demostrate prrelimiary data for several such applications.
A2 – Contributed Paper USE OF FLAGELLIN AND MANNOSAMINE NANOPARTICLES AS 58 BIOADHESIVE CARRIERS FOR MUCOSAL VACCINATION
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Carlos Gamazo1 , H.H. Salman2, J.M. Irache2 , Immunoadjuvant Unit, Departments of Microbiology1 and Pharmaceutical Technology2, University of Navarra, Pamplona, Spain; [email protected]
Oral immunization using polymeric vectors including nanoparticles has been considered as a suitable oral antigen delivery system. However, conventional nanoparticles are considered as non-specific bioadhesive carriers, since they did not display the capacity to target or interact specifically with specific regions within the gut, including Peyer’s patches. The result is their rapid eliminated due to the continuous mucus turnover and intestinal movements gastrointestinal tract. In order to overcome this drawback has been proposed the design of new carriers with specific distribution profiles within the gut mediated by the use of ligands able to bind to specific receptors. For this purpose, we have designed and evaluated a polymeric vector resulting from the association between Gantrez® AN [poly (methyl ethyl ether-co-maleic anhydride)] nanoparticles and mannosamine or flagellin (from Salmonella Enteritidis) in order to obtain bioadhesive nanoparticles able to interact specifically with the mucosa of the gastrointestinal tract. The gut distribution profile of mannosylated nanoparticles or flagellin nanoparticles indicated a strong bioadhesive capacity and an important ileum tropism. Moreover, fluorescence microscopy demonstrated a strong uptake of these carriers by Peyer’s patches. Immunization studies were performed in BALB/c mice using ovalbumin (OVA) as antigen model to be loaded in nanoparticle formulations. A single dose of OVA (20 µg for S.C. route or 100 µg for oral route) were administered in the free form or loaded in modified (flagellin or mannosamine) nanoparticles. The use of the linkers increased the systemic specific antibody response (IgG1 and IgG2a) and the intestinal secretory IgA levels compared to unmodified nanoparticles. In conclusion, we propose that mannosylated nanoparticles of flagellin nanopartciles as promising non-live vectors for oral delivery strategies.
G1 – Keynote Paper STRUCTURE-FUNCTION RELATIONSHIPS OF NANOMATERIALS 59 AND THEIR TOXICITY - A GENERAL INTRODUCTION TO NANOMATERIALS AND THEIR USES IN MEDICINE Vicki Colvin, Center for Biological and Environmental Nanotechnology, Rice University, 6100 Main Street, MS-63, Houston, Texas 77005, USA; [email protected]
Traditionally, nanotechnology has been motivated by the growing importance of very small (d < 50nm) medicinal, computational, and optical elements in diverse technologies. Our effort has considered broadly how these features can be leveraged in environmental technologies. We consider how the nano/bio interface manifests itself in cell culture systems, thus giving an indication of the toxic effects of a nanoparticle. For example, we consider the environmental chemistry and biological interactions of a model nanostructure, carbon-60 (C60). Though a hydrophobic substance, fullerenes form stable, sub-micron, colloidal clusters which can persist in water for long times under a variety of conditions. This behavior makes fullerenes more accessible to biological systems and permits them to
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have a marked effect on bacteria and cells in culture. However, it is the structure of the C60 aggregate that determines its function in a biological entity. Specifically, the more water-soluble derivatives are covalently attached to the surface of the C60 cage, the less toxic it becomes. The ability to change the structure of the nanomaterial, thus tune its toxicity, will indeed greatly advance the current research in drug delivery, diagnosis, and patient recovery.
G1 – Invited Paper NANOPARTICLE-INDUCED BIOLOGICAL RESPONSES: EFFECT OF 60 SIZE, SHAPE AND SURFACE CHEMISTRY Hong Yang, Xiaowei Teng, Department of Chemical Engineering, University of Rochester, 206 Gavett Hall, Rochester, NY 14627; and Alison C. Elder, Nancy Corson, Department of Environmental Medicine, University of Rochester School of Medicine, 575 Elmwood Avenue, Rochester, NY 14642; [email protected]
The property of nanomaterials can depend heavily on the size, shape and surface chemistry. It is not unexpected that these structural properties are important factors which can affect the response biological systems upon the exposure to nanomaterials. In this talk, I will first present several methods for controlling the size and shape of colloidal metal and metal oxide nanoparticles. Monodisperse iron oxide nanoparticles and platinum shapes have been used as model systems for understanding the cellular responses, following the exposure to nanometer-sized materials. Surface of these nanoparticles has been modified and their compatibility in culture medium has been examined accordingly. The toxicity of nanoparticles was followed in cultured lung epithelial cells (A549) based on the lactate dehydrogenase (LDH) release or luciferase reporter activity. For platinum nanoparticles, different shapes (particle, multipod and porous particle) have been synthesized in solution phase (Teng, Yang, Nano Lett., 2005, 5, 885; Teng, Liang, Maksimuk, Yang, Small, 2006, 2, 249). The shape-depedent the oxidative stress and toxicity in cultured epithelial and endothelial cells have been studied. I will present the observed difference in shape-related biological response and discuss the possible mechanism.
G1 – Invited Paper PHYTOTOXICITY OF SOME MANUFACTURED NANOPARTICLES 61 Daniel J. Watts, Otto H. York Center for Environmental Engineering and Science, New Jersey Institute of Technology, University Heights, Newark, NJ 07102; and Ling Yang, Department of Environmental Engineering, Beijing University of Chemical Technology, 15 North Sanhuan East Road, Beijing 100029, P.R. China; [email protected]
The rapid development of nanotechnology and the growing application of nano-sized substances are bringing large quantities of manufactured nanomaterials into the environment. Epidemiological studies have suggested that fine particles with aerodynamic diameter smaller than 2.5 μm have a relative association with various adverse health effects
71 Oral Abstracts in humans. Studies have indicated that manufactured nanomaterials may impose harmful effects on human health. Results suggesting harm were also observed in aquatic organisms. Little or no investigation though has been reported on the effects of nano-sized materials in plant systems. In this study, toxicities of manufactured nanoparticles including 13-nm alumina from Degussa, 14-nm hydrophilic silica from Cabot, and 21-nm titania from Degussa, are evaluated by means of a root elongation test. Six plant species, Zea mays (corn), Avena sativa (oat), Cucumis sativus (cucumber), Glycine max (soybean), Brassica oleracea (cabbage), and Daucus carota (carrot) were used in this study of the phytotoxicity of nanoparticles. For comparison purposes, phytotoxicity of larger particles, including 161.2-nm hydrophilic silica (produced at NJIT), 1.0-μm alumina (Atlantic Equipment Engineers, Inc), 667.6-nm hydrophilic silica (Anonymous Manufacturer), and 0.96-μm titania (Atlantic Equipment Engineers, Inc), were investigated. It was found that 1) the 667-nm silica, the 0.96-μm titania, and the 1.00-μm alumina showed no toxic effects on plant root elongation, 2) among the nanoparticles, the 13-nm alumina could retard the seedling development, whereas the 14-nm silica increased root elongation, and no effects were found for the 21-nm titania, 3) the 161-nm silica had an inhibitory effect. Results of further studies on toxicity mechanisms induced by the 13-nm alumina nanoparticles suggested that phytotoxicity of the nanoparticles seemed to be related to 1) surface properties, not size properties of the nanoparticles, 2) oxidation reaction between the nanoparticles and the plant organisms, and 3) the ability of nanoparticles to enter the root.
G1 – Invited Paper COMPLEMENT-MEDIATED HYPERSENSITIVITY REACTIONS TO 62 NANOPARTICULATE DRUGS AND SOLVENT SYSTEMS Janos Szebeni, US Military HIV Research Program, 1600 E Gude Drive, Rockville, MD 20850 and Nephrology Research Center, Semmelweis University, Hungarian Academy of Sciences, Budapest 1089 Nagyvarad ter 4, Hungary; [email protected]
One of the new trends in modern pharmacotechnology is to apply nanoparticulate drug carriers, vehicles or solvents in order to increase the therapeutic index of existing drugs. Beside all demonstrated clinical benefits or promising preclinical data, this approach unavoidably entails at least one problem, the induction of acute hypersensitivity (HSR, or anaphylactoid, or idiosyncratic) reactions. The phenomenon is due to the physical and/or chemical similarity of drug particles to many natural pathogens (viruses, bacteria and other microbes) against which the immune system is normally sensitized. However, this state of sensitization differs from that mediated by IgE, as it arises as a consequence of activation of the complement (C) system. Hence the new term, “C activation-related pseudoallergy” (CARPA). Drugs and agents causing CARPA include radiocontrast media (RCM), liposomal drugs (Doxil, Ambisome and DaunoXome) and micellar solvents containing amphiphilic lipids (e.g., Cremophor EL, the vehicle of Taxol). These agents activate C through both the classical and the alternative pathways, giving rise to C3a and C5a anaphylatoxins that trigger mast cells and basophils for secretory response that underlies HSRs. Recent studies suggest that it might be possible to screen candidate drugs or drug carriers for C activation and subsequent CARPA, using C split product (SC5b-9 ELISA)
72 Oral Abstracts assays and the porcine “cardiovascular distress” model, wherein quantification of the hemodynamic, cardiopulmonary and ECG changes allows for the assessment of the reactogenic potential of particulate drugs and agents. In conclusion, our studies suggest that the CARPA concept and the in vitro and in vivo assays that we use may help in the prediction, prevention and treatment of the acute immune toxicity of numerous state-of-the-art drugs.
G1 – Contributed Paper AQUEOUS DISPERSIONS OF CARBON NANOTUBES FOR 63 BIOLOGICAL TESTING AND APPLICATIONS Natalia Varaksa, Tammy Oreskovic, and Paul Rice, National Institute of Standards and Technology; Materials Reliability Division, Boulder, Colorado 80305, USA; [email protected]
Due to their exceptional structural, mechanical, and electrical properties, as well as their chemical inertness, carbon nanotubes (CNTs) are often envisioned as candidates for biological and medical materials and devices. Some of the possible applications include drug and vaccine delivery, tissue engineering (neural and orthopedic tissue implants, vascular grafts), biosensors, and biocomposites. However, the high hydrophobicity of CNTs and their tendency to remain in bundles make them difficult to form homogeneous and stable dispersions in liquids, especially in aqueous solutions. Aqueous solutions and cell culture media are of particular interest in biological and medical studies, which prompted our investigation into aqueous dispersion of CNTs. One way to make CNTs more hydrophilic and thus to facilitates their dispersibility in aqueous solutions is to immobilize certain surfactants or polymers on CNT walls and/or edges. In addition a critical consideration, for biological applications, is that these surfactants and polymers should be nontoxic and biocompatible. In our work, we focused on the selection and testing of various surfactants considering their dispersive power of multi-walled carbon nanotubes (MWCNTs), and also their biocompatibility with living cells. These suspensions were evaluated by optical (due to the size of MWCNTs) and scanning electron microscopies. Their stability was evaluated by visual inspection over the course of several days. For biocompatibility tests the response of vascular smooth muscle cells (VSMCs) and fibroblasts to MWCNTs was studied. The viability of cells cultured in the presence of MWCNT suspensions was evaluated using optical and electron microscopies as well as Alamar Blue (a cell proliferation assay). Results of the Alamar Blue indicated that liquid suspensions of MWCNTs did not present cytotoxic activity against VSMCs and fibroblasts. These results permit us to claim that MWCNTs coated with nontoxic surfactants are biocompatible with VSMCs and fibroblasts, and can be considered as candidates for biomedical applications.
G1 – Contributed Paper TOXICOLOGICAL IMPACT STUDIES BASED ON ESCHERICHIA COLI 64 BACTERIA IN AN ULTRAFINE ZnO NANOPARTICLES COLLOIDAL MEDIUM
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Roberta Brayner and Fernand Fiévet, Interfaces, Traitements, Organisation et Dynamique des Systèmes (ITODYS), UMR-CNRS 7086, Université Paris 7-Denis Diderot, case 7090, 2 place Jussieu, 75251 Paris Cedex 05, France; Roselyne Ferrari-Iliou and Marc F. Benedetti, Laboratoire de Géochimie des Eaux, UMR-CNRS 7047, Université Paris 7 & IPGP - 2 place Jussieu, 75251 Paris Cedex 05, France ; [email protected]
We report here preliminary studies of toxicological impact of ZnO nanoparticles on Escherichia coli bacteria. ZnO nanoparticles were synthesized in diethylene glycol (DEG) medium by forced hydrolysis of ionic Zn2+ salts. Particle size and shape were controlled by addition of small and macromolecules such as tri-n-octylphosphine oxide (TOPO), sodium dodecyl sulfate (SDS), polyoxyethylene stearyl ether (Brij-76) and bovine serum albumin (BSA). Transmission electron microscopy (TEM) and X-ray diffraction (XRD) analyses were used to characterize particle structure, size and morphology. Bactericidal tests were performed in Luria-Bertani (LB) medium on solid agar plates and in liquid systems with different concentrations of small and macromolecules and also with ZnO nanoparticles. TEM analyses of bacteria thin sections were used to study biocidal action of ZnO materials. The results confirmed that E. coli cells after contact with DEG and ZnO were damaged showing a Gram-negative triple membrane disorganization. This behavior causes the increase of membrane permeability leading to accumulation of ZnO nanoparticles in the bacterial membrane and also cellular internalization of these nanoparticles.
H1 – Keynote Paper PHOTODYNAMIC THERAPY IN THE CHORIOALLANTOIC 65 MEMBRANE MODEL: COMBINATION THERAPY USING ASPIRIN ENHANCES PHOTODYNAMIC SELECTIVE DRUG DELIVERY AND INFLUENCE OF THE SIZE OF TCPP-LOADED NANOPARTICLES ON PHOTOTOXICITY Hubert van den Bergh, Elodie Debefve, Bernadette Pegaz, Jean-Pierre Ballini; Swiss Federale Institute of Technology (EPFL), LPAS- Station 6, CH-1015 Lausanne, Switzerland; [email protected]
Vascular hyperproliferation characterises several diseases, including cancer and age-related macular degeneration (AMD). One therapeutic approach that is based in part on the closure of the vascular networks associated with these diseases is photodynamic therapy (PDT). The final result is vascular collapse and blood flow stasis leading to the occlusion of the pathological irradiated neovascularization. Two studies in the chorioallantoic membrane model (CAM), intending to improve the outcome of it are described here: 1) Combination therapy using aspirin enhances photodynamic selective drug delivery. The aim of the present study was to develop a novel combination therapy approach, in which we take advantage of the transient enhanced vascular permeability in PDT using aspirin, to selectively release at the location where PDT is inducing the vascular response, chemotherapeutic agents in the case of treatment of a malignant tumor or antiangiogenic factors to prevent growth/regrowth of the neovasculature in the case of AMD. The synergy
74 Oral Abstracts of the highly localised chemotherapy with local closure of the blood vessels that supply the tumour then leads to new and more effective treatment possibilities for various neoplasms. 2) Influence of the size of TCPP-loaded nanoparticles on phototoxicity. To be useful as a therapeutic regimen, some selectivity between the target tissue and the neighbouring healthy structures must be achieved by choosing a suitable delivery system for the photosensitizer (PS). Hence, the main goal of the present study was to develop a polymeric carrier system such as nanoparticles (NP) for PS intended to be intravenously administrated, capable of increasing the therapeutic index of the PS. To assess the effect of the size of NP in terms of photothrombic activity, four sizes of TCPP-loaded NP (121-343nm) were synthetized using the salting-out technique with biodegradable polymer. The PDT-induced vacular damage proportionaly decreases with the size of the TCPP-loaded NP.
H1 – Invited Paper NANOPARTICLE SELF-LIGHTING PHOTODYNAMIC THERAPY FOR 66 CANCER TREATMENT Wei Chen, Nomadics Inc., 1024 S. Innovation Way, Stillwater, OK 74074, USA; [email protected]
The purpose of this new design is to find a more efficient treatment for cancer while reducing the risk of radiation damage. To reach this goal, we proposed a novel method for cancer treatment that combines radiotherapy and photodynamic therapy (PDT). Under this concept, scintillation or persistent luminescence nanoparticles with attached photosensitizers, such as porphyrins, are used as a new type of agent for photodynamic therapy. Upon exposure to ionizing radiation such as X-ray, scintillation or persistent luminescence will emit from the nanoparticles to activate the photosensitizers; as a 1 consequence, singlet oxygen ( O2) is produced to augment the killing of cancer cells by ionizing radiation. With this novel therapeutic approach, no external light is necessary to activate the photosensitizing agent within tumors. Thus, this new modality is called nanoparticle self-lighting photodynamic therapy.
H1 – Contributed Paper TIME-RESOLVED SPECTROSCOPY STUDY OF THE PHOTOTHERMAL 67 PROPERTIES OF GOLD NANOCAGES Min Hu, Jingyi Chen, Xingde Li, Younan Xia Department of Chemistry and Department of Bioengineering, Univ. of Washington, Seattle, WA 98195; Hristina Petrova, Gregory V. Hartland, Department of Chemistry and Biochemistry, Univ. of Notre Dame, Notre Dame, IN 46556; Manuel Marquez, INEST Group, Research Center, Philip Morris USA Inc., Richmond, VA 23234; [email protected]
Gold nanocages were synthesized from silver nanocube templates via a galvanic replacement reaction. The Surface Plasmon Resonance (SPR) peak of the nanocages can be tuned into the near-IR region, which makes them promising candidates for photothermal
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therapy and biological imaging applications. Gold nanocages of different sizes dispersed in water solution have been studied by ultrafast time-resolved spectroscopy. The experimental results show that the energy relaxation time increases as the size of the particles increases, and the relaxation time is independent of the initial particle temperature with different excitation powers. Modulations due to coherently excited vibrational modes were also observed in the transient absorption traces. The period of the excited vibrational motion is proportional to the dimensions of the particles. For a specific size of nanocage, experiments with different excitation powers of the pump laser were performed (from 2 µJ to 17 µJ), we found that the period of the vibrational mode increased with the laser intensity. This was compared to experiments on spherical gold nanoparticles, which allow us to roughly estimate the temperature of the nanocages when the electrons and the phonons reach the equilibrium. We estimate that the 17 µJ pump energies produce a lattice temperature of 1100 ± 100 K in the nanocages, which is near the melting point of the bulk metal, while the particles maintain their integrity. This makes the gold nanocages potentially useful for photothermal therapy applications.
H1 – Invited Paper LASER IRRADIATED GOLD NANOPARTICLES: A NEW TOOL FOR 68 SELECTIVE MAINPULATION OF CELLS AND BIOMOLECULES Gereon Hüttmann, Marco Bever, Institute of Biomedical Optics, University Luebeck, Germany; Cuiping Yao, School of Life Science and Technology, Xi’an Jiaotong University; China, Ramtin Rahmanzadeh, Johannes Gerdes, Research Center Borstel, Germany; Florian Lewolth, Elmar Endl, Institute of Molecular Medicine and Experimental Immunology, University. Bonn, Germany
The strong absorption of gold nanoparticles in the visible spectral range allows the localized generation of heat in a volume of only a few tens of a nanometer. Irradiation with a pulsed lasers can easily heat up the particles above the melting temperature of gold. Due to their small volume cooling by heat diffusion is very efficient. Therefore temperatures above 1000°C can be confined to the surroundings of the nanoparticles with heating times in the sub-nanosecond range. Pulsed irradiation of gold nanoparticles is therefore expected to cause very localized chemical, thermal or mechanical modifications to cells and biomolecules. We demonstrate the selective destruction of proteins, the permeabilization of the cell membrane and selective killing of cells by laser-irradiated gold nanoparticles. Conjugates made from the different enzymes with nanoparticles served as a model system for investigating the effect of laser-irradiated nanoparticles on proteins. We were able to show, that an inactivation of the enzymes is possible with very high spatial confinement. Permeabilization was studied systematically in different cells lines. Antibodies against membrane proteins were conjugated to gold nanoparticles. The conjugates than were bound to the cell membrane and subsequently irradiated by nanosecond and picosecond laser pulses. Transient permeabilization was observed for 10 kDa Dextran. Efficacies of more than 60% were attained under optimal conditions with only 27% cell death. When irradiated with higher pulse energies, cells to which the gold particles were bound, were effectively killed. In mixed cell cultures of different lymphoma cells, the targeted cells
76 Oral Abstracts were killed with over 95% efficacy without significantly affecting the non-targeted cells. In combination with selectively binding antibodies, laser-irradiated gold nanoparticles allow a precise and effective destruction of biomolecules and cells, which can be used for a new kind nanoparticle mediated cell surgery (NPCS). Possible application are protein knock-out, cell purging or a selective depletion of cells from cell or tissue culture.
H1 – Invited Paper ANGIOGENIC VESSEL-TARGETED CANCER THERAPY 69 Naoto Oku, Tomohiro Asai, Kosuke Shimizu, Sei Yonezawa, Univ. of Shizuoka, Dept. of Medical Biochem., Yada, Suruga-ku, Shizuoka 422-8526 Japan; [email protected]
Since angiogenic endothelial cells have growing character, these cells are expected to be degenerated by anticancer drugs like as cancer cells. We have developed a new modality of cancer treatment by use of DDS technology, in which anticancer drugs are effectively delivered to the angiogenic endothelial cells of a solid tumor. Anti-neovascular therapy (ANET) may cause indirect lethal damage of tumor cells through the damage of newly formed blood vessels with reduced side effects. Moreover, this therapy overcomes drug-resistance tumors. To develop liposomal DDS drugs that target angiogenic vessels, we previously isolated peptides specific for tumor angiogenic vasculature using a phage-displayed peptide library, and thus obtained APRPG peptide was used for liposomal modification. APRPG-PEG-modified liposomes encapsulating adriamycin or entrapping a hydrophobized prodrug of CNDAC, a novel anticancer drug, caused strong tumor growth suppression through possible damaging of angiogenic endothelial cells. The accumulation of APRPG-PEG-liposomes was quite similar to that of control PEG-liposomes. However, intratumoral distribution of APRPG-PEG-liposomes was quite different from the control liposomes: The former colocalized with endothelial cells, and the latter accumulated the surrounding of the blood vessels when the intratumoral distribution of fluorescence-labeled liposomes was examined by confocal laser scanning microscopy. By the way, pancreatic cancer is intractable, and shows poor development of angiogenic vessels suggesting a large number of tumor cells are controlled by a small number of vessel cells. Therefore, we applied ANET to pancreatic cancer model prepared by orthotopical implantation of human pancreatic tumor line cells, and observed the superior growth suppression of the tumor by the treatment of angiogenic vessel-targeted liposomal adriamycin. The present study provides a novel modality of cancer treatment.
H1 – Invited Paper DEVELOPMENT OF TUMOR TARGETING MAGNETIC 70 NANOPARTICLES FOR CANCER THERAPY Robert Ivkov1, Sally J. DeNardo2, Laird A. Miers2, Arutselvan Natarajan2, Allan R. Foreman1, Cordula Gruettner3, Grete N. Adamson2, and Gerald L. DeNardo2; 1Triton BioSystems, Inc., Chelmsford, MA; 2Radiodiagnosis and Therapy, Molecular Cancer Institute, University of California, Davis, Sacramento, CA; 3Micromod Partikelteknologie GmbH, Rostock, Germany;
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Magnetic nanoparticles will rapidly heat, via hysteresis losses, when activated by an alternating magnetic field (AMF). Systemic delivery of magnetic nanoparticles conjugated to a monoclonal antibody (MAb) offers potential for selective and minimally invasive cancer treatment by heating the microenvironments of cells containing particles embedded in cancer tissue. One to two MAb’s per nanoparticle were conjugated using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide HCl (EDC), 111In-7,10-tetraaza cyclododecane-N, N’, N’’, N’’’-tetraacetic acid-ChL6 (111In-DOTA-ChL6) to carboxylated polyethylene glycol (PEG-COOH) on dextran-coated iron oxide 20 nm particles. After magnetic purification and sterile filtration, pharmacokinetics, histopathology, and AMF/bioprobe therapy were done using 111In-ChL6 bioprobe intravenous doses (20μg/2.2mg ChL6/ bioprobe) with 50μg ChL6 in athymic mice bearing HBT3477 xenografts. A 153 kHz AMF was applied 72 hr post injection (pi) for therapy with amplitudes of 1,300, 1,000 or 700 Oersteds. Weights, blood counts and tumor size were monitored and compared with control mice receiving nothing, or AMF or bioprobes alone. 111In-ChL6 bioprobe binding in vitro to HBT3477 cells was 50 to 70% of that of 111In-ChL6. At 48 hours, tumor, lung, kidney, and marrow uptake of the 111In-ChL6 bioprobes were not different from that observed in prior studies of 111In-ChL6. Significant therapeutic responses from AMF/bioprobe therapy were demonstrated with up to eight times longer mean time to quintuple tumor volume with therapy compared with no treatment (P = 0.0013). Toxicity was only seen in the 1,300 Oe AMF cohort, with 4 of 12 immediate deaths and skin erythema. Electron micrographs showed bioprobes on the surfaces of the HBT3477 cells of excised tumors and tumor necrosis 24 hours after AMF/bioprobe therapy. This study shows that MAb-conjugated nanoparticles (bioprobes), when given intravenously, escape into the extravascular space and bind to cancer cell membrane antigen. Bioprobes can be used in concert with externally applied AMF to deliver thermoablative cancer therapy.
H1 – Contributed Paper METHACRYLAMIDE-OLIGOLACTATES AS BUILDING BLOCKS FOR 71 TARGETED BIODEGRADABLE POLYMERIC MICELLES TO DELIVER PHOTOSENSITIZERS Cristianne J.F. Rijcken, Jan-Willem Hofman, Femke van Zeeland, Aissa Ramzi, Wim E. Hennink and Cornelus F. van Nostrum, Deptartment of Pharmaceutics, Utrecht Inst. for Pharmaceutical Sci. (UIPS), Utrecht Univ., P.O. Box 80082, 3508TB, Utrecht, The Netherlands; [email protected]
A class of generally very hydrophobic drugs is the group of photosensitizers, which are currently under investigation in photodynamic therapy (PDT) to treat cancer. Upon irradiation with light of a specific wave length, photosensitizers generate a variety of reactive oxygen species. The radical compounds damage various cell components and ultimately lead to cell death. However, the hydrophobicity of many photosensitizers results in an unfavorable biodistribution (e.g. prolonged skin toxicity) and aggregation with
78 Oral Abstracts subsequent quenching of the excited state, lower radical production, etc. These side effects necessitate the photosensitizers to be encapsulated in an adequate carrier system to deliver them specifically to the tumor regions, preferably in a solubilized state. It is the aim of this study to apply biodegradable thermosensitive polymeric micelles as a delivery vehicle for photosensitizers and to target the carrier specifically to neck/head cancer cells. The polymers used in this study are based on methacrylamide-oligolactates, which display thermosensitive behavior that is transient under physiological conditions due to hydrolysis of the lactate side chains. Amphiphilic block copolymers synthesized using a PEG-macroinitiator, self-assemble in aqueous media into highly condensed spherical micelles above the cloud point (CP) of the thermosensitive block. Cleavage of the lactic acid side chains causes a gradual increase of the critical micelle temperature (CMT) and leads to controlled destabilization and eventually dissolution of the thermosensitive biodegradable polymeric micelles. The CP and the destabilisation time period can be tailored by varying the methacrylamide backbone and the length of the oligolactate chain. A degradation-controlled release of a novel, highly potent photosensitizer (a substituted silicon phthalocyanine) was observed in vitro. The hydrophobic core of the micelle and physiological destabilization can be exploited for a controlled delivery of drugs.
H1 – Invited Paper PORPHYRIN-NANOPARTICLE SENSORS FOR DETECTING 72 CELLULAR RESPONSE(S) TO OXIDATIVE STRESS ASSOCIATED WITH REACTIVE OXYGEN SPECIES AND PDT Leanne B Josefsen, Ross W Boyle, Photobiology and Photomedicine Group, Departments of Chemistry and Clinical Biosciences, The University of Hull, Cottingham Road, Hull, East Yorkshire, HU6 7RX, UK; [email protected]
The sensor is based on a porphyrin and a polyacrylamide nanoparticle, which bear specific functionalities to allow conjugation of the two components. The conjugates are designed to allow real-time monitoring of intra-cellular signals in response to oxidative stress caused by reactive oxygen species, such as those generated during PDT. By selectively functionalising the porphyrin to carry an isothiocyanato group and the nanosensor free primary amines, an isothiourea bond is formed between the porphyrin and nanosensor conjugate. The role of the porphyrin is (i) to promote localisation to specific sub-cellular compartments and (ii) to generate reactive oxygen species, similar to those released in porphyrin-mediated PDT. The first of these two criteria is met by imparting a net cationic charge on the porphyrin. Reactive oxygen species are then generated by exciting the porphyrin at a specific wavelength. The subsequent cellular response can then be monitored by the nanosensor. The nanosensor works by encapsulating an ion-selective fluorophore within the sensor matrix. The fluorophore selectively interacts with specific analytes/ions that have passed through surface pores into the sensor matrix, allowing real-time monitoring of the cells response. Insertion of these porphyrin-loaded sensors into cells and subsequent irradiation with light of an activating wavelength, generates reactive oxygen species. By controlling the amount of porphyrin attached to the sensors and the light dose absorbed by the porphyrin, the amount of reactive species generated can be
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varied and the cellular response to oxidative stress measured. The nanosensors desrcibed here thus have potential for studying cell signaling events occuring in PDT between photosensitiser activation and cell death, and also for elucidating mechanisms of cytotoxicity associated with the intracellular generation of reactive oxygen species in general.
D2 – Keynote Paper QUANTUM DOTS AS IMMUNOASSAY PROBES 73 Roger M. Leblanc, University of Miami, Department of Chemistry, 1301 Memorial Drive, Cox Science Center, Coral Gables, FL, USA; [email protected]
Peptides were incorporated with organic fluorophores and luminescent quantum dots (QDs) for molecular recognition studies as well as imaging of biological processes. Fluorescent peptides containing the Gly-His or Gly-Gly-His Cu2+ binding motifs selectively detected the metallic ion at the µM level in solution. Divalent copper was also selectively detected by similar peptide lipids at the air-water interface in 2D and Langmuir-Blodgett films. Photosensitive peptide systems and hydrogels were utilized to prepare Cu2+ membrane probes. Surface functionalization of cadmium chalcogenides QDs with peptides allowed detection of Cu2+ with nM detection potential. The photosensitive hydrogels can also physically immobilize QDs for preparation of hydrogel membrane probes. The layer-by-layer assembly technique helped to build ultra thin films of QDs and organophosphorus hydrolase (OPH) on quartz for detection of paraoxon, a potent neurotoxin. Paraoxon was detected at the nM levels. The results are compared to QD/OPH systems in solution. Staining of ß-amyloid (fragments and full length) aggregation process in solution, dry films, and at the air-water interface was monitored with QDs. Langmuir films were further utilized to characterize and manipulate the 2D self-assembly of QDs at the air-water interface. Self-assembled QD monolayers can be deposited onto solid substrates utilizing the LB film deposition technique. Practical applications include fabrication of analytical solid devices with controlled nanoparticle organization.
D2 – Invited Paper DEVELOPMENT OF FRET-BASED QD-BIOCONJUGATE SENSORS 74 FOR DETECTING ENZYMATIC ACTIVITY Hedi Mattoussi, Igor L. Medintz, Aaron R. Clapp, U.S. Naval Research Laboratory, Washington, DC 20375; Optical Sciences Division and Center for Bio/Molecular Science and Engineering; and Florence M. Brunel, Philip E. Dawson, Departments of Cell Biology & Chemistry, The Scripps Research Institute, La Jolla, CA 92037; [email protected]
We have previously demonstrated the use of self-assembled QD-protein-dye bioconjugates to design sensing assemblies based on Förster resonance energy transfer - (FRET) for signal transduction. Solution-phase sensors specific for the detection of the nutrient maltose and the explosive TNT were developed. The sensor design exploited a few
80 Oral Abstracts inherent benefits of using QDs over conventional organic fluorophores for FRET, including: 1-the ability to size-tune fluorescent emission as a function of QD core size to better match overlap with a particular acceptor; and 2-the option of arraying multiple acceptors around a QD to increase the overall FRET efficiency. In the present investigation, we apply these QD-bioreceptor assemblies to detect the activity of several proteolytic enzymes in vitro. In particular, we focus on the digestion of protein and cleavage of peptide substrates by specific proteolytic enzymes. Assembling QD-peptide-dye and QD-protein-dye conjugates brings the dye in close proximity to the QD and results if efficient FFET between dots and dye, which translates in loss of QD photoemission. Digestion of the dye-labeled protein or peptide by the target enzyme displaces the free dye from the QD surface and reduces the FRET efficiency. Measuring changes in the FRET efficiency allows monitoring of the specific protease activity. A calibration curve derived from an increasing number of acceptors per donor allowed transformation of the FRET changes into units of enzymatic activity. We applied this sensing concept/design to detect activity of endopeptidases proteinase K or papain and several peptidases (e.g., caspace-1 and thrombin). These peptidases are involved in inflammation and blood coagulation, respectively. Changes in FRET efficiency were also monitored when inhibitors were added to the peptide containing assay solutions. Analysis of the data was carried out within the framework of the Michaelis-Menten treatment using different mechanisms of inhibition. Practical applications for this technology will also be discussed.
D2 - Contributed Paper GIANT MAGNETIC QUANTUM DOTS AS INDIVIDUALLY 75 ADDRESSABLE HIGH SENSITIVE OPTICAL BAR CODES FOR DIAGNOSTICS AND BIOANALYTICS D. Müller-Schulte, MagnaMedics GmbH, Martelenberger Weg 8, D-52066 Aachen, Germany, [email protected]; P.Borm, Hogeschool Zuyd, NL-6400 Heerlen, The Netherlands
Spherically-shaped nano- and micro particles on the basis of silica gels were synthezised using a newly designed inverse suspension technology. Total transparency, no autofluorescence and varied bead sizes from 0,5 to 3000 µm are some of the unique features of these prepared beads. In addition, the new silica bead technology for the first time allows the simultaneous encapsulation of quantum dots, fluorescence dyes and magnetic colloids. By encapsulating a huge number of diverse quantum dots into the silica matrix, extremely strong luminescent micro and nano carriers (“giant quantum dots “) are obtained covering the whole visible spectral range. The newly designed particles can be ideally used for individually optical coding of biomolecules and cells. The application is exemplarily demonstrated for tumor cell screening and proteomics.
D2 – Contributed Paper SYNTHESIS OF ULTRA-STABLE BARE CDS NANOCRYSTALS WITH 76 STRONG LUMINESCENCE BY HYDROTHERMAL SYNTHESIS
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Yongxian Wang, Ronghui Xu, Wanbang Xu, Radiopharmaceutical centre of Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China; and Guangqiang Jia, Instrumental Analysis and Research Centre of Shanghai University, Shanghai 200444,China; [email protected]
Ultra-stable bare CdS quantum dots (QDs) with a size between 1.5 nm and 8 nm and strong luminescence were synthesized directly by hydrothermal synthesis method. The nanocrystals emit highly symmetric fluorescence whose full width of half-maximum (fwhm) is as narrow as 80 nm. The bare QDs retain robust keeping in reacting mixtures for at least 45 days. The parameters influencing stability, size and fluorescence are discussed in detail. Having used a combination of X-ray Diffraction (XRD), Atomic Force Microscopy (AFM), High Resolution Electron Microscopy (HRTEM), Fluorescence/UV-VIS spectroscopy, EDS to analyze the chemical composition, size, shape, and crystal structure etc, we found that the semiconductor quantum dots of CdS produced via hydrothermal method were not only ultra-stable but also highly luminescent, mainly owing to the perfect structure of QDs.
D2 – Invited Paper COLLOIDAL SEMICONDUCTOR QUANTUM DOTS IN SELECTIVE 77 LABELING OF PATHOGENIC BACTERIA Todd D. Krauss and Megan A. Hahn, University of Rochester, Department of Chemistry, Rochester, New York, 14627-0216, Joel S. Tabb, Agave BioSystems, Ithaca, NY 14850, [email protected]
Having diameters of only a few nanometers, colloidal CdSe semiconductor quantum dots (QDs) are highly emissive, spherical, inorganic particles that exhibit size-tunable physical properties due to the effects of quantum confinement. Typically, these materials are synthesized with relatively inert hydrocarbon surface-groups that must first be modified if they are to be compatible with biological systems. To that end, a variety of different strategies have been established to make these hydrophobic surfaces of CdSe QDs applicable for use in biology. We have demonstrated that CdSe/ZnS core/shell QDs functionalized with streptavidin bind specifically to pathogenic Escherichia coli O157:H7 cells labeled with biotinylated antibodies. Using fluorescence microscopy of individual bacterial cells and standard fluorimetry of bacterial cell solutions, we will present results comparing E. coli O157:H7 cells labeled with QDs to cells similarly labeled with a standard dye, fluorescein isothiocyanate (FITC). We will also present the results of using flow cytometry to analyze bacterial cell mixtures, which contained pathogenic E. coli O157:H7 cells amid increasing concentrations of innocuous E. coli DH5α cells. With their numerous advantages over traditional organic dyes, semiconductor QDs should also be advantageous in high-throughput techniques such as flow cytometry. Indeed, use of QDs to label the pathogenic serotype showed greater detected percentages of labeled cells, higher fluorescence intensities, and lower limits of detection (1%) compared to an analogous labeling done with FITC. The particular biochemical interactions and surface
82 Oral Abstracts functionalizations incorporated in these methods can easily be generalized to allow for the rapid and selective detection of other common pathogens.
D2 – Invited Paper A GENERIC APPROACH TO BIOMOLECULAR FUNCTIONALITY ON GOLD 78 AND SILVER NANOPARTICLES Robert T. Tshikhudo,a,b Jennifer L. Brennan,a Claire Rees,a Zhenxin Wang a and Mathias Brust a; aNanoscale Science Centre, Chemistry Department, The University Of Liverpool, Liverpool, L7 7ZD, UK, bProject AuTEK, Advanced Materials Division, Mintek, Private Bag X 3015, Randburg, 2125, South Africa; [email protected]
The exploitation of monolayer protected clusters (MPC s) of gold and to a lesser extent silver, is rapidly advancing both in applied and fundamental research due to their ease of preparation, extreme stability and tunable optical properties. It is generally understood that the extreme stability of these materials is conferred by the thiolate-ligands, which form a protective shell around the particles to which they are attached via strong Ag/Au-S interaction. The optical properties of the particles are controlled by particle size, shape and state of aggregation. Since most MPCs are not water-soluble, significant effort to overcome this limitation has recently been made with the aim to make them suitable for biological applications. To explore the usefulness of these materials, particularly in biomedical and/or pharmaceutical areas, it is vital to introduce specific functionalities, which will address potential applications in these areas. To combine excellent water solubility, stability and functionality of MPCs, we have developed a generic approach to introduce biomolecular functionality such as proteins and oligonucleotides on Au and Silver nanoparticles. Here we present the facile synthesis of these hydrophillically functionalised Au and Ag nanoparticles, with special emphasis on their ease of preparation and functionalisation. The versatility of these functional MPCs is highlighted. This includes the ability to demonstrate that the attached biomolecules retain their functionality, for example, in the case of attaching enzymes to an MPC ligand shell. These particles are ideal candidates for use as drug and/or gene carrier, as bio-labels and for large-scale diagnostic purposes, and it is possible to introduce specific recognition capabilities of choice.
D2 – Invited Paper EPITOPE PRESENTATION ON GOLD NANOPARTICLES FOR 79 IMMUNOASSAY DEVELOPMENT David E. Cliffel, Aren Gerdon, Brian Huffman, David W. Wright, Department of Chemistry, Vanderbilt University, Nashville, TN 37235-1822, USA; [email protected]
Monolayer protected clusters provide a unique nanoparticle structure for the exploration of the biological interactions. With a central metal core surrounded by a protecting thiolate ligand, the clusters allow for a combination of functions similar to that of many biomacromolecules like many proteins. One of the key chemical functions of biological systems that we can hope to mimic is antibody-antigen interactions using epitope
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presentation on the nanoparticle. This talk will present our recent progress in these areas by creating antigenic nanoparticles for antibody recognition. Initial studies with glutathione protected gold clusters demonstrated binding of polyclonal anti-glutathione antibodies to the nanoparticle using the quartz crystal microbalance technique. Progress includes successful efforts to create nanoparticles that show strong binding to monoclonal antibodies against specific peptide target epitopes recreated on the nanoparticle surface. These peptide-functionalized nanoparticles simulate the antibody binding site, i.e. the epitope for two specific diseases. The first epitope, hemaggluttatin, is abundantly present on the surface of influenza virus and is a linear epitope of 6 amino acids. The second epitope is based on the protective antigen protein of bacillus anthracis infection. This second epitope has been explored in both a linear and loop configuration the cluster, with the stronger binding observed in the loop configuration suggesting a conformational epitope that involves creation of peptide secondary structure off of the cluster surface. An immunoassay using the quartz crystal microbalance and epitope presenting clusters has been developed for antibody diagnostics in patient sera.
D2 – Contributed Paper SURFACE MODIFICATION OF PARTICLES ENHANCES 80 INTRACELLULAR UPTAKE Volker Mailänder, Myriam Kern, Karin Nothelfer, Institute for Clinical Transfusion Medicine and Immunogenetics Ulm, Department of Transfusion Medicine, University of Ulm, 10 Helmholtzstr., Ulm 89081, Germany; Anna Musyanovych, Verena Holzapfel, Katharina Landfester, University of Ulm, Department of Organic Chemistry III / Macromolecular Chemistry, 11 Albert-Einstein-Allee, Ulm 89081, Germany; [email protected]
Labeling of cells with nanoparticles for in-vivo detection is interesting for various biomedical applications e.g. labeling of cells with superparamagnetic nanoparticles. The objective of this study was to evaluate the feasibility and efficiency of labeling cells with particles without using transfection agents. We hypothesized that surface charge would influence cellular uptake. The particles were synthesized by the miniemulsion process. A hydrophobic fluorescent dye was embedded in these nanoparticles and served as reporter. As most transfection agents are positively charged molecules we synthesized particles with different surface charge by adjusting the amount of copolymerized monomers with amino groups thus enabling to study the cellular uptake in correlation to this parameter. Uptake of the particles or attachment of nanoparticles were detected by FACS measurements. This was performed in four different cell lines: mesenchymal stem cells (MSC), HeLa, Jurkat, and KG1a. These cell lines were chosen as they can serve as models for clinically interesting cell types. For these cell lines - with the exception of MSCs - a clear correlation of surface charge and fluorescence intensity was shown. Most interestingly no transfection agents which are not approved for in-vivo application in humans were needed for an efficient uptake of the particles. As FACS cannot differentiate between extracellular and intracellular localization and subcellular localization we used confocal laser scanning microscopy (LSM) and transmission electron microscopy (TEM) to reveal differences in subcellular localization of the particles. In MSCs and HeLa particles were mostly located
84 Oral Abstracts inside of cellular compartments resembling endosomes, while in Jurkat and KG1a, particles were predominantly located in clusters on the cell surface. Scanning electron microscopy showed microvilli to be involved in this process.
I1 –Keynote Paper POLYMERIC MICELLES: POTENTIAL AS PHARMACEUTICAL 81 NANOCARRIERS Vladimir P. Torchilin, Department of Pharmaceutical Sciences, Northeastern University, Boston, MA 02115, USA; [email protected]
Micelles represent an important class of nanocarriers for poorly soluble pharmaceuticals. Among polymeric micelles, micelles formed by amphiphilic polymer-lipid conjugates (such as polyethyleneglycols or polyvinylpyrrolidones conjugated at one terminus with lipid residues) are of a special interest because they form very stable micelles with low CMC values. These micelles maintain stability upon dilution and in biological fluids, demonstrate prolonged circulation times in vivo (mice, rats, rabbits), and can be effectively loaded (from 5 to 50 % by weight) with various poorly soluble pharmaceuticals, such as various anticancer drugs (taxol, tamoxifen, camptothecin, porphyrins) or hydrophobic and amphiphilic diagnostic agents. Drug-loaded micelles demonstrate an increased accumulation in experimental tumors and infarcts via the enhanced permeability and retention (EPR) effect. The potential of polymeric micelles as pharmaceutical carriers may be still further improved by attaching targeted ligands including specific antibodies, to the micelle surface. Using polyethylene glycol-phosphatidyl ethanolamine (PEG-PE) conjugate activated at its distal water-exposed end with the p-nitrophenylcarbonyl (pNP) group, pNP-PEG-PE, as a micelle component, we have been able to attach to micelles various monoclonal antibodies (monoclonal tumors-specific 2C5 anti-nucleosome antibody and monoclonal infarct-specific 2G4 anti-myosin antibody). Anticancer drug (taxol)-loaded 2C5 antibody-bearing micelles provided a sharp increase in killing cancer cells in vitro and in tumor models in mice. Preferential accumulation of plain and targeted micelles loaded with various therapeutic or diagnostic agents in experimental tumors and areas of experimental myocardial infarction in animals was also demonstrated. Polymer-lipid-based micelles and immunomicelles represent a new family of convenient nanocarriers for poorly soluble pharmaceuticals.
I1 – Invited Paper SELF-ASSEMBLY OF SURFACTANT AIDED DELIVERY SYSTEMS 82 Lise Arleth, Rex P. Hjelm, Los Alamos Neutron Science Center, Los Alamos National Laboratory; Beena Ashok and Hyat Onyuksel, Department of Biopharmaceutical Sciences, College of Pharmacy, University of Illinois, Chicago; [email protected]
Advanced drug delivery systems based on self-assembled surfactant particles require detailed understanding of the factors controlling size and shape, to tailor the particles and predict their behavior with drug loading and in vivo. Among the factors that may control
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particle morphology is the inherent interfacial curvature of surfactants and the free energy required to bend the interface. A key idea underlying considerable theoretical and modeling work is that inherent interfacial curvature can be controlled by mixing surfactants with different inherent curvature. Although this idea is in qualitative agreement with observation, there is not sufficient experimental work to validate the models quantitatively. We studied aqueous suspensions of mixed surfactants with different inherent curvature, egg yolk phosphaticylcholine (PC) and poly(ethyleneglycol) (PEG) modified distearoyl phospatidylethanolamine (DSPE), at molar mixing ratios from 0:100 to 30:70 (DSPE-PEG:PC) using PEG with molar mass of either 2 or 5 kDa,. We used neutron, x-ray and light scattering data on particle morphology for comparison with theoretical predictions. Considerable latitude in controlling the particle shape was found. At high DSPEPEG:PC ratios, spheroid micelles were formed. As the PC content was increased, the micelles became elongated to tri-axial prolate ellipsoids. At a DSPEPEG:PC molar ratio of 30:70 rodlike micelles longer than 1000 Å were formed. In all cases the micelle internal structure consisted of a hydrophobic core, surrounded by a dense hydrophilic layer of PC heads condensed with part of the PEG chain. The outer layer consisted of the remaining part of the PEG chains in overlapping Gaussian random coils. The data gave quantitative agreement with an existing model, but showed that the mushroom to brush transition of PEG, suggested by other work, is not a likely determinant of interfacial curvature. We discuss the implications of this work for tailored particle delivery of sparingly soluble drugs.
I1 – Invited Paper POLYMERIC MICELLES FOR TUMOR TARGTED DELIVERY OF 83 P-GLYCOPRTEIN INHIBITORS Afsaneh Lavasanifar, Hamidreza Montazeri Aliabadi, Dion R. Brocks, Faculty of Pharmacy & Pharmaceutical Sciences, University of Alberta, Edmonton, AB, Canada T6G 2N8; [email protected]
One of the major causes of failure in cancer treatment is multi-drug resistance (MDR), where cancer cells simultaneously become resistant to different anticancer drugs. Over-expression of membrane efflux pumps like P-glycoprotein (P-gp) that recognizes different chemotherapeutic agents and transports them out of the cell, plays a major role in MDR. P-gp inhibitors have shown little benefit in overcoming drug resistance in clinic because systemic administration of P-gp inhibitors will reduce the elimination of P-gp substrates (e.g., anticancer drugs) from healthy cells that overexpress P-gp leading to intolerable toxicities by anticancer drugs. The objective of this research is to develop a tumor targeted carrier that permits specific delivery of P-gp inhibitors to tumorogenic P-gp. Polymeric micelles have shown promise in tumor targeted delivery of hydrophobic drugs. Although P-gp inhibitors are structurally diverse, they share the physicochemical property of lipophilicity necessary for plasma membrane penetration. This makes them ideal candidates for incorporation in polymeric micelles. We have focused on self-associating poly(ethylene oxide)-b-poly(ε-caprolactone) (PEO-b-PCL) block copolymers as biodegradable polymeric nanocarriers for tumor targeted delivery of P-gp inhibitors. PEO-b-PCL copolymers were assembled to carriers of 80-100 nm, caused a 100
86 Oral Abstracts fold increase in the aqeous solubility of CsA and retained 94 % of their drug content after 24 h. In healthy rats, a 6.1 fold increase in the blood AUC was observed for polymeric micellar CsA compared to commercial formulation of CsA. PEO-b-PCL micelles reduced uptake of encapsulated CsA by kidney and caused a reduction in the nephrotoxicity of CsA. The creatinine clearance was lowered (1.9 fold) by the commercial formulation but did not change when polymeric micellar CsA was injected for 7 days. Polymeric micelles caused 1.7 and 2.5 fold increases in the blood and tumor concentrations of CsA, respectively, in Lewis Lung Carcinoma (LLC1) bearing mice. Further studies are underway to define whether tumor targted delivery, would lead to an increased therapeutic benefit for P-gp inhibitors.
I1 - Invited Paper ENGINEERING COPOLYMER MATERIALS AND MICELLES FOR 84 TARGETED DELIVERY OF HYDROPHOBIC DRUGS C. Allen, Faculty of Pharmacy, University of Toronto, 19 Russell St., Toronto, ON, M5S 2S2, [email protected]
Block copolymer micelles have gained increasing attention as a viable formulation strategy for hydrophobic drugs. The micelles may serve as solubilizers and/or true drug carriers depending on their stability and drug retention properties in vivo. Polymeric micelle systems have been shown to increase the aqueous solubility of hydrophobic drugs up to 30 000 fold. Micelle systems have also been shown to enhance the circulation lifetime and reduce the systemic toxicity of some drugs. The full exploitation of micelles as a formulation and/or carrier technology requires a firm understanding of the relationships between the properties of the copolymer, the physico-chemical properties of the micelles and their performance (i.e. drug loading capacity, drug retention properties, pharmacokinetics profile, biodistribution). Recent research efforts in our laboratory have demonstrated that block copolymer micelles have great potential for use in the targeted delivery of hydrophobic drugs. Firstly, it has been found that micelles may be designed to retain their stability in vivo even at copolymer concentrations below the critical micelle concentration of the material. Our studies have characterized the in vivo distribution kinetics and fate of copolymer following i.v. administration of 1) copolymer assembled as thermodynamically stable micelles, 2) copolymer assembled as thermodynamically unstable micelles and 3) copolymer single chains. Secondly, targeted micelles may be successfully prepared by conjugation of specific ligands to the surface of micelles. Specifically, the epidermal growth factor (EGF) was attached to the micelle surface and the EGFR-targeted micelles were found to localize in the perinuclear region as well as the nucleus of EGFR-overexpressing breast cancer cells. The EGFR-targeted delivery system may not only serve to increase the overall intracellular accumulation of an encapsulated agent, but may also allow for the delivery of agents to the perinuclear or nuclear regions of the cell. Overall, findings from these studies demonstrate the ability to tailor block copolymer micelles for effective targeted delivery of specific hydrophobic drugs.
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I1 – Invited Paper NANODELIVERY USING BLOCK COPOLYMERS: A PROPOFOL 85 SOLUTION François Ravenelle, Labopharm Inc., 480 Blvd Armand-Frappier, Laval, Québec H7V 4B4, Canada; [email protected]
A novel bacterio-static and lipid free, nanocarrier [poly(N-vinyl-2-pyrrolidone)-block- poly(D,L-lactide) copolymer] formulation of propofol has been developed that addresses the deficiencies of currently available versions. The formulation is presented as a lyophilized cake which reconstitutes instantly in all aqueous media to produce a clear liquid that is both filterable and compatible with commonly used infusion media. The reconstituted solution is stable over a wide range of ionic strengths, pH and drug concentrations and has been shown to remain stable for more than 5 days after reconstitution. Non-clinical studies in rats have shown the formulation possesses pharmacokinetic and pharmacodynamic properties equivalent to Diprivan(r).
I1 – Invited Paper TUMOR TRIGGERED ACTIVATION AND RELEASE OF DRUGS FROM 86 LIPID BASED DRUG AND PRODRUG NANOCARRIERS Kent Jørgensen, LiPlasome Pharma A/S, Technical University of Denmark, Building 207, DK-2800 Lyngby, Denmark; and Thomas L. Andresen, LiPlasome Pharma A/S, Technical University of Denmark, Building 207, DK-2800 Lyngby, Denmark; [email protected]
The selectivity of anticancer drugs in targeting the tumor tissue presents a major problem in cancer treatment. We report a new generation of liposomal nanocarriers that can be used for enhanced anticancer drug and prodrug delivery to tumors. The liposomes are engineered to be particularly degradable to secretory phospholipase A2 (sPLA2), which is a phospholipid hydrolyzing enzyme that is significantly upregulated in the extracellular microenvironment of cancer tumors. Thus, when the long circulatory liposomal nanocarriers extravasate and accumulate in the interstitial tumor space, sPLA2 will act as an active trigger resulting in the release of cytotoxic drugs in close vicinity of the target cancer cells. The local degradation of the phospholipids by sPLA2 in the tumor results in the generation of lysolipid and fatty acid hydrolysis products, which advantageously can function as locally released membrane permeability promoters facilitating the intracellular drug uptake. In addition, the liposomal membrane can be composed of a novel class of prodrug phospholipids that can be converted selectively to active anticancer agents by sPLA2 in the tumor. The integrated drug discovery and delivery technology offers a promising way to rationally design novel tumor activated liposomal nanocarriers for better cancer treatment.
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J1 – Invited Paper NON-CROSSLINKING AGGREGATION OF DNA-CARRYING 87 NANOPARTICLES FOR GENE DIAGNOSIS Mizuo Maeda, RIKEN Institute, Bioengineering Lab., Hirosawa, Wako 351-0198, Japan; [email protected]
We developed a reliable method for detecting single nucleotide mutation by using spontaneous aggregation of a DNA-carrying colloidal nanoparticle, which was made from poly(N-isopropylacrylamide) (polyNIPAAm) grafted with probe DNA chains. The particle with the diameter of 50 nm was formed through self-assembly of polyNIPAAm-graft-DNA copolymers above the phase transition temperature (ca. 36 degree C) of polyNIPAAm part. The particle was determined to consist of 20 graft copolymers each of which has 10 graft chains of the probe DNA, suggesting that one particle has 200 probes on it. When adding the complementary DNA of the equimolar amount into the colloidal dispersion, the particles were found to aggregate within a minuite in buffer (pH 7.4) containing 0.5M NaCl through the salt concentration-dependent aggregation. In contrast, they kept dispersing in the presence of the one point-mutated DNA under the identical conditions. Interesting and important is that the phenomenon is extraordinary sensitive to the terminal mismatch of duplex formed on the particle. The results demonstrated that the non-crosslinking aggregation of DNA-carrying nanoparticles is useful for analyzing various SNPs when coupled with the primer extention assay. The core material is not restricted to PNIPAAm; DNA-functionalized gold nanoparticle (15 nm diameter) was found to show a similar aggregation phenomenon induced only by the fully-complementary DNA, resulting in rapid color change within 3 min at ambient temperature. This methodology is general in principle and applicable for wide variety of clinical diagnosis.
J1 - Invited Paper GLUATATHIONE DEGRADADABLE AND pH MODULATABLE 88 POLYCATION/DNA NANOPLEXES FOR GENE DELIVERY Cameron Alexander,a Jon A Preeceb and Len Seymourc, a School of Pharmacy, University of Nottingham; bSchool of Chemistry, University of Birmingham; cDepartment of Clinical Pharamacology, University of Oxford, United Kingdom; [email protected]
The potential benefits of successful gene therapy cannot yet be fully realised, mainly because of difficulties of gaining adequate delivery and expression of therapeutic transgenes. This research aims to develop an efficient, fully defined and safe system for delivery of DNA and RNA to cells, based on novel self-assembled polyplexes (colloidal polycation/nucleic acid particles). The polyplexes will be bio-responsive, designed to combine stability during the extracellular delivery phase with triggered intracellular destabilisation to maximise activity of the contained nucleic acids. The work will encompass a number of aspects vital to the development of new vectors for gene delivery, including modulation of synthetic vector-nucleic acid binding, transport of polyplexes
89 Oral Abstracts through cell membranes and triggered release of nucleic acids within the cytoplasm and/or nucleus. Specifically, we will: (i) Synthesise a series of cationic polymers based on basic poly(amino acids) and correlate their chemical properties with the physicochemical properties of the polyplexes they form with nucleic acids, with a view to maximising their endosomolytic activity, (ii) Evaluate the design of polyplexes for triggered intracellular destabilisation, by reduction, to enable intracellular release of the nucleic acid and achieve maximal therapeutic activity, (iii) Examine the incorporation of thiol-bearing targeting agents (to endow endothelial cell-selectivity) and endosome-escape agents (e.g. melittin-SH) to maximise cell-specific biological activity. The proposed programme requires a multidisciplinary approach, including organic and polymer synthesis, biomolecular recognition, molecular biology and pharmacology, and combines the research of three complementary academic groups. The results from this project will have relevance in fields varying from pharmaceutical chemistry to cell biology and medicine. The presentation will detail progress made to date.
J1 – Contributed Paper# LIPID PHASE CONTROL OF DNA DELIVERY 89 Rumiana Koynova, Li Wang, Robert C. MacDonald, Department of Biochemistry, Molecular & Cell Biology, Northwestern University, Evanston, IL 60208, USA; [email protected]
Cationic lipids form nanoscale complexes (lipoplexes) with polyanionic DNA, and can be used to deliver DNA to cells for transfection. Lipid-mediated DNA delivery (lipofection) is now considered highly promising, but its mechanism is still largely unknown, and the transfection efficiency is unsatisfactorily low for many cell types. Understanding the mechanism of gene delivery by cationic liposomes is thus of utmost importance for effective gene therapy. Here we report a correlation between the delivery efficiency of cationic phospholipid DNA carriers and the mesomorphic phase behavior of their mixtures with negatively charged membrane lipid compositions. Specifically, formulations that are particularly effective DNA carriers, form nonlamellar phases of highest negative interfacial curvature (micellar, hexagonal, cubic) when mixed with negatively charged membrane lipids, whereas less effective formulations form phases of lower curvature; moreover, this is true when the mixed cationic/membrane lipid aggregates exhibit the lamellar-nonlamellar phase transition within the physiological temperature range, ~37°C. Lipid vehicles are well known to exhibit maximum leakiness and contents release in the vicinity of phase transitions, especially those involving nonlamellar phase formation. Structural evolution of the carrier lipid/DNA complexes upon interaction with cellular lipids is hence proposed to be a controlling factor in lipid-mediated DNA delivery. A strategy for optimizing lipofection is deduced: Lamellar lipoplex formulations with compositions close to the lamellar-nonlamellar phase transition, which could easily undergo such a transition upon mixing with membrane lipids, are especially promising lipoplex candidates. The ability to rationally assess critical structural characteristics and
90 Oral Abstracts phase behaviors of lipoplex components would represent an unprecedented step forward for this form of gene therapy vector.
J1 – Invited Paper GENE DELIVERY AND QUANTUM DOT ASSISTED INTRACELLULAR 90 TRAFFICKING Charudharshini Srinivasan,1 Jeunghoon Lee2, Fotios Papadimitrakopoulos2, Lawrence K. Silbart3, and Diane J. Burgess1. 1Dept of Pharmaceutical Sciences, 69 North Eagleville Road, Unit 3092, 2Institute of Materials Science, 97 North Eagleville Road, Unit 3136, 3Dept. of Animal Science, 1390 Storrs Road, Unit 4163, University of Connecticut, Storrs, CT 06269, USA] [email protected]
Despite their low transfection efficiencies, non-viral vectors (such as, cationic and anionic lipids, cationic polymers and polymeric nanoparticles) are actively being pursued as a means of gene delivery due to their low immunogenicity and low toxicity in comparison to viral vectors. To better understand and improve transfection efficiency, it is essential to quantify the processes involved in cellular uptake and trafficking of exogenous DNA into nucleus. Semiconductor nanocrystal quantum dots (QDs) are excellent candidates for fluorescent tagging in biological applications since they display long-term resistance to photobleaching. A novel method of QD-DNA conjugation is presented that allows long-term tracking of DNA within the cellular environment. Cadmium selenide/zinc sulphide QDs were encapsulated in maleimide functionalized PEG-PE2000. Plasmid DNA labeling with QDs was achieved using a SPDP-PNA linker. Photostability of QD-DNA conjugates were compared to DNA complexes with the organic dye, rhodamine. Time lapse imaging experiments were performed using a confocal laser scanning microscope (Leica SP2 Spectral Confocal Microscope). Transfection of QD-DNA conjugates (enhanced green fluorescent protein (EGFP)) was conducted in CHO-K1 cells in the presence of LipofectamineTM2000. QD-DNA labeling was confirmed by AFM imaging. Clusters of QDs were observed on the plasmids presumably at the PNA binding site. The QD-DNA conjugates were photostable throughout a 100 min continuous laser excitation study in in vitro cell culture. The QD-DNA conjugates were capable of expressing the reporter protein (EGFP) allowing simultaneous tracking of plasmid DNA at the subcellular level. Quantification of EGFP expression revealed 68% transfection efficiency for the QD-DNA conjugates which is comparable to control plamid DNA (72% transfection efficiency), indicating no loss of activity on conjugation. Acknowledgements: The authors thank Minhua Zhao and Shafiuddin Shafiuddin, University of Connecticut for technical assistance. This research was supported in part by a Parenteral Drug Association Foundation Schering-Plough Grant.
J1 – Invited Paper MULTIFUNCTIONAL NANOPARTICLES FOR IMAGING ASSISTED 91 GENE THERAPY Yong Zhang, Wee Beng Tan, Division of Bioengineering, Faculty of
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Engineering, National University of Singapore, 9 Engineering Drive 1, Singapore 117576; [email protected]
There has been immense interest in luminescent nanocrystals due to their ideal optical properties and promising applications in fields such as biological labelling. In particular, these luminescent nanocrystals have been exploited for their superb optical properties, such as excellent photostability and high quantum yields, in their use as biological labels over conventional organic flourophores. However, as most of them are prepared in an organic medium and are not soluble in water, they are not suited for any bio-applications. Furthermore, these nanocrystals are not biocompatible. To overcome these problems, different polymerization techniques to incorporate the nanocrystals into polymer microbeads have been used and reported. While these microbeads are generally very useful for multiplexed immunoassays, they are not suitable for the staining or labeling of subcellular components or intracellular measurements as they are relatively large in size. In this work, biocompatible chitosan nanobeads encapsulating luminescent nanocrystals were prepared, which can be used as fluorescent markers for immunoassay/cell labeling. Chitosan is a biopolymer with amino and hydroxyl groups on the surfaces that can be used for further attachment of biomolecules. Furthermore, as chitosan beads are very small, they can be used to label subcellular components such as intracellular proteins or to study the intracellular delivery of nanoparticles. At the same time, chitosan has also been widely used to deliver therapeutic genes by intravenous, oral and mucosal administration. However, the biodistribution of nanoparticles and the mechanism of their delivery to cells and tissues remain unclear. Using the multi-functional chitosan nanobeads developed, imaging techniques can be used to study the biodistribution of nanoparticles and their intracellular pathway, and the same results can be used for the delivery of genes or other drugs, using chitosan as a carrier.
J1 – Contributed Paper CLUSTER ASSEMBLED NANOSTRUCTURED TIO2 FILM MEDIATES 92 EFFICIENT AND SAFE RETROVIRAL GENE TRANSDUCTION IN PRIMARY ADULT HUMAN MELANOCYTES FOR EX-VIVO GENE THERAPY Roberta Carbone, Ida Marangi, Andrea Zanardi, Luisa Lanfrancone, Saverio Minucci, Pier Giuseppe Pelicci, European Institute of Oncology, Via Ripamonti 435, I-20141 Milan, Italy; and Luca Giorgetti, Elisabetta Chierici, Gero Bongiorno, Paolo Piseri, Paolo Milani, CIMAINA and Dipartimento di Fisica, Università di Milano, Via Celoria 16, I-20133 Milan, Italy; [email protected]
Primary human melanocytes represent the precursor cells to melanoma and also specific targets of inherited and acquired pigmentation disorders. The possibility to genetically manipulate these cells for biological studies or therapy has not been fully exploited for the lack of efficient and safe stable gene transfer techniques. We have developed a new gene transfer technology on primary adult human melanocytes by means of retroviral infection in absence of any toxic polycations currently employed to improve infection efficency (i.e.
92 Oral Abstracts polybrene). We demonstrate that this polycation induce apoptosis and DNA damage through ATM phosphorylation, causing a further genetic perturbation to cells. We have cultured primary human melanocytes on a biocompatible nanostructured TiO2 film, obtained by the deposition of a supersonic beam of titania clusters, coated by retroviral vectors expressing GFP. By means of a “reverse infection “ mechanism we achieved 80% of infection in absence of any toxic effect. This high efficiency is related to the interaction of retroviral vectors and cells with the nanoparticle-assembled substrates. We expect that such technology based on nanostructured surfaces will allow efficient and safe genetic manipulation of primary cells for ex-vivo gene therapy.
J1 – Contributed Paper JC VIRUS-LIKE PARTICLES: SIZE DEPENDENT IN VITRO DNA 93 PACKAGING AND GENE DELIVERY Oliver Ast, Andrezj Citkowicz Linda Cashion, Brent Larsen, Richard N. Harkins, and Harald Petry, Departments of Gene Therapy Research and Pharmacology, Berlex Biosciences, Richmond, CA, USA; [email protected]
Virus-like particles (VLP) are known to be a promising alternative to the existing viral and non-viral DNA delivery systems but also have been used to deliver RNA and small molecules. VLPs are self-assembled structures composed only of proteins necessary for capsid formation and DNA packaging. They are replication deficient and exhibit a similar morphology and cell tropism compared to their viral origin. One of the major differences between VLPs and traditional viral vector systems is the manner in which DNA is packaged. For viral delivery systems the use of packaging cell lines is indispensable whereas the DNA packaging into the VLP can be accomplished by controlled and reproducible in vitro methods. In this study we present an in vitro protocol to package plasmid DNA into JCV-derived VP1-VLP. JCV is a non-enveloped virus with a 50nm icosahedric capsid containing three proteins VP1, VP2 and VP3 enclosing the viral genome of 5kb. It has been shown that the expression of the major structural protein VP1 is sufficient to generate VLPs with the same size and morphology as the original JCV. VP1-VLPs were dissociated into VP1 pentamers before DNA packaging. In the presence of DNA the pentamers reassembled efficiently into VLPs in a CaCl2 containing buffer. VLPs packaged with a 5 kb EGFP expression plasmid were successfully used for cell transfection. Analytical characterization of the VLPs indicated a 2.5 fold increase in size compared to JC-virus. After DNase treatment however, the particles showed the expected size of 50nm but transfection efficiency was reduced. A 1.6 kb DNA fragment expressing GFP was then used to investigate if smaller DNA molecules are packaged more efficiently. The 1.6 kb fragment was efficiently packaged, the DNA was DNase resistant and VLPs showed the expected size. The transfection efficiency of VLPs packaged with the 1.6kb fragment was higher compared to the VLPs packaged with the original 5kb plasmid. The packaging and DNA delivery efficiency of the 5kb EGFP expression plasmid could be significantly increased by condensing the DNA used for packaging with histones. In this study we describe a simple and reproducible in vitro method to efficiently and quantitatively package DNA into JCV derived VP1-VLPs that results in high transfection efficiency.
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J1 – Invited Paper WELL-DEFINED CORE-SHELL NANOPARTICLES WITH BRANCHED 94 POLY(ETHYLENIMINE) SHELLS AS EFFICIENT GENE DELIVERY CARRIERS Lijun Li, Man Fai Leung, Junmin Zhu,1 Angie Tang, 1 Lai Pang Law,1 Min Feng1, Kin Man Ho1, Daniel K. L. Lee1, Frank W. Harris,2 Pei Li1 Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, P. R. China; [email protected]; 2Maurice Morton Institute of Polymer Science, University of Akron, Ohio, USA
Spherical, well-defined core-shell nanoparticles that consist of poly(methyl methacrylate) (PMMA) cores and branched poly(ethylenimine) shells (b-PEI, 25kDa) were prepared in aqueous in the absence of surfactant. The PMMA-PEI core-shell nanoparticles were approximately 150 nm in diameters, and displayed zeta-potentials above +40 mV. Plasmid DNA (pDNA) could effectively complex onto the nanoparticles in various biological mediums, and average diameter of the complexed particles was approximately 100 to 120 nm with high homogeneity. The complexing ability of the nanoparticles was strongly dependent on the molecular weight of the PEI and the thickness of the PEI shells. The stability of the complexes was influenced by the loading ratio of the pDNA and the nanoparticles. The condensed pDNA in the complexes was significantly protected from enzymatic degradation by DNase I. Cytotoxity studies suggested that the PMMA-PEI core-shell nanoparticles were three times less toxic than the b-PEI polymer (25 kDa). Transfection efficiencies of both PMMA-PEI nanoparticles and b-PEI polymer were evaluated at different N/P ratio, transfection time, cell density and cell line. Furthermore, investigation of the intracellular behavior of FITC-labeled pDNA/nanoparticles using a flow cytometric analysis indicated that the PMMA-PEI nanoparticles were effective carriers to deliver the pDNA into cells.
Plenary 2 PROGRESS IN PHOTONIC CRYSTAL CLINICAL SENSORS 95 Sanford A. Asher, Jeremy P. Walker, Kyle W. Kimble, Michelle M. Muscatello and Matti Ben-Moshe, University of Pittsburgh, Department of Chemistry, Pittsburgh, PA 15260, USA; [email protected]
We have been developing a new clinical sensing technology based on crystalline colloidal arrays embedded in hydrogels. These photonic crystal materials diffract light. We add molecular recognition agents to these materials which actuate volume phase transitions that shift the spacing between particles which shifts the wavelength of the diffracted light. We will review our progress in developing new sensors which can be used in point-of-care bedside clinical chemical applications.
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A3 – Invited Paper CHARACTERIZATION AND BIOLOGICAL APPLICATIONS OF 96 COLLOIDAL NANOPARTICLES Wolfgang J. Parak, Center for Nanoscience, Ludwig Maximilians Universität München, Amalienstraße 54, 80799 München, Germany; [email protected]
Colloidal quantum dots are semiconductor nanocrystals well dispersed in a solvent. The optical properties of quantum dots, in particular the wavelength of their fluorescence, depend strongly on their size. Because of their reduced tendency to photobleach, colloidal quantum dots are interesting fluorescence probes for all types of labeling studies. In this review we will discuss the biological relevant properties of quantum dots and focus on the following topics: 1) How can individual nanoparticles be modified with an exactly controlled number of biological molecules. 2) How can nanoparticles of different materials be used for drug delivery. Also biocompatibility issues will be discussed.
A3 – Invited Paper BIOMIMETIC PROCESSING METHODS FOR ORGANIC-INORGANIC 97 PARTICLES Vishal M. Patel, Chelsea M. Magin, and Laurie B. Gower, Department of Materials Science & Engineering, University of Florida, Gainesville, FL, USA; [email protected]
“Soft”-“Hard” Core-shell particulate systems are being developed for use in pharmaceutical and other controlled release and uptake applications. The technology is based on the ability to deposit coatings of a “hard” mineral shell composed of calcium carbonate onto “soft” emulsion colloids. These robust composite particles are prepared using a biologically-inspired mineralization process, in which soluble acidic polymers act as process-directing agents to generate droplets of a fluidic mineral precursor which selectively adsorb onto the charged surface of surfactant stabilized oil droplets. The amorphous mineral precursor encapsulates the oil-filled colloid, and then solidifies and crystallizes into a uniform crystalline shell of calcite. Core-shell particles on the order of 1 to 4 μm have been successfully synthesized. The metastable structure of the calcium carbonate makes it biodegradable, as well as biocompatible, providing a novel system for controlled release applications. The particles bear a striking resemblance to our biological inspiration, dinoflagellate cysts, in which the unicellular organism encapsulates itself within a porous CaCO3 shell. We are now seeking to mimic the organism’s ability to regulate porosity in the mineral shell, which would be valuable for controlled uptake applications, such as in toxicity reversal of drug overdose, which requires rapid removal of toxins from the bloodstream. In this case, the mineral shell needs to be a porous molecular filter, such that when the particles are introduced into the bloodstream, the oil-based core can sequester toxic levels of lipophilic drugs. Using a phase-segregated binary amphiphile system, we have been able to demonstrate that porosity can be templated on flat free-standing mineral films deposited at the air-water interface of Langmuir monolayers.
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Next, we hope to transfer this organic-inorganic templating approach to the core-shell particles, ultimately enabling the high degree of morphological control found in biomineral systems.
A3 – Contributed Paper HYALURONAN-BASED HYDROGEL MICROSPHERES FOR VOCAL 98 FOLD REGENERATION Xinqiao Jia, Dept. of Mater. Sci. & Eng., University of Delaware, Newark, DE 19716; Yoon Yeo, Robert Langer, Dept. of Chem. Eng., Massachusetts Institute of Technology, Cambridge, MA 02139; Rodney J. Clifton, Tong Jiao, Div. of Eng., Brown University, Providence, RI, 02912; Daniel S. Kohane and Steven M. Zeitels, Massachusetts General Hospital, Boston, MA 02114; [email protected]
Vocal fold vibration depends critically on the viscoelasticity of the connective tissue of the vocal fold mucosa. Voice abuse or overuse can lead to scarring that disrupts the natural pliability of the lamina propria and results in vocal dysfunction. An estimated 5-10% of the population is affected by some degree of vocal fold scarring. Despite a well-recognized clinical need for a material to replace missing or damaged vibratory connective tissue of the vocal fold, materials have yet to be engineered specifically for this purpose. We have developed hyaluronan (HA)-based soft hydrogel microspheres (microgels) as an injectable remedy for vocal fold regeneration. Chemical modification of HA led to quantitative introduction of mutually reactive functional groups (hydrazide HA-Hdz and aldehyde HA-Ald) along its backbone. Alternatively, polyethylene glycol dialdehyde (PEG-DiAld, Mw 3400) was employed as the second component in place of HA-Ald. Simple mixing of the respective components resulted in bulk hydrogels that were degraded rapidly in the presence of hyaluronidase. On the other hand, in situ cross-linking within inverse emulsion droplets generated microgels with improved stability and defined functionality. These hydrogel microspheres are interesting in that they exhibit residual functional groups on their surfaces which can be used as reactive handles for covalent conjugation of therapeutic molecules. The presence of surface functional groups also allows for subsequent cross-linking of the microgels with other reactive polymers, giving rise to macroscopic hydrogels with tunable viscoelasticity. Mechanical measurements indicate that these microgel networks exhibit similar viscoelastic responses as the natural vocal fold lamina propria at phonation frequency range. In vitro cytotoxicity studies using vocal fold fibroblasts indicate that microgels synthesized from HA-Hdz/HA-Ald are highly biocompatible, whereas microgels derived from HA-Hdz/PEG-DiAld exhibit certain adverse effects to the cultured cells at high concentration (≥ 2 mg/ml). These HA-based microgel systems hold great promise for the treatment of vocal fold scarring, not just as an inert filler material, but instead as smart entities that can repair focal defects, smooth the vocal fold margin, and soften and dissolve scar tissue.
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A3 – Contributed Paper MINIEMULSIONS FOR BIOMEDICAL APPLICATIONS 99 Anna Musyanovych, Katharina Landfester, University of Ulm, Department of Organic Chemistry III / Macromolecular Chemistry, 11 Albert-Einstein-Allee, Ulm 89081, Germany; and Volker Mailänder, Institute for Clinical Transfusion Medicine and Immunogenetics Ulm, Department of Transfusion Medicine, University of Ulm, 10 Helmholtzstr., Ulm 89081, Germany; [email protected]
Polymeric particles in the sub-micron size range with advanced and controlled properties have been synthesized by the miniemulsion process. Miniemulsions consist of liquid nanodroplets in the size range of 50 to 500 nm and can be easily prepared by shearing the system consisting of oil, water and a surfactant. The growth of the nanodroplets can be effectively suppressed by using a strong hydrophobe and effective surfactant. The hydrophobe acts as an osmotic agent which stabilizes the system against Ostwald ripening. Such a system contains about 1018 droplets and each droplet behaves as an independent reaction vessel that can be identified as a “nanoreactor”. Different types of reactions can be performed inside the droplets or at their interface in order to design biologically interesting materials according to the medical needs and application. For example, labeling of cells with nanoparticles for in-vivo detection was performed by using fluorescent amino- or carboxyl-functionalized polystyrene nanoparticles. The particles of 100-180 nm were synthesized by miniemulsion copolymerization of styrene and a functional monomer in the presence of a fluorescent dye. The cell uptake was visualized using fluorescent microscopy. The presence of functional groups on the surface allows the covalent coupling of biomolecules and used the obtained nanoparticles for bio-specific cell recognition. Polymer nanocapsules with oil or aqueous core and various wall thicknesses were successfully prepaid by performing the reactions at the interface of miniemulsion droplets. By appropriately choosing the capsule wall material and reaction conditions, biocompatible/biodegradable nanocapsules can be synthesized and used as the potential carriers in drug delivery system. The miniemulsion technique was applied to prepare “nanoreactors” and perform the multiplication of DNA molecule via polymerase chain reaction (PCR) within these “nanoreactors”. When the droplets are in the size range smaller than 300 nm it is possible to obtain a single molecule chemistry situation.
A3 – Invited Paper TARGETING MONOCYTES BY NANO-PARTICULATED 100 FORMULATIONS AND VASCULAR HEALING Gershon Golomb, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel; [email protected]
Inflammation plays a key role in the progression of post-injury neointimal hyperplasia. Local inflammatory activation occurs in a timely fashion following injury. We validated the hypothesis that systemic and transient depletion of monocytes and macrophages inhibits restenosis. Monocytes/macrophage depletion was achieved with a systemic
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injection of liposomes or polymeric nanoparticles (NP) containing bisphosphonates (BP) formulated for effective phagocytosis. Following phagocytosis the vesicles discharge their encapsulated drug, inactivating the cell with no effect on non-phagocytic cells. We investigated the effect of different bisphosphonates (alendronate and clodronate), NP size (0.1, 0.2, 0.4 and 0.8nm), and phospholipids and polymer type on the formulation and bioactivity properties. The pharmacokinetics and pharmacodynamics have been evaluated by utilizing 67Ga encapsulated in liposomes. The bioactivity of the nanoparticulated dosage forms was assessed in cell cultures of RAW 264 macrophages and primary SMC of rat, rabbit and human. Inhibition of blood monocytes was measured by flow cytometry (FACS) following IV injection of different formulations, and in vivo therapeutic efficacy was studied in rat and rabbit restenosis models. Monocytes count was reduced by 70%, 24-48 hrs. after a single injection of alendronate liposomal formulation, at a dose 10-fold lower than that of clodronate. Monocyte blood levels returned to basal values after 7 days. A significant reduction of stenosis (>70%) was achieved in both rat and hypercholesterolemic-rabbit models of restenosis, without side effects. The NP dispose mainly to the liver and spleen with high uptake in the injured artery transported by circulating monocytes. It can be concluded that macrophage depletion by NP encapsulated BP is a promising therapeutic tool against restenosis after angioplasty.
A3 – Invited Paper OCULAR DRUG DELIVEY USING LIPID, POLYMER AND 101 SUSPENSION FORMULATIONS Ron Boch, Lina Hong, Yuan Huang, Ruihong Li, Philippe Margaron, Jasbinder Sanghera, Luis Sojo ; QLT Inc. 887 Great Northern Way, Vancouver, British Columbia, Canada V5T 4T5; [email protected]
The treatment of eye diseases such as age-related macular degeneration (AMD) necessitates drug delivery to the back of the eye. The in-vivo delivery of ocular drugs is achievable by the systemic route of administration or by directly injecting compounds into the eye. Drug candidates were screened in cellular screening assays. Poorly water soluble small molecule compounds were formulated for intravenous injection or for local administration to deliver test compounds to the back of the eye. Aqueous formulation strategies for liquid solutions and suspensions included a variety of approaches to enhance, or sustain the delivery to improve efficacy. One class of drugs, derived from Visudyne® (used in photodynamic therapy of certain eye diseases) called benzoporphyrin derivatives were administered intravenously. These drug formulations were assessed in vivo for systemic delivery. Alternatively, suspension formulations of poorly soluble small molecule angiogenesis inhibitors were administered locally into the eyes of anaesthetized animals. Delivery to the back of the eye was assessed by extracting the ocular tissues for active substance. Aqueous formulation strategies for poorly soluble candidates included the use of surfactants, lipids, polymers and cyclodextrins. Following intravenous administration into animals, the systemic delivery was found to be compound and formulation dependent. Suspension formulations that were locally injected into the eye achieved high levels of active substance in ocular tissue. Local and intravenous routes of administration effectively delivered poorly soluble test substances to the back of the eye.
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A3 – Invited Paper IMAGING CELL SURFACE RECEPTORS WITH LIGAND CONJUGATED 102 NANOCRYSTALS Sandra J. Rosenthal, Department of Chemistry, Vanderbilt University, Nashville TN 37235, USA; [email protected]
The simultaneous localization of several different proteins in situ is has been limited by the wide emission spectra and low photostabilities of fluorescent dyes traditionally used to study cell surface receptors, ion channels, and transporters. It has been demonstrated that core/shell semiconductor nanocrystals can overcome these limitations. We have developed small molecule, peptide, and antibody nanoconjugates for targeting cell surface receptors. The structure of the core/shell nanocrystals has been determined with exquisite resolution by atomic number constrast scanning transmission electron microscopy. The biological activity of the nanoconjugates is determined by competitive uptake assay. Imaging and detection applications in neuroscience and immunology will be discussed.
A3 – Contributed Paper ORAL INSULIN DELIVERY WITH BIODEGRADABLE AND 103 BIOCOMPATIBLE NANOCAPSULES Zhengmao Li, Robert Gyurik, Bentley Pharmaceuticals, Inc., 2 Holland Way, Exeter, NH 03833, USA; and Jerome Claverie, Materials Science Program, University of New Hampshire, Durham, NH 03824, USA; [email protected]
In protein and peptide delivery, researchers and consumers are always seeking viable alternatives to painful and bothersome injections. Among pulmonary, intranasal, transdermal and other options, oral delivery is the most preferred route and is also the most challenging route. In order to protect the hydrophilic therapeutic proteins and peptides from degradation in the intestine via oral delivery, we have developed a Nanocaplet™ delivery system, using poly(glutamic acid)-poly(lactic acid)-poly(ethylene glycol) triblock copolymers via self-assembly. The size of a Nanocaplet™ is as small as 100 nm. Due to the biodegradable and biocompatible nature of the copolymer, the safety concerns over nanotechnology can be resolved by our delivery system. Human insulin is used as a model protein. The pharmacokinetic animal (rat) study results show that insulin can be successfully delivered in Nanocaplet™ form through the intestine. The fact that the human insulin concentration in the blood peaks around 90 minutes after the administration, indicats protection and degradation of the Nanocaplet™ after uptaken by the epithelial cells in the intestine.
K1 –Keynote Paper THE NANOSCALE IN INHALATION DELIVERY 104 Philippe G. Rogueda, AstraZeneca R&D Charnwood, Bakewell Road, Loughborough, LE11 2HD, UK; [email protected]
Inhalation delivery covers 4 formulation platforms: dry powder inhalers (DPIs), pressure
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metered dose inhalers (pMDIs), nebulisers and nasal devices. There are currently no products on the market claiming the use of nanoparticles or nanodroplets for inhalation delivery, although some may unknowingly benefit from their presence. There is however a range of patents and delivery platforms available claiming the use of the nanoscale. Deposition of particles/droplets in the lung is normally associated with sizes between 1-5 μm. Particles below 100 nm are also known to reach the deep lungs, and these open numerous possibility for nanoparticle inhalation delivery. There have been some concerns about the toxicity of nanoparticles in the lungs, but these must be seen in the context of the chemical entity used. Most nebulisers are formulated as aqueous solutions, therefore nanodroplets production is a matter of fact, and can be achieved through adequate hardware. DPIs (dry powder inhalers) are yet to fully exploit nanoparticle delivery. The size distribution of a pMDI aerosol cloud is bimodal by nature, with 2 peaks: at 600 nm and 2 μm. Therefore a solution pMDI will deliver nanoparticles to the lungs. For suspension pMDIs, the delivery of nanoparticles necessitates the availability of dry, stable, individual nanoparticles. These are not available in commercially viable quantities yet. If it is assumed that nanoparticles can be made available, their fate in suspension will need to be considered, in particular their stabilisation. In addition, their fate in the aerosol droplets will need to be studied, as theories predict that agglomeration will occur, leading to the delivery of micron sized particles.
K1 – Invited Paper PARTICULATE SYSTEMS FOR PULMONARY DRUG DELIVERY: 105 SAFETY, SUSTAINED RELEASE, AND EFFICACY Uday B. Kompella and Devender Dhanda. College of Pharmacy, University of Nebraska Medical Center, Omaha, NE 68198-5840; [email protected]
Ou purpose in this work was to determine whether intratracheally administered large porous microparticles are safe and sustain celecoxib delivery in mice and to further assess their effectiveness in a mouse model for lung tumors. Celecoxib-PLGA (75/25) large porous microparticles were prepared using a modified solvent evaporation method followed by supercritical fluid processing. The pulmonary safety of celecoxib and PLGA particles (conventional and large porous) with and without celecoxib was assessed by determining lactate dehydrogenase (LDH) levels, total protein levels, and total and differential cell counts in the bronchoalveolar lavage fluid (BAL) and soluble collagen levels in the lung tissue of A/J mice 21 days following intratracheal administration. Lung sections were stained with hematoxylin and eosin (H&E) and Masson’s Trichrome stain for gross histological evaluations to assess the inflammatory and/or fibrotic response. To assess sustained drug delivery, particle formulations were administered in normal A/J mice and the plasma, BAL, and lung tissues were isolated on day 21 and the drug levels were quantified using an LC-MS assay. For efficacy studies, female A/J mice were administered 2 mg B[a]P orally in 0.2 ml of cottonseed oil on days 1, 4, and 7. Dry powder formulations of celecoxib, and PLGA microparticles with or without celecoxib (~90 µg of celecoxib) were administered intratracheally using a dry powder insufflator one week after the last dose of B[a]P. The animals were sacrificed on day 14 post-administration and prostaglandin E2 (PGE2) and vascular endothelial growth factor (VEGF) levels were
100 Oral Abstracts determined in the BAL. There was no significant hyperplasia, granulomatous tissue, and collagen deposition in the lung in the formulation treated groups. In addition, the measures of cellular injury (lactate dehydrogenase levels), vascular leakage (total protein levels), and pulmonary fibrosis (soluble lung collagen levels) were not significantly different between the untreated and the formulation treated groups. The drug levels at the end of 21 days in the lung tissue for large porous celecoxib-PLGA microparticles were 5- and 50-fold higher, when compared to conventional celecoxib-PLGA microparticles and celecoxib powder, respectively. The drug levels were 15- and 96- fold higher in the BAL for large porous microparticles when compared to conventional particles and celecoxib powder, respectively. B[a]P treatment significantly increased the PGE2 and VEGF levels and single doses of dry powder formulations of conventional and large porous celecoxib-PLGA microparticles significantly reduced these elevations. We thereby can conclude that large porous celecoxib-PLGA microparticles are safe and do not cause any significant lung inflammation, cellular injury, or pulmonary fibrosis. These particles better sustain pulmonary drug delivery compared to conventional microparticles or drug powder, and reduce B[a]P induced elevations in PGE2 and VEGF levels in a lung tumor model.
K1 – Contributed Paper AN INGREDIENT-SPECIFIC METHOD FOR PARTICLE SIZE 106 CHARACTERIZATION OF CORTICOSTEROID NASAL SPRAYS Oksana Klueva, Matthew P. Nelson, Patrick J. Treado, and Amy M. Waligorski, ChemImage Corporation, 7301 Penn Ave., Pittsburgh PA 15208, USA; and Changning Guo, William H. Doub, John A. Spencer, Food and Drug Administration, St. Louis, MO, 63101 USA; [email protected]
FDA Draft Guidance on bioavailability and bioequivalency studies for nasal aerosols and sprays (2003) advocates in vitro methods for establishing bioequivalency for solution formulations of locally acting nasal sprays and aerosols, based on the assumption that in vitro studies are more sensitive indicators of drug delivery to nasal sites of action compared to clinical studies. Particle size is an important physico-chemical property of nasal sprays as it influences bioavailability, delivery rate, stability and, ultimately, safety and efficacy of the inhaled drug. Currently there is no validated in vitro method to characterize API particle size distribution (PSD) in formulated aqueous nasal spray suspensions. Availability of such a method would provide pharmaceutical scientists with a reliable assessment of API-specific PSD of drug substances in finished aqueous suspension nasal spray products. Raman chemical imaging (RCI) technology is being investigated as a methodology for ingredient-specific PSD determination for several corticosteroid aqueous suspensions. A Raman spectral library has been developed for the active and inactive compounds in the several corticosteroid formulations (Rhinocort AQ™, Flonase™, Beconase AQ™, Nasonex AQ™). Raman and optical imaging techniques have been applied over hundreds of particles fields of view to characterize PSD of active ingredients. Raman imaging obtained from corticosteroid API in 1600-1700 cm-1 spectral range allowed ready distinction between the drug and excipient. The Raman images can be used to calculate particle size of those active pharmaceutical ingredients (API) independent of excipients of the mixture as well as adhered to excipient particles. From these and on-going
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studies, RCI has been able to differentiate active ingredient within complex aqueous nasal spray samples and allow measurement of the PSD of active drug components. In summary, RCI technique shows promise as a method for characterizing particle size of corticosteroids in aqueous suspension nasal sprays. The current state of validation of RCI for particle analysis will be presented.
K1 – Contributed Paper A STUDY OF LIQUID PROPERTIES AND DROPLET SIZE 107 DISTRIBUTIONS OF SOME COMMON NASAL DECONGESTANT SPRAY FORMULATIONS Mark Bumiller, Henrik Krarup and Chuck Rohn, Malvern Instruments, Inc., 10 Southville Road, Southboro, MA 01772; [email protected]
Laser diffraction is recommended for in vitro testing of droplet size distribution characteristics in bioequivalence and bioavailability studies of nasal sprays and nasal aerosols. The droplet size distribution obtained from the nasal pump is a function of factors ranging from actuation characteristics, pump design to the rheological properties of the formulation liquid. In particular microcrystalline cellulose and carboxymethylcellulose (CMC) is used as a viscosity modifier to enhance the residence time in the nasal cavity. This study investigates the viscoelasticity of two common nasal decongestants and compares rheological properties to the droplet size distribution obtained from laser diffraction. Results indicate that the long relaxation time described by the low frequency storage modulus G’ that relates to the extensional rheological behavior of the material. Formulation A has higher G’ than formulation B, at lower frequencies, indicating that formulation A is more elastic and thus more capable of forming larger droplets.
K1 – Invited Paper NEW POLYMER ENABLES ZERO-ORDER RELEASE OF DRUGS 108 James R. Benson, Nai-Hong Li, William Landgraf, Polygenetics, Inc., P.O. Box 33115, Los Gatos, CA 95031, [email protected]
Scientists at Polygenetics, Inc. have developed a highly porous polymer material (Cavilink™) that can be used to contain drugs, and release those drugs following near zero-order kinetics. The polymers can have up to 90% porosity so that drug-polymer combinations are 90% pure. The zero-order release feature is due to the unique, patented polymer morphology and is independent of drug composition. Constant blood levels can be maintained for twenty-four hours, making these polymers ideal for oral drug delivery applications. Cavilink™ polymers are not metabolized and pass through the GI tract unchanged. These polymers are particularly suited for medical problems in which constant blood levels are particularly important, such as pain management, cardiovascular intervention, anti-inflammatory and certain antimicrobial treatments. Cavilink™ polymers can also be used effectively in taste masking applications.
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K1 – Invited Paper ADVANCES IS LIQUID AEROSOL DELIVERY SYSTEMS 109 Jeffrey Schuster and Jim Blanchard, Aradigm Corporation, 3929 Point Eden Way, Hayward, CA, 94545, USA; [email protected]
Aerosol drug delivery presents a significant opportunity for topical lung treatment and non-invasive delivery of systemically active compounds. Because the lung is an anatomic portal of entry to the systemic circulation, there is the possibility of much higher efficiency and more rapid onset as compared to other non-invasive methodologies such as trans-dermal or nasal. At Aradigm corporation, we have developed a highly efficient technology for the delivery of aqueous aerosols via the lung, the AERx® system. The AERx system generates a fine particle aerosol from a liquid formulation which is packaged in a unit dose container with an integrated single use disposable nozzle. An all electronic version, with features such as dose titration and compliance monitoring, is in Phase III clinical trials with insulin. We have developed a smaller and more cost effective platform based on this technology, AERx Essence™. This system is all mechanical, and includes features that ensure proper inhalation rate and proper coordination of delivery with inhalation. Results of a Gamma-scintigraphic study utilizing SPECT, and a user study, will be presented. Additionally, there are two new inhaler technologies under development at Aradigm. One is a single use disposable version of our AERx technology, AERx Ease™. Because AERx Ease utilizes components from AERx Essence and our Intraject® needle-free injector platform that are scaled up for high volume manufacture, development times and cost should be minimal. The second technology in development is our Pulmoshield™ platform, based on an aerosolization technology licensed from the University of Seville. The Pulmoshield technology will deliver very high density aerosols, minimizing the long treatment times that are associated with presently available nebulizers.
K1 – Invited Paper EMBRACING PARTICLE ENGINEERING FOR INHALATION DRUG 110 PRODUCTS Robert Cook, Thomas Armer, MAP Pharmaceuticals, Inc. 2400 Bayshore Parkway, Mountain View, CA 94043, USA; [email protected]
Particle engineering offers potential to enhance inhaled product performance and provide differentiated patient benefits; e.g. formulation of drugs that could otherwise not be delivered by inhalation, more consistent dosing, rapid onset of therapeutic action and dosage reduction. Budesonide is a corticosteroid indicated for bronchial asthma. For patients unable to use pressurised metered dose inhalers (pMDIs) or dry powder formulations (e.g. young children), an aqueous suspension of micronised budesonide (nebuliser delivery) is a clinical alternative - delivery of which is typically inefficient. A novel, particle engineered submicron aqueous dispersion of budesonide was formulated for improved performance. At equivalent clinical dose (1 mg) and strength (0.5 mg/mL), the submicron formulation delivered twice the fine particle dose (FPD, mass deposited in an Andersen Cascade Impactor <4.7 μm): 145±8 mcg compared to 69±6 mcg for the existing commercial budesonide preparation (Pulmicort Respules, AstraZeneca).
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Therefore, potential for reduced corticosteroid doses and faster delivery times may exist for this engineered product. Dihydroergotamine mesylate (DHE) is used for effective migraine treatment, by IV injection or nasal delivery. DHE was processed using supercritical fluid technology into highly respirable crystals (<3 μm). Suspension of these crystals in hydrofluoroalkane propellant in pMDI canisters (500 mcg/actuation) yielded a FPD of 162±5 mcg. Combining this formulation with a next generation inhaler (Tempo™), with advanced aerosol plume control and breath synchronous delivery, elevated FPD to 220±15 mcg, and halved throat deposition (80.6 vs. 156.5 mcg). The respirable dose achieved in humans resulted in 250 mcg/actuation delivered to the plasma. Plasma profiles of this inhaled DHE formulation, from Tempo, closely matched IV administration, reaching faster peak concentrations than nasal preparations (10 vs. 45 min) thus offering injection-free potential for rapid migraine relief. These two products, under development, provide noteworthy examples of where particle engineering can significantly benefit both local and systemic inhalation drug therapy.
K1 – Invited Paper DESIGN AND PRODUCTION OF ULTRAFINE ACTIVE 111 PHARMACEUTICAL INGREDIENTS UNDER HIGH GRAVITY ENVIRONMENT Jimmy Yun1, Jian-Feng Chen1,2, Hak-Kim Chan3, William Glover1, 1Nanomaterials Technology, Singapore; 2Sin-China Nanotechnology Center, Beijing University of Chemical Technology, Beijing, China; 3Faculty of Pharmacy, University of Sydney, Australia; [email protected]
Currently there is an increasing trend in pharmaceutical aerosol development to investigate highly dispersive formualtions to improve drug delivery to the lungs. The reality of these formulations may be realised using engineered nanoparticles. There is a variety of methods of preparation, however the production scalability and cost associated with these processes represent significant constraints for these technologies to conquouer. High gravity precipitation has the potentially to overcome these obstacles based on its simplistic design around crystalisation kinetics, nucleation events occur rapidly in a turbelent zone and are seperated from the crystal growth region of the process. This in turn can lead to the production of extremely small particles (10 to 1000nm). A feasibility study was performed on a common inhalation drug to assess the suitability of high gravity precipitation for producing active ingredients for aerosol drug delivery. Two separate precipitation methods i) a standard antisolvent precipitation and ii) a reactive precipitation were designed around the high gravity precipitation platform. Optermization trials were carried out by varying solvent/antisolvent ratios, concetrations of drug and rates of addition. The purpose of the optermization trials was to reduce the primiary particle size below 500nm x 1µm. Suspensions of the precipitated particles were spray dried in order to evaporate excess solvent and form dry powders. The morphology of the powders consisted of uniform needle shaped nanoparticles which appear moderately agglomerated. X-ray diffraction analysis indicated that powders produced from both methods were crystaline in nature. The in vitro aerosol dispersion results (FPFloaded under 5µm) for nanonised salbutamol sulphate was 52% and 83% for the antisolvent and reactive precipitations
104 Oral Abstracts respectively. This is considerably better than the commercially available micronized raw material with 16%. In conclusion, nanonising active pharmaceutical ingredients using high gravity precipitation has the potenital to further improve physical characteristics of pharmaceutical aerosols which may lead to increased delivery performances of drug compounds.
L1 – Keynote Paper NEW POLYMER-LIPID HYBRID NANOPARTICLES FOR ENHANCED 112 ANTICANCER DRUG DELIVERY Xiao Yu Wu, H.L. Wong, R. Bendayan, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada M5S 2S2; A.M. Rauth, Ontario Cancer Institute, Toronto, Ontario, Canada M5G 2M9; [email protected]
New polymer-lipid hybrid nanoparticles (PLN) were developed for the first time for enhanced delivery of water-soluble, ionic anticancer drugs and chemosensitizing agents to treat multidrug resistant (MDR) cancer. PLN containing drug-polymer complex and lipid were produced after sonication and cooling. The size and morphology of the particles were characterized by photon correlation spectroscopy and transmission electron microscopy. The drug loading and release kinetics were measured by spectrophotometry. A trypan blue exclusion assay and a clonogenic assay were carried out on a P-glycoprotein (P-gp) over-expressing human breast cancer cell line (MDA435/LCC/MDR1) to determine the effects of the formulation on cell survival. The influence of the formulations on the rates of cellular uptake and efflux, and cellular distribution of doxorubicin (Dox) was also studied. Dox-PLN with 60-80% drug encapsulation efficiency and particle diameters of 80-350 nm were obtained. Drug release was faster in the first two hours followed by slower release which lasted over 16-70 hours. Neither the polymer alone or blank PLN altered membrane permeability. In the clonogenic assay, Dox-PLN resulted in nearly an additional log kill on MDA435/LCC6/MDR1 cells, while did not lead to different activity against wild-type cells as compared to the conventional Dox treatment. Prolonged accumulation of Dox-PLN in the MDR cell line was observed. The PLN formulation containing both Dox and GG918, a Pgp inhibitor, was more effective against Pgp-overexpressing cancer cells than the two drugs in solution form or separate PLN form. This result suggests that it may be advantageous to simultaneously deliver the drug combination in a single carrier system. The present study indicates that Dox-PLN possess significant MDR reversal activity. The elevated activity is likely a result of the intracellular accumulation of the Dox-PLN, which are difficult to remove by the drug efflux transport mechanism and may serve as extended intracellular sources of Dox.
L1 – Invited Paper A VERSATILE TUMOR TARGETING NANOADELIVERY PLATFORM 113 FOR CANCER DIAGNOSIS AND THERAPY Esther Chang and Kathleen F. Pirollo, Georgetown University Medical
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Center, 3970 Reservoir Rd NW, TRB/E420, Washington, DC 20057-1469, USA; [email protected]
Many issues need to be addressed before the promise of tumor-targeting diagnosis and therapy for cancer can be realized. Foremost among these is the efficient and selective delivery of diagnostic and/or therapeutic molecules to the site(s) in the body where the target tumor cells reside. Of particular relevance to cancer is the ability to target cells that have metastasized from the site of the primary tumor to distant sites in the body. Our laboratory has developed a platform nanotechnology comprising a cationic liposomal nanocomplex bearing molecules that home to the surface of tumor cells. When systemically administered, this tumor targeting nanocomplex can efficiently and selectively deliver not only nucleic acid-based therapeutics, but also diagnostic contrast agents and small molecules to primary tumors and metastases in animal models of a variety of human cancers. The nanodelivery of imaging agents results in a significant improvement in the sensitivity and resolution in detecting metastatic lesions. Moreover, the various nucleic acid-based therapeutics have been shown to dramatically synergize with conventional radio- and chemotherapies. This approach is now entering clinical trials.
L1 – Contributed Paper DNA MESOSPHERES AS DRUG DELIVERY VEHICLES FOR 114 LOCALIZED CANCER CHEMOTHERAPY Iris V. Enriquez1,3, Eugene P. Goldberg1,2,3 1Department of Materials Science & Engineering, 2Department of Biomedical Engineering, 3Biomaterials Center, University of Florida, PO Box116400, Gainesville, FL 32611-6400; [email protected], [email protected]
There is a growing need to find alternative delivery methods for cytotoxic chemotherapeutic agents. Traditional systemic chemotherapy is often limited in effectiveness and may severely impact the quality of life for many patients. Recent research has shown that intratumoral therapy may be a useful alternative to conventional systemic chemotherapy. The potential benefits of intratumoral therapy are localized delivery of an extraordinary drug dose to the tumor site, greatly reduced systemic toxicity, and the opportunity to prolong the local effectiveness of the drugs. Previous research in our lab with various protein nano-meso-microspheres in a variety of animal cancer models has been promising and suggests unique opportunities for the use of deoxyribonucleic acid (DNA) as a mesosphere drug carrier. The research reported here was therefore devoted to the synthesis and properties of new DNA mesospheres (DNA/MS). DNA has been shown to complex with many cancer drugs and Trouet investigated the systemic delivery of a complex with daunorubicin. It was observed that the DNA-drug complex had reduced cardiotoxicity and provided a longer blood half life. The therapeutic value of the DNA complex was also comparable to that of free drug. These studies further suggested to us that crosslinked but biodegradable DNA mesosphere compositions, which had not been previously reported, might have interesting and useful drug delivery properties. DNA/MS were synthesized using a steric stabilization process developed in our lab. In brief, the process involved dispersing an aqueous DNA solution in an organic phase solution
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composed of 5% cellulose acetate butyrate in 1,2-dichloroethane. After vigorous mixing, the resulting MS suspension was crosslinked in a novel fashion, i.e. with chromium (Cr3+) or gadolinium (Gd3+) ions via the phosphate groups of the DNA molecule. After crosslinking, the DNA/MS were purified by acetone wash and dried at room temperature. Resulting DNA/MS were characterized for particle size, morphology, and elemental composition. Uniform spherical particles were readily synthesized with yields of >70% and with dry vs. hydrated diameters of 4um vs. 13um. Drug loading studies with various cancer drugs, using in situ and post-loading methods, are in progress.
L1 – Invited Paper LIGAND-MEDIATED TARGETING OF NANOPARTICLES AND LOW 115 MOLECULAR WEIGHT DRUGS TO CANCER CELLS AND INFLAMMATORY IMMUNE CELLS Philip S. Low, Department of Chemistry, 560 Oval Drive, Purdue University, West Lafayette, IN 47907, USA; [email protected]
We have been developing methods to target drugs specifically to pathologic cells, thereby avoiding the collateral toxicity associated with drug uptake by healthy cells. In the case of cancer, we have exploited the strong upregulation of the folate receptor on malignant cells to target: i) protein toxins, ii) chemotherapeutic agents, iii) gene therapy vectors, iv) oligonucleotides, v) radioimaging agents, vi) nanoparticles, vii) liposomes with entrapped drugs, viii) radiotherapeutic agents, ix) immunotherapeutic agents, and x) enzyme constructs for prodrug therapy to tumor tissue by linking the various agents to the vitamin folic acid. Current clinical trials of folate-linked drugs demonstrate that the folate-targeting strategy holds great promise for significantly improving the tumor specificity of therapeutic reagents. Folate-linked drugs must obviously extravasate and penetrate the tumor tissue before they can bind to FR-expressing cancer cells. Consequently, size, shape, and water-solubility influence a folate conjugate’s tumor targeting ability. Data showing the real time penetration, binding and internalization of various fluorescent folate conjugates by tumor cells in live mice, as monitored by multiphoton intravital microscopy, will be presented to illustrate the impact that particle size has on its penetration into solid tumors. We are also developing drug targeting strategies for the imaging and therapy of rheumatoid arthritis, Crohn’s disease, atherosclerosis, lupus, glomerulonephritis, atherosclerosis, and multiple sclerosis. Methods associated with these technologies will also be discussed.
L1 – Invited Paper SINGLE WALL CARBON NANOTUBE NANOBOMB AGENTS FOR 116 CANCER THERAPEUTICS Balaji Panchapakesan, Department of Electrical and Computer Engineering, University of Delaware, Newark, DE 19716; [email protected]
Although there have been outstanding advances in fundmental cancer biology, these have not translated into comparable advances in the clinic. Inadequacies in the ability to
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administer therapeutic agents with high selectivity and with minimum collateral damage have largely accounted for such discrepancies in cancer therapeutics. Furthermore it is most astonishing to recognize that only 1 to 10 parts per 100,000 of intravenously injected monoclonal antibodies reach their targets in vivo. Keeping these in mind, in this paper, we report the applications of single wall carbon nanotubes as potent threpautic nanobomb agents for killing breast cancer cells. By adsorbing water molecules in loosely packed nanotubes that are adsorbed on top of cell clusters, potent nanobombs were created when nanotubes were exposed to 808 nm laser light at light intensities between 50-200 mW/cm2 that completely destroyed the cells that were in contact with the nanotubes. Conversion of optical to thermal energy and subsequent thermal energy confinement in nanotubes caused such explosions in water and saline solutions. Co-localized nanobombs killed human BT474 breast cancer cells in physiological phosphate buffered saline solution. Cells that were treated with nanobombs exploded into fragments while the surrounding cells that were not treated with the nanobombs were healthy and viable showing the selective nature of this cell killing technique. Nanobombs coated with Her2 specific antibodies were highly selective in killing BT474 cancer cells (overexpress Her2) compared to non-specific antibodies paving the way for immunotargeted nanobombs for cancer therapeutics. Nanobombs can find wide variety of use in cancer therapeutics, immunotargeting, drug delivery and minimally invasive surgical applications.
L1 – Invited Paper FOLATE RECEPTOR-TARGETED LIPOSOMES AND NANOPARTICLES 117 FOR CANCER THERAPY Robert J. Lee, Division of Pharmaceutics, College of Pharmacy, NCI Comprehensive Cancer Center, NSF Nanoscale Science and Engineering Center, The Ohio State University, Columbus, OH 43210, [email protected]
Targeted delivery through malignancy-associated cellular markers is a promising strategy for cancer therapy. Folate receptor (FR) is a cell surface receptor selectively amplified in a significant fraction of human neoplasms. Folate binds to FR with high affinity, displays high specificity and enters cancer cells through receptor-mediated endocytosis. FR, therefore, represents a very attractive selective target for cancer-specific delivery of therapeutic agents. FR-targeted liposomes have been constructed through incorporation of a lipophilic derivative of folate. FR-targeted liposomes have been loaded with a variety of bioactive reagents. It has been evaluated for the targeted delivery of anticancer agents to cancer cells and was shown to be efficiently internalized and selectively cytotoxic to FR positive cells. In addition, FR-targeted liposomal doxorubicin has been shown to overcome Pgp-mediated drug resistance in vitro and to have superior antitumor activity to non-targeted control liposomes in several preclinical murine tumor models. Furthermore, strategies have been developed to selectively up-regulate FR-beta expression in human acute myelogenous leukemia and FR-alpha expression in solid tumors, which might further enhance the translational potential of this targeting strategy. FR-targeted liposomal drug delivery thus represents a particularly attractive strategy for future development.
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D3 - Keynote Paper SUPERPARAMAGNETIC IRON OXIDE NANOPARTICLES AS A DRUG 118 DELIVERY SYSTEM FOR IN VIVO APPLICATION IN THE JOINT Brigitte von Rechenberg, Musculoskeletal Research Unit, Equine Hospital, Vetsuisse Faculty Zurich, University of Zurich, Switzerland; [email protected]
Joint diseases are one of the most common orthopedic problems in daily patient care. They may be either related to the rheumatoid complex including polyarthritis or to degenerative processes caused through traumatic lesions and/or mechanical overload. Degenerative joint diseases are becoming more important as a consequence to a society, where the age expectation has increased considerably, sports play a significant role in daily life of young and middle aged persons and where overweight has become the rule rather than the exception. Although much research has been devoted to understand or inhibit chronic inflammation and mechanisms leading to osteoarthritis, the “magic bullet” or drug has not been found yet, let alone a drug that would be locally effective while not having major systemic side effects (kidney disease, gastric ulceration, stroke, heart disease, etc.). Drugs that are given systemically and slow down the process of osteoarthritis are usually directed towards inhibiting joint inflammation (eg.cox-2 inhibitors), resp. down regulate degradation of the cartilage matrix through local enzymes, such as matrix metalloproteinases (MMPs). When given systemically, it may prove difficult to achieve effective local concentrations of the drugs within the joint because of chronic indurations and fibrosis of the joint capsule and ligaments. In the other hand, if drugs are given locally through intraarticular injections, the maintenance of effective concentrations may also be impossible, since drugs may be filtered and transported away from the joint within a very short time period (ca. 1hour) and before full action can be reached. Superparamagnetic iron oxide nanoparticles (SPION) seem be a very attractive drug delivery system to overcome these problems, since they can be injected intraarticularly and maintained at the location by means of an external magnetic field while protracted release of drugs could take place. Therefore, our collaborative research efforts have focused on the use of SPIONs as drug delivery system for drugs, or as non-viral gene transfection system for plasmids (DNA). Since no previous studies about the use of SPIONs within a joint were available, our work concentrated first on in vitro work with primary cell lines of synoviocytes (cells lining the joint capsule), where issues as particle uptake, dosage and toxicity were addressed. For all our work, iron oxide particles with a core between 6-10nm were used that were either coated with polyvinyl alcohol alone (PVA) or polyvinyl alcohol /vinyl amine copolymer functionalized with Cy3.5 or Texas Red fluorescence dye. For gene transfection the same type of SPIONs were used but coated with Polyethyleneimide (PEI) and functionalized with plasmids encoding green fluorescent protein (GFP). Animal experiments were conducted in sheep, where functionalized SPONs were injected intraarticularly (i.a) either into the stifle or the radiocarpal joints. Immediately after SPION application external magnets were locally fixed directly at the joint and kept in place for 12 hours in case of plain PVA, or PVA-NH3-Cy3.5 coated SPIONs. With PEI-coated and with GFP-plasmid functionalized particles a static magnet was first applied before a pulsating magnet was applied for 5 minutes, after which the static magnet was reapplied and kept there for 12 hours. Animals were sacrificed at 24, 72 and 120 hours after injection, and after
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macroscopic assessment of the joints, tissue was harvested for histology. Apart from routine HE sections special staining for detection of SPIONs within the tissue were applied (Prussian Blue (PB), Turnbulls (TB)). Confocal microscopy was used for the detection of the fluorescent dyes or GFP synthesis. Immunhistochemistry with antibodies against GFP were additionally used for the gene transfection experiments. To follow the systemic elimination of SPIONs samples of all organs except the brain were collected and investigated with PB or TB stainings.Plain PVA-SPIONs were well biocompatible and taken up by the synovial membrane within 24 hours. They were detectable for 120 h at which time they had already penetrated into the deeper layers of the joint capsule. The same was true for PVA-NH3-Cy3.5 SPIONs, although a mild inflammation was noticed at 72 h after injection. The Cy3.5 fluorescence occurred parallel to the particle uptake within the synovial membrane. Systemic elimination of SPIONs could be followed and occurred via the local lymphnodes into the systemic vascular system, where the particles were filtered through the glomerular system of the kidney, were reabsorbed in the proximal tubuli and were then captured in the reticuloendothelial system (RES) of the spleen and liver. Cy3.5 fluorescence was noted only in the joint capsule, faintly also in the local lymph nodes but not in the other organs. The PEI-coated and with GFP-plasmid functionalized SPIONs also caused a moderate joint irritation already at 24h. Particle agglomeration was noticed within the synovial fluid. GFP expression could not be found within the synovial membrane, but in few cells of the local lymph nodes by means of immunohistochemistry. Aggregation of the SPIONs seemed to elicited by the synovial fluid, and there, was attributed to the negative charge of the proteoglycans and hyaluronic acid. In summary we concluded that drug delivery with PVA-coated particles could be confirmed using Cy3.5 as proof of principle, gene transfection of synoviocytes in vivo was not possible by means of PEI-coated SPIONs. Future research will focus on drug delivery of anti-inflammatory drugs for acute and chronic joint inflammations by usingf PVA-coated SPIONs. For gene transfection by means of SPIONs new coatings and methods of plasmid functionalization will be investigated.
D3 – Invited Paper GENERALIZED AND LARGE-SCALE SYNTHESIS OF MONODISPERSE 119 NANOCRYSTALS OF FERRITES AND OXIDES AND THEIR APPLICATIONS AS MRI CONTRAST AGENTS AND IN DRUG DELIVERY Taeghwan Hyeon, Jongnam Park, Kwangjin An, and Hyon Bin Na, National Creative Research Initiative Center for Oxide Nanocrystalline Materials and School of Chemical and Biological Engineering, Seoul National University, Seoul 151-744, South Korea, [email protected]
We developed a new generalized synthetic procedure to produce monodisperse ferrite nanocrystals without a size selection process. Highly-crystalline and monodisperse nanocrystals were synthesized from the thermal decomposition of metal-surfactant complexes. We synthesized monodisperse spherical nanocrystals of metals (Fe, and Ni), and metal oxides (γ-Fe2O3, Fe3O4, CoFe2O4, MnFe2O4, NiO, and MnO). We report the
110 Oral Abstracts ultra-large-scale synthesis of monodisperse ferrite nanocrystals by the thermolysis of metal-oleate complexes. We synthesized as much as 40 grams of monodisperse magnetite nanocrystals using 1 L reactor. Furthermore, the current synthetic procedure is very general, and was successfully used to produce the nanocrystals of MnO, CoO, MnFe2O4, CoFe2O4, and cube-shaped iron. By controlling the nucleation and growth processes, we were able to synthesize monodisperse magnetite nanoparticles with particle sizes of 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16 nm. We developed a new generalized synthetic route to produce uniform-sized nanorods of Fe2P, FeP, Co2P, Mn2P, Ni2P from the thermal decomposition of syringe-pump-delivered metal-surfactant complexes. The synthesized organic dispersable nanocrystals were transformed to hydrophilic water-dispersable nanocrystals by treating with phospholipids or PEG’s. The resulting water dispersable and monodisperse nanocrystals of ferrites and oxides were successfully employed as new MRI contrast agents. We fabricated monodisperse nanoparticles embedded in uniform pore-sized mesoporous silica spheres. Monodisperse magnetite and CdSe/ZnS nanocrystals (quantum dots) dispersed in organic media were transferred to aqueous phase using CTAB stabilization. The subsequent sol-gel reaction of TEOS generated monodisperse nanocrystals embedded in spherical mesoporous silica particles with an average particle size of ~150 nm. The magnetic separation (guiding) was demonstrated for the spheres. These mesoporous silica spheres embedded with nanocrystals were investigated as drug delivery carriers using ibuprofen as a drug model and the controlled release rate of ibuprofen by the surface modification was demonstrated.
D3 – Contributed Paper SILICA-POLYPEPTIDE COMPOSITE PARTICLES 120 Paul S. Russo, Sibel Turksen, Erick Soto-Cantu, Jianhong Qiu, Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803; [email protected]
Core-shell composite particles have been prepared, each consisting of a silica-coated cobalt center to which a homopolypeptide shell, either poly (ε-carbobenzyloxy-L-lysine) or poly (γ-benzyl-L-glutamate), is attached covalently. Core particles were coated with a mixture of reactive amino and passivating moieties through silylation reactions. The amino groups initiated the polymerization, with attachment, of N-carboxyanhydride monomers, resulting in a homopolypeptide shell. Core size and shell thickness can be varied readily. Characterization by dynamic light scattering confirmed the helix-coil transition of the polypeptide shell, reminiscent of the coat proteins of certain viruses, through repeated heating and cooling cycles in an organic solvent. The living nature of the polypeptide shell has also been confirmed. The particles have a size and uniformity that leads to formation of colloidal crystals, and they modify phase relationships of solutions containing rodlike polymers. Magnetometer measurements suggest the particles are superparamagnetic. The sidechains of the shell polypeptides can be deprotected, rendering the particles dispersible in aqueous media, where pH-induced size transitions are observed.
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D3 – Contributed Paper SURFACE FUNCTIONALIZED NANO-MAGNETIC PARTICLES FOR 121 ADSORPTION/DESORPTION OF BIO-MOLECULES Mohammad S Uddin, Kus Hidajat, Liang Hong, Zanguo Peng, Nabila Shamim and Nguyen T K Thuyen; Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117576, SINGAPORE; [email protected]
Surface funcionalized nano-sized magnetic particles are recently gaining interest for its application in the field of bio-separation. Due to their size, nano-particles provide large specific surface area for adsorption of solutes and moreover the magnetic properties of these particles offer an excellent tool to handle these particles in suspension by the use of magnetic field. This paper deals with our work on nano-magnetic particles for purification of protein and enzyme. The nano-magnetic particles (Fe3O4) were synthesized by chemical precipitation of Fe2+ and Fe3+ salts at 80oC at alkaline condition and inert atmosphere and the average particle size was about 10 nm. The particle surface was then functionalized in two different ways: surface charge (electrostatic interaction) and coating with thermosensitive polymer (hydrophobic-hydrophylic interaction). The particle surface charge was controlled through the pH of the solution at isoelectric point of the solute. For hydrophobic-hydrophylic interaction, the particles were coated with thermosensitive polymer, Poly (N-isopropylacrylamide) (PNIPAM). Its lower critical solution temperature is 32oC in water and changes from hydrophylic to hydrophobic above this temperature. Adsorption and desorption of bovine serum albumin (BSA) and lysozyme were carried out on these sruface functionalized particles. It was found that at pH of 4.7 (isoelectric point of BSA) the adsorption of BSA was maximum (about 400 mg/g of dry solids at equilibrium condition) compared to those of other pH values. At equilibrium, PNIPAM coated particles adsorbed about 275 mg BSA/g dry solids and upon cooling to 25oC about 9% of the adsorbed BSA was desorbed. The conformational and activity changes of BSA and lyszyme due to the adsorption/desorption processes were studied. Analytical equipments such as Transmission Electron Microscopy (TEM), Fourier Transform Infrared Spectroscopy (FTIR), X-ray Photoelectron Spectroscopy (XPS) were used for particle characterization and confirmation of adsorption/desorption processes.
D3 – Invited Paper STRATEGIES FOR THE DESIGN AND READOUT OF ULTRAHIGH 122 DENSITY IMMUNODIAGNOSTIC PLATFORMS H.-Y. Park, J. Driskell, K. Kwarta, B. Yakes, J. Uhlenkamp, R. Millen, N. Pekas, J. Nordling, R. J. Lipert, and M. D. Porter, Departments of Chemistry and Chemical Engineering, Institute for Combinatorial Discovery, and Ames Laboratory-USDOE, Iowa State University, Ames IA 50011, USA; [email protected]
The drive for early disease detection, the growing threat of bioterrorism, and a vast range of
112 Oral Abstracts challenges more generally in biotechnology have markedly amplified the demand for ultrasensitive, high-speed diagnostic tests. This presentation describes efforts to develop platforms and readout methodologies that potentially address demands in this arena through a coupling of nanometric labeling with surface enhanced Raman spectroscopic, micromagnetic, and scanning probe microscopic and readout concepts. Strategies will be described for both the fabrication and readout of chip-scale platforms that can be used with each novel readout modality. Examples will focus on the use of protein arrays as platforms targeted for immumoassays in early disease diagnosis and the rapid, ultralow level detection of cancer markers and viral pathogens. Each example will also discuss challenges related to sensitivity and nonspecific adsorption and to fluid manipulation at micrometer length scales.
D3 – Invited Paper MAGNETIC NANOPARTICLES: APPLICATIONS BEYOND DATA 123 STORAGE Andreas Hütten, Forschungszentrum Karlsruhe GmbH, Institute for Nanotechnology, Hermann-von-Helmholtz-Platz, P.O. Box 3640, D-76021 Karlsruhe, Germany, [email protected], and Inga Ennen, Günter Reiss, University of Bielefeld, Dept. of. Phys., and Klaus Wojczykowski, Peter Jutzi, University of Bielefeld, Dept. of. Chem., Universitätsstr. 25, D-33615 Bielefeld, Germany, [email protected]
Paramagnetic carriers, ranging from micro sized particles to nano sized colloids, which are linked to antibodies enable highly specified biological in vitro cell separations as well as therapeutic in vivo applications such as drug-targeting, cancer therapy, lymph node imaging or hyperthermia. Ensuring biocompatibility and non-toxicity so as to meet the requirements for these applications, iron oxide based particles, e.g. magnetite, are commonly used as the magnetically responsive component of commercially available magnetic microspheres. With the colloidal synthesis of superparamagnetic or ferromagnetic Co and FePt nanocrystals with superior magnetic moments the question arises whether these nanomagnets could replace the iron oxide particles as magnetic carriers in in vitro separation and therapeutic in vivo technology. The potential of these nanocrystals can directly be evaluated, comparing their magnetophoretic mobility under identical experimental conditions. Chemical and physical aspects of their synthesis, the resulting microstructure and magnetic properties will be discussed in detail. The synthesis kinetics is modeled using a consecutive decomposition and growth model and is compared to experimental data. It allows to uncover the three dimensional composition gradients of these nanoparticles ranging from 1 to 10 nm. Potential in vitro and in vivo application will be addressed as a function of the nanoparticles microstructure and morphology..
D3 – Contributed Paper FUNCTIONALIZATION OF MAGNETIC NANOPARTICLES WITH 124 BIOMOLECULES Shy Chyi Wuang, Koon Gee Neoh*, En-Tang Kang, Department of Chemical
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and Biomolecular Engineering, National University of Singapore 10 Kent Ridge, Singapore 119260, Daniel W. Pack, Deborah E. Leckband, Department of Chemical and Biomolecular Engineering, University of Illinois, Urbana-Champaign, Urbana, IL 61801, USA; [email protected]; [email protected]
Modifications were made on magnetic nanoparticles to tailor them for various biomedical applications such as targeted drug delivery, hyperthermia and magnetic resonance imaging. Superparamagnetic magnetite nanoparticles (Fe3O4) with average diameters ~6 nm were surface-functionalized with poly(N-isopropylacrylamide) (P(NP)) via atom-transfer radical polymerization (ATRP) followed by the immobilization of heparin, an anti-coagulant. The heparinized nanoparticles are able to delay phagocytosis by macrophages and are effective in preventing thrombosis as demonstrated by their ability to prevent blood clotting in vitro, for up to at least 24 hr, as compared to the normal clotting time of about 4 min. Such nanoparticles are potentially very useful where targeted delivery of heparin is desired, e.g. in the prevention or reduction of restenosis. On the other hand, effective uptake of magnetic nanoparticles into cancer cells would be desirable for hyperthermia treatment. In our project, magnetite nanoparticles are encapsulated inside a conductive polymer, polypyrrole (PPY), via a miniemulsion polymerization. Fe3O4–PPY nanospheres of about 100 nm in diameter and containing over 30% by weight of Fe3O4 can be prepared by this technique. These nanospheres retained high levels of magnetization (>20 emu/g) and electrical conductivity (~ 1 S/cm) and hence are potentially useful for hyperthermia by deploying both the magnetic and conductive heating capacities. Transmission electron microscopy (TEM) images have shown the distribution of Fe3O4 in the PPY nanospheres to be roughly uniform. It was found that the choice of surfactant affects the cell uptake of nanospheres. In particular, hyaluronic acid (HA) enhances the adhesion of the Fe3O4–PPY nanospheres to cells and hence increases their cell uptake. The Fe3O4–PPY nanospheres can be further functionalized to increase their potential for the detection of cancer cells and treatment therapy.
D3 – Contributed Paper PARTICLE POPULATION CLASSIFICATION BY MICRO-FLOW 125 IMAGING Dave Thomas, David King, Peter Oma, Brightwell Technologies Inc., 195 Stafford Road West, Ottawa, Ontario, Canada, K2H 9C1; [email protected]
The paper will describe a new particle analysis technology which employs automated digital image analysis to classify particulate/cell suspensions. The technology involves drawing sample through a sheathed or unsheathed micro-fluidic cell. Sections of the flowing fluid are illuminated with a pulsed high-intensity source, magnified and imaged frame-by-frame on the camera’s pixel array. The system software identifies, in real time, pixels in each frame which are wholly or partially contained within a particle image. This information is used to isolate particles and measure particle parameters such as count, diameter, area, perimeter, circularity, and transparency. The volume of fluid sampled in each frame is accurately known, therefore particle concentrations are determined
114 Oral Abstracts absolutely. The database of images and parameter values is interrogated by the user to produce histograms, scattergrams and to isolate sub-populations of interest. Micro-Flow Imaging (MFI) technology will detect and classify particles ranging from 0.8 to 100 microns in size over a concentration range of 0 to 100K’s per ml. The technique is insensitive to the particle’s optical properties and therefore offers distinct advantages over laser diffraction and light obscuration in terms of anlyzing heterogenous populations. The data set is intermediate between flow cytometry and manual microscopic analysis providing the automation, statistical validity and reproducability of the former while preserving the visual insight gained by the latter. Continuous sample introduction and time-resolved image analysis permitting high resolution reaction monitoring and trend charting. The paper will present experimental results applying MFI technology for general purpose particle fabrication process control with particular emphasis upon characterization and optimization of specific cell-related bio-processes including fermentation, viability, purification, dissolution, preservation, and protein aggregation. Application of the technology for detecting unwanted particulate within process fluids will also be presented, as well as use of the technology for realtime chemical reaction monitoring.
D3 – Invited Paper DEVELOPMENT OF MAGNETIC NANOPARTICLES FOR 126 BIOMEDICAL APPLICATIONS Nguyen Thi Kim Thanh, Ian Robinson and David G Fernig, Centre for Nanoscale Sciences, Department of Chemistry and School of Biological Sciences, University of Liverpool, Crown Street, Liverpool L69 7ZD, United Kingdom; [email protected]
Interest in metallic nanoparticles has grown rapidly in recent years due to their diverse applications in biomedicines and novel materials for engineering devices. Magnetic nanoparticles are of particular interest because of their potential application in areas such as biosensing, targeted drug delivery and hyperthermia treatment of solid tumours as well as high density magnetic storage. Magnetic nanoparticles composed of iron oxides are currently used as image enhancers in magnetic resonance imaging (MRI). However iron oxides have a relatively low saturation magnetisation, requiring these of larger particles. Transition metal nanoparticles, e.g. those made from cobalt, have a much higher saturation magnetisation value, allowing the use of small particles, without compromising their magnetic response/sensitivity. Using the thermal decomposition of organometallic compounds [Co2(CO)8 and Fe(CO)5] we have synthesised cobalt, iron and iron oxide nanoparticles. By adjusting the reaction conditions, magnetic nanoparticles of various sizes have been produced and by coating the particles with a variety of capping ligands, their stability in aqueous solution and biological systems can be investigated. Gold has also been used to coat iron and cobalt nanoparticles with a shell which, while protecting the tranision metal core from oxidation and corrosion can also easily be functionalised, for example by attaching peptides. Rationally selecting amino acids with structural and functional groups possessing different properties can give an insight of the behaviour of magnetic nanoparticles in aqueous and biological solutions. In particular, by changing the terminal amino acid of the peptide chain the biofunctinalisation of the nanonparticles can be finely
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tuned, with the ultimate aim of producing a useful therapeutic tool. The synthesised particles were characterised using TEM, SQUID and powder XRD.
E2 – Keynote Paper LIPOSOMAL BUPIVACAINE: AN ULTRA-LONG ACTING LOCAL 127 ANESTHETIC Gilbert J. Grant, Department of Anesthesiology, New York University School of Medicine, 550 First Avenue, New York, NY 10016; [email protected]
Pain after surgery and trauma results in considerable suffering and has deleterious effects on many physiological functions. Current pain management options have many drawbacks, and pain is notoriously under-treated. Direct numbing of the painful site by infiltration of local anesthetics is a treatment option that has many potential advantages, and avoids the complications that may occur with currently available approaches. A key drawback of using existing local anesthetics for infiltration is that the analgesia is of limited duration, because the small local anesthetics (MW ~300) are rapidly redistributed from the site of administration. To date, achieving prolonged analgesic duration requires repeated injections or the use of an indwelling catheter, but insertion and management of indwelling catheters requires highly skilled personnel, and are associated with a variety of complications (e.g., infection). Thus, there is a compelling need for a safe and effective local long-lasting analgesic. Liposomal bupivacaine was developed to meet this need. The formulation is based on proprietary techniques that enable efficient loading of drug into the liposome, resulting in a favorable drug-to-lipid ratio. In vivo assessment in animals and humans has demonstrated that liposomal bupivacaine produces ultra-long acting analgesia. The liposomes function as a depot from which the local anesthetics are slowly released, and the mass of free drug in the extra-liposomal milieu is sufficient to inhibit axonal conduction in the adjacent nerves. Furthermore, the slow drug release permits the safe administration of a relatively large dose. Unlike standard local anesthetics, which are quickly redistributed after injection, the liposomes tend to remain at the injection site. These characteristics make liposomes promising agents for the safe and effective treatment of pain. Their introduction into clinical practice is expected to revolutionize the way in which pain is managed, and to provide relief for many millions of patients suffering from painful conditions.
E2 – Invited Paper APPLICATION OF LIPID BASED DRUG DELIVERY SYSTEM FOR 128 WATER INSOLUBLE DRUGS Indranil Nandi, Sandoz, 2400 Route 130 N, Dayton, NJ 08810, USA;. [email protected]
In recent times up to 50% of new chemical entities are poorly water-soluble and therefore largely show poor bioavailability. Lipid-based formulation approach is an effective tool to improve oral absorption upon lipid digestion. Lipid based formulations, such as
116 Oral Abstracts microemulsions are increasingly recommended for such candidates. Microemulsion is an optically isotropic and thermodynamically stable system, which consists of water, oil, surfactant and co-surfactant. Microemulsions are used as drug delivery systems because of their solubilization capacity of poorly water-soluble compounds and enhancement of topical and systemic drug availability. The presentation will cover formulation design of lipid formulations. It will also covers issues related such as bioavailability, stability and manufacturability. This presentation will also focus on the application and success of microemulsion based formulations in the area of drug delivery. It also includes the application of microemulsion for various routes of administration and provides insight on the selection of pharmaceutically acceptable excipients for a stable lipid based delivery system.
E2 – Invited Paper PREPARATION OF THERMOSENSITIVE LIPOSOMES BY USE OF 129 AMPHIPHILIC BLOCK COPOLYMERS AND THEIR APPLICATION TO ANTICANCER DRUG DELIVERY Kenji Kono, Osaka Prefecture University, Department of Engineering, Sakai, Osaka 599-8531, Japan; [email protected]
Liposomes that release anticancer drug, doxorubicin (DOX), a few degree above physiological temperature were designed by use of block copolymers of (2-ethoxy)ethoxy ethyl vinyl ether (EOEOVE) and octadecyl vinyl ether (ODVE). The poly(EOEOVE) block behaves as a thermosensitive moiety with the lower critical solution temperature of about 40oC, whereas the poly(ODVE) moiety acts as anchor for the copolymer fixation onto liposomal membranes. The DOX-loaded egg lecithin-choresterol liposomes modified with the copolymer were prepared by hydration of a mixture of the lipids and copolymer and subsequent loading of DOX into the liposomes by the pH-gradient method. It was found that the copolymer-modified liposomes strongly enhanced the release of DOX above the LCST of poly(EOEOVE): the release was completed withn 1 min at 45oC. Next, distribution of the copolymer-modified liposomes in the body was examined by using radioisotope-labelled liposomes. The liposomes were injected into teil vein of mice and their distibution in the body was followed. Although the copolymer-modified liposomes exhibited slightly longer blood circulation time than bare liposomes, these liposomes were quite qickly taken up by liver. However, incorporation of poly(ethylene glycol) (PEG)-bearing lipid effectively increased their circulation time. Finally, antitumor activity of the DOX-loaded liposomes modified with the copolymer and PEG was investigated. The copolymer-PEG-modified liposomes containing DOX were injected into teil vein of mice bearing tumor. Tumor growth was slighly suppressed by the DOX-loaded liposomes when the tumor was not heated. However, when the tumor was heated at 45oC for 10 min, their efficacy for the suppresion of tumor growth increased dramatically. The same heat treatment hardly affected tumor growth when temperature-insensitive PEG-modified, DOX-loaded liposomes were injected. Therefore, it is likely that the copolymer-PEG-modified, DOX-loaded liposomes accumulated at the tumor site efficiently and released the anticancer drug responding to heat application, resulting in the strong suppression of its growth.
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E2 – Invited Paper PROMAXX MICROSPHERE DRUG DELIVERY TECHNOLOGY 130 Terrence L. Scott, Epic Therapeutics, A Wholly-Owned Subsidiary of Baxter Healthcare Corporation, 220 Norwood Park South, Norwood, MA 02062 USA, [email protected]
Epic/Baxter hasdeveloped a simple, scaleable, water based process of protein microsphere (PROMAXX) formation via controlled phase separation of the active agent in the presence of water soluble polymers that generates microspheres with narrow size distributions. The technology offers the capability for the fabrication of labile therapeutic proteins into microspheres for pulmonary delivery via dry powder formulations with ideal characteristics.The technology allows careful control of particle mean diameter as well as the size ditribution of the microspheres. The microsphere fabrication technology has been applied to human insulin, alpha-1-antitrypsin, human growth hormone as well as several other proprietary therapeutic proteins. In all cases protein microspheres were fabricated in an appropriate size range for pulmonary delivery (1-3 microns). The protein microspheres were produced with narrow particle size distribution, high retention of integrity, and high protein loading by a simple, cost efficient and scaleable process. The presentation will provide case studies of the aerodynamic characteristics, the stability and the bioavailability of several therapeutic proteins fabricated in the PROMAXX microsphere technology for delivery of dry powders via the pulmonary route.
E2 – Invited Paper DISLOCATION LOOP MEDIATED SMECTIC MELTING 131 Laurence Navailles, Patrick Moreau, Joanna Giermanska-Kahn, Olivier Mondain-Monval, Frédéric Nallet and Didier Roux. Centre de Recherche Paul Pascal, CNRS, avenue A. Schweitzer, F-33600, France ; [email protected]
The role of topological excitations in driving phase transitions has long been a matter of debate in several analogous three-dimensional condensed matter systems, namely superfluids, superconductors, and smectics. These topological excitations are borne by vortex (superfluids), magnetic flux (superconductors), and screw-dislocation (smectics) lines. The underlying microscopic mechanisms have been discussed theoretically by several authors and, as pointed out by most of them, analogous transitions should be driven by analogous mechanisms. In spite of the universality of "topological melting", such a mechanism has not yet been described experimentally. Here, we report the direct observation of topological melting in an unusual lyotropic system in the vicinity of a smectic – cholesteric (N*) phase transition. The proliferation of dislocations leads to at least one, and possibly two intermediate phases, characterised by orientational ordering of the dislocation loops and the subsequent unbinding of the screw-dislocation lines.
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M1 – Keynote Paper PARTICLE ENGINEERING AND FORMULATION FOR ENHANCED 132 BIOAVAILABILITY OF POORLY WATER SOLUBLE DRUGS Keith P. Johnston1, Xiaoxia Chen1, Jason McConville2, Jason Vaughn2, Kirk Overhoff2, Michal Matteucci1, Matthew Todd Crisp1 and Robert O. Williams III2; 1Dept. Chemical Engineering, Univ. of Texas Austin, 1 University Station C0400, Austin, TX 78712; 2College of Pharmacy, Univ. of Texas Austin, Austin, TX 78712
For poorly water soluble drugs, the clinical utility of available pharmaceutical dosage forms can be limited. Novel technologies have been developed to form amorphous and crystalline nanostructured particles of water insoluble drugs to enhance dissolution: evaporative precipitation into aqueous solution (EPAS), spray freezing into liquid (SFL), ultra-rapid freezing (URF) and controlled precipitation. The mechanisms of particle formation and stabilization, as well as the enhancement in dissolution rates, are discussed. The goal is to control nucleation and growth from solution to design particles with desirable in vitro dissolution behavior to achieve high bioavailability in animal studies. Strategies are reported for achieving high drug potencies up to 90%, yet with rapid dissolution rates. With these technologies, the ability to arrest drug particle growth to achieve high surface areas results from rapid phase separation and stablization of the resulting particles with polymers and surfactants. A transport model has been developed to predict dissolution from the micron to sub-micron size scale with a size-independent interfacial rate constant for incorporation of the drug into micelles. Bioavailabilty results are reported and analyzed in terms of in vitro data for mice, for oral and pulmonary administration. These solution based approaches to produce nanostructured particles offer several advantages over mechanical homogenization/milling processes including reduced handling of solids, high yields, ease of incorporation of stabilizing excipients, ease of scale-up, and control of crystallinity.
M1 – Invited Paper PARTICLES FOR DRUG DELIVERY AND MEDICAL APPLICATIONS: A 133 SUPERCRITICAL APPROACH Steven M. Howdle and Kevin M. Shakesheff∆ School of Chemistry and School of Pharmacy∆, University of Nottingham, University Park, Nottingham. NG7 2RD, UK; Vladimir K. Popov, Institute of Laser Technologies, Troitsk, Moscow, Russia; Martin J. Whitaker, Critical Pharmaceuticals, BioCity, Pennyfoot St., Nottingham NG1 1GF, UK; [email protected]
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Supercritical carbon dioxide (scCO2) is a versatile and clean processing aid for biodegradable polymers. We describe the use of scCO2 to prepare micron scale polymeric particles loaded with proteins for application in drug delivery, and in the preparation of novel laser fabricated surgical implants. Under mild conditions (ca. 35oC and 3000 psi) scCO2 can very effectively plasticise (liquefy) a wide range of biodegradable polymers, including poly(d,l-lactic acid), poly(lactide-co-glycolide), poly(caprolactone) and poly(ethyleneglycol). Using suitable high pressure mixing apparatus, a wide range of proteins can be dispersed throughout the liquefied polymer and by controlled pressure release can be processed into monolithic blocks, fibres or particles of protein loaded polymer. We will illustrate the principle using two protein based drug candidates; human growth hormone (hGH) and granulocyte macrophage colony stimulating factor (GMCSF), demonstarting that protein activity is retained and that the particles show sustained release in-vitro of protein over a period of one month. We have further utilised such supercritically prepared particles to create 3-D scaffolds with precise shape for tissue engineering applications in cranio-facial surgery. Rapid prototyping via slective laser sintering has been employed to develop protein loaded biodegradable scaffolds which also retain protein activity.
M1 – Contributed Paper INVESTIGATION INTO PARTICLE SIZE VARIATIONS WITH PROCESS 134 PARAMETERS USING NEKTAR SUPERCRITICAL FLUID TECHNOLOGY J. A. Green and A. Kordikowski, Nektar Therapeutics UK Ltd, 69 Listerhills Science Park, Bradford, BD7 1HR; [email protected]
In the past, various supercritical fluid antisolvent techniques have been used to achieve particle size reduction. However, conflicting results have been reported in the literature to how operating parameters influence the particle size of a crystalline material. In this investigation supercritical CO2 together with Nektar Supercritical Fluid Technology was used for the precipitation of a pharmaceutical active from organic solution. The influence of key process parameters (e.g. temperature, pressure, solution flow rate, etc) on the particle size was investigated. Dry powder particle size was determined by Sympatec analyser and particle morphology was studied by SEM. To determine the effect of the key operating parameters on the particle size it is vital that the precipitated particles have a small aspect ratio, as more lath and blade like particles tend to fracture during particle sizing giving erroneous results. Further, the supercritical technique should show efficient mixing of solvent and antisolvent in order to avoid the generation of erroneously large particles. Additionally, variations in temperature and pressure during the precipitation step should be kept to a minimum (i.e. no pressure fluctuations). Nektar Supercritical Fluid Technology was used to precipitate a solution containing hydrochlorothiazide (HCT). Operating pressures were varied between 75-200 bar and the temperatures between -1 298-353 K. Flow rates of CO2 were kept constant at 20 ml·min and the solution flow rates -1 were varied between 0.1-1.6 ml·min . Further the solubility of HCT in modified CO2 and solvent was measured under operating conditions to elucidate the influence of the residual solubility in the fluid and the saturation level of the solution on the particle size.
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M1 – Invited Paper NEW PARTICLE TECHNOLOGIES FOR DRUG DELIVERY 135 Boris Y. Shekunov and Pratibhash Chattopadhyay, Ferro Pfanstiehl Laboratories, Pharmaceutical Technologies, 7500 E. Pleasant Valley Rd, Independence, OH 44131, USA; [email protected]
The majority of drug products and drug delivery systems include particulate pharmaceuticals. Consistent production of particulate materials, in relatively pure physical forms as well as composite drug-excipient particles of various size, morphology and structure can be described as “particle engineering”. In the recent years new therapeutic applications, combined with increasing requirements for efficacy and safety of drug delivery, call for a diverse “tool-box” of manufacturing techniques. The aim is to create advanced manufacturing processes in terms of product consistency and quality. Additionally the new processes developed should also provide means for enhanced drug formulations not achievable using more conventional methods. Although many techniques can be applied in laboratory, large-scale production presents many development hurdles. This presentation will address the technology specifics in such application areas as: • New technologies of supercritical fluid extraction of emulsions, polymer plasticization and spray-freeze drying; boundaries of different methods in the context of drug-loading mechanisms, analytical issues and scalability. • Nanotechnology of water-insoluble drugs, polymer particles and lipid-based formulations. • Optimized formulations for pulmonary drug delivery. Composite particles of stable biological molecules immediate and controlled release.
M1 – Invited Paper FORMATION OF DRUG NANOPARTICLES AND MICROPARTICLES 136 USING SUPERCRITICAL CO2 Ram B. Gupta, Department of Chemical Engineering, Auburn University, Auburn, AL 36849-5127, USA; [email protected] Over past two decades, supercritical carbon dioxide (above 31.1 oC and 73.7 bar) has emerged as a medium for the formation of micro- and nano-particles of pharmaceutical compounds, due to the its adjustable solvent properties, high diffusivity, non-flammability, and non-toxicity. Depending upon the solubility in supercritical CO2, two classes of processes have emerged: (a) rapid expansion of supercritical solution (RESS) for CO2 soluble materials, and (b) supercritical antisolvent (SAS) for CO2-insoluble materials. In RESS process, the drug material is first dissolved in supercritical CO2 and then expanded through a nozzle to rapidly precipitate as particles. Since the expansion occurs as fast as the speed of sound, the material comes out as small microparticles. But due to the limited solubility of drugs in supercritical CO2, RESS process had limited utility, so far. In our recently developed RESS-SC process by using menthol solid co-solvent, the solubility has been enhanced by several hundred fold. The presence of the solid cosolvent also hinders the particle growth; hence the particles in nanometer size range are easily obtained.
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Menthol is later removed by sublimation, yielding pure drug nanoparticles. In SAS process, the drug material is first dissolved in an organic solvent. The solution is then injected into supercritical CO2, resulting in the extraction of solvent by supercritical CO2 and precipitation of the material. Since, the speed of extraction is fast due to the high (gas-like) diffusivity of supercritical CO2, the small microparticles of the material are obtained. We have further enhanced the extraction speed by ultrasonic mixing which results in nanoparticles. In the new process, the particle size is easily controlled by the extent of ultrasonic power supplied. The strong extraction ability of supercritical CO2 allows the production of pure drug nanoparticles, free of any residual solvent or additives.
M1 – Invited Paper DRUG DELIVERY APPLICATIONS OF PARTICLE TECHNOLOGY 137 USING CAN-BD PROCESSING Robert E. Sievers, Brian P. Quinn, Jim A. Searles, Pradnya Bhagwat, Lia G. Rebits, Aktiv-Dry, 6060 Spine Road, Boulder, CO 80301; Nurhan T. Dunford, Dept. of Plant and Soil Sciences, Oklahoma State Univ., Stillwater, OK 74078; Dexiang Chen, Debra Kristensen, PATH, 1455 NW Leary Way, Seattle, WA 98107; Mark T. Hernandez, Lowry Lindsay, Civil, Environmental and Architectural Engineering, Univ. of Colorado, Boulder, CO 80309-0428; and Stephen P. Cape, Chad S. Braun, David H. McAdams, Jessica A. Best, Jessica L. Burger, Center for Pharmaceutical Biotechnology, Dept. of Chemistry and Biochemistry, and CIRES, Univ. of Colorado, Boulder, CO 80309-0214; [email protected]
Formation of microparticles and nanoparticles by Carbon dioxide Assisted Nebulization with a Bubble Dryer® (CAN-BD) permits aerosol pulmonary delivery of pharmaceuticals, nutraceuticals, vaccines, antibiotics, and anti-virals. In addition, the creation of very finely divided nanoparticles leads to faster rates of dissolution and enhanced bioavailability. CAN-BD of phytosterols dissolved in ethanol, acetone or hexane leads to particles averaging 100 nm in diameter that are rapidly dissolved in soybean oil. Aqueous vaccines such as Hepatitis B surface antigen (HBsAg) adjuvanted with aluminum hydroxide sometimes suffer freeze damage or heat damage. When stabilized with trehalose or trehalose plus PVP, then dried by CAN-BD at 50 °C, the powders stored at temperatures ranging from -20 °C to 66 °C for 43 days showed no loss of activity in vitro (ELISA) or in vivo (mice) upon reconstitution in water for analysis or injection. Creating improved dry powder vaccines for transport and use in developing countries where storage temperature control is difficult is one of the main goals of the Grand Challenges in Global Health initiative. Another goal is the creation of needle-free delivery systems. We are involved in a program that will hopefully make contributions to both of these goals and have initiated studies of the stabilization, micronization, and drying by CAN-BD of inhalable live-attenuated measles virus vaccine. Initial results indicate powder stabilization with trehalose and/or sucrose. Respirable particles of anti-tuberculosis drugs including capreomycin, amikacin, ciprofloxacin, and moxifloxacin have been made by CAN-BD from aqueous solutions. For rifampin, ethyl acetate was the solvent of choice and supercritical nitrogen was the nebulizing dispersant. All five antibiotics maintained full
122 Oral Abstracts activity after preparation by CAN-BD. The anti-viral pharmaceutical, Relenza®, was dissolved in water and processed by CAN-BD to yield microparticles with MMAD of 2.4 µm and the respirable mass fraction was 73% < 5 µm.
M1 – Invited Paper NANOPARTICLES FOR ENHANCED SOLUBILITY AND 138 BIOAVAILABILITY OF POORLY-WATER SOLUBLE DRUGS USING SUPERCRITICAL FLUID TECHNOLOGY Sibeum Lee, Min-Soo Kim, Jeong-Soo Kim, Hee Jun Park, and Sung-Joo Hwang, National Research Lab of Pharmaceutical Technology, College of Pharmacy, Chungnam National University, Yuseong-gu, Daejeon, 305-764, Korea, [email protected]
Among many techniques for the preparation of solid dispersions, including milling, spray drying, freeze-drying and solvent evaporation, supercritical fluid technology (SCF) provides a novel alternative for formulating nano-sized co-precipitates that may be lower in residual organic solvent and overcomes some of the problems faced in conventional methods. In this study, solid dispersion nanoparticles containing felodipine, cefuroxime axetil (CA) and itraconazole, respectively as poorly water-soluble drugs were prepared using SCF process in an effort to increase its dissolution rate of drug and subsequently the bioavailability. Their physicochemical properties in solid-state were characterized by particle size, zeta potential, DSC, powder x-ray diffraction (PXRD), Fourier transform infrared spectroscopy (FT-IR) and SEM. In addition, equilibrium solubility and dissolution study were also carried out. The mean particle size of the felodipine solid dispersions was 200-250 nm; these had a relatively regular spherical shape with a narrow size distribution. Pure CA was presented as irregular and flaky particles (size range ca. 5-10 μm) whereas all solid dispersion nanoparticles prepared using SCF process at various ratios were presented as regular and spherical particles (size range ca. 200-300 nm) with narrow particle size distributions (PSDs). CA solid dispersion nanoparticles existed in the amorphous or non-crystalline form. Dissolution studies indicated that the dissolution rates were remarkably increased in solid dispersion nanoparticles, compared with those in physical mixture and drug alone. The particle size of itraconazole solid dispersions ranged from 100 to 500 nm. The nanoparticles prepared at 60℃/10 MPa showed approximately 610-fold increase in equilibrium solubility when compared to pure itraconazole. In conclusion, SCF process could be a promising technique to reduce particle size and/or prepare amorphous solid dispersion of drugs in order to improve the solubility and bioavailability of poorly water-soluble drugs.
A4 – Invited Paper DOUBLE DIRECT TEMPLATING OF PERIODICALLY 139 NANOSTRUCTURED ZNS HOLLOW MICROSPHERES Paul V. Braun, Alejandro Wolosiuk, Onur Armagan, Univ. of Illinois at Urbana-Champaign, Department of Materials Science and Engineering,
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Beckman Institute, and Frederick Seitz Materials Research Laboratory, Urbana, Illinois 61801, USA; [email protected]
Hollow capsules are both technologically and scientifically interesting. ‘Smart’ nano and micro containers could lead new catalysts, structures for the controlled release of chemicals, and materials that may even mimic some properties of living cells. Here we present a ‘double direct templating’ approach to obtain hollow ZnS 500 nm diameter microspheres perforated with a periodic array of uniform 2-3 nm diameter pores and demonstrate the entrapment of Au nanoparticles and biological macromolecules within these microspheres. In double direct templating, a lyotropic liquid crystal templates the mineralization of ZnS on the surface of a silica or polystyrene colloidal template. Removal of the templates results in a periodically mesostructured ZnS hollow capsule. Lyotropic liquid crystal templating is in part useful for this process because of its simplicity and ability to control structure on the nanoscale, using only the self-assembled structure formed by the mixture of an amphiphilic molecule (e.g. a nonionic surfactant) with water to template the formation of a mineral phase. The regular controlled pore structure of the shell wall opens the possibility for the encapsulation of nanoparticles, polymers, and biological macromolecules.
A4 – Invited Paper SIGNAL BIOMOLECULE-RESPONSONSIVE GELS 140 Takashi Miyata, Faculty of Engineering, Kansai University, Suita, Osaka 564-8680, Japan; PRESTO, Japan Science and Technology Agency (JST); [email protected]
Stimuli-responsive gels that exhibit volume changes in response to environmental changes such as pH and temperature have many future opportunities as suitable materials for designing smart systems in biomedical fields. We have prepared a variety of biomolecule-responsive gels by using biomolecular complexes as stimuli-responsive cross-linking points. This paper describes two types of signal biomolecule-responsive gels that undergo volume changes in response to signal biomolecules, which were prepared using biomolecular complexes such as antigen-antibody complexes, saccharide-lectin complexes and DNA duplexes. One is a biomolecule-crosslinked gel and the other is a biomolecule-imprinted gel. The biomolecule-crosslinked gels can swell in the presence of a targel biomolecule due to the dissociation of biomolecular complexes that play a role as reversible crosslinking points. For example, the bioconjugated gels having antigen-antibody bindings at cross-linking points swelled in the presence of a specific antigen, but did not in the presence of other proteins. The antigen-responsive gels controlled drug permeation in response to the antigen concentration. Furthermore, we have prepared glucose-responsive gels and DNA-responsive gels as biomolecule-crosslinked gels. On the other hand, biomolecule-imprinted gels can shrink in response to a target biomolecule due to the complex formation between ligands and the target molecule. As biomolecule-imprinted gels, we prepared glycoprotein-responsive gels and DNA-responsive gels by biomolecular imprinting. Glycoprotein-responsive gels were prepared by using lectin and antibody as ligands for a template tumor-marker
124 Oral Abstracts glycoprotein in biomolecular imprinting. The glycoprotein-imprinted gels having both lectin and antibody shrunk in a buffer solution containing the target glycoprotein. Furthermore, biomolecular imprinting using single strand DNAs as ligands enabled us to prepare DNA-responsive gels that can recognize SNPs. Thus, biomolecule-responsive gels have many potential applications as smart biomaterials in biomedical fields. This paper focuses on synthetic strategy of biomolecule-responsive gels and their responsive behavior.
A4 – Contributed Paper NANOPARTICLE PREPARATION OF α-LIPOIC ACID SUSCEPTIBLE TO 141 POLYMERIZATION Chul ho Park, Jongwhi Lee; Department of Chemical Engineering and Materials Science, Chung-Ang University, 221, Heukseok-dong, Dongjak-gu, Seoul 156-756, Korea (South); [email protected]
Among various antioxidants, the redox system of alpha-lipoic acid (ALA) and dihydrolipoic acid (DHLA) is an excellent antioxidant because both the reduced and oxidized forms retain their functions of protecting defects related to blood flow and neurotrophins. Recent medical studies confirmed their efficacy of not only anti-obesity but also appetite suppression, as an overdose of ALA is taken (over 600 mg/kg). The dose might be reduced into an acceptable range by improving the bioavailability of ALA. The reduction of crystal sizes to nanometers in a drug delivery system can directly improve its bioavailability. There are several restrictions in utilizing the general processes to prepare ALA nanoparticles such as high shear homogenizing and ball milling, because cyclic five-membered disulfide is susceptible to free radical ring-opening polymerization by friction heat. In wet comminution, ALA of less than 0.01 M was reduced to DHLA whose thiol (-SH) can attack the cyclic disulfide (SN2 reaction) and act as the initiator for ionic polymerization. Therefore, this study focuses on how to prepare the nanoparticles of ALA without any side chemical reactions like the polymerization. The temperature of surroundings during ball milling was preserved at 4 °C in order to control that of ALA particles below 20 °C for 4 hours. As a result, we could produce ALA nanoparticles having the mean particle size of 300 nm with using a stabilizer, PVP (polyvinylpyrrolidone). To prove the possible bioavailability improvement, nanoparticles and microparticles of ALA were injected to standard rats. After 6 hours, the average amount of cumulative food intake of rats on ALA nanoparticles was half as much as that of rats on ALA microparticles.
A4 – Contributed Paper POLYELECTROLYTE MICROCAPSULES FOR BIOMEDICAL 142 APPLICATIONS Bruno G. De Geest, Stefaan C. De Smedt and Jo Demeester, Department of Pharmaceutics, Ghent University, 72 Harelbekestraat, 9000, Ghent, Belgium; and Gleb B. Sukhorukov, IRC/Materials, Queen Mary University, Mile End Road, E1 4NS, London,UK; [email protected]
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Due to it’s simplicity in preparation and multifunctionality, the advent of the layer-by-layer (LbL) technique has led to an accelerated discovery in the field of organic thin films. In brief, this technique is based on the sequential adsorption of charged species, such as polyelectrolytes, onto an oppositely charged substrate. Using the LbL technique it is possible to fabricate hollow microcapsules by coating a colloidal substrate followed by the decomposition of the substrate. These microcapsules hold high potential for biomedical applications. However, until now there are very few reports on polyelectrolyte microcapsules which can decompose under conditions applicable in vivo. Here we present three types of polyelectrolyte microcapsules sensitive to stimuli which can be evoked by the human body: (1) Self-exploding microcapsules consisting of a biodegradable microgel core surrounded by a polyelectrolyte membrane. When the microgel core degrades a swelling pressure is created which will finally rupture the surrounding polyelectrolyte membrane leading to the release of the encapsulated material. Such microcapsules could be highly interesting for single-shot vaccination as it could repleace the booster injections which are required to generate sufficient immunity. (2) Degradable microcapsules consisting of biopolymer polyelectrolyte multilayers which can be degraded enzymatically or polyelectrolyte multilayers containing a polycation whose cationic sidechains can be hydrolyzed. We show that these microcapsules can be taken up and digested by living cells whereas microcapsules from synthetic polyelectrolytes cannot be digested. These types of capsules hold potential for the targetted deliery of therapeutics. (3) Glucose responsive microcapsules fabricated using a novel synthesized polycation containing phenylboronic acid as glucose sensitive moiety. We show that upon addition of glucose the microcapsules are able to disassemble. This type of microcapsules could be interesting to deliver insulin to diabetic persons only at that moment the glucose concentration in the blood exceeds a critical value.
A4 – Invited Paper ORAL NANOPARTICLE DELIVERY 143 James Talton, Nanotherapeutics, Inc., 12085 Research Drive, Alachua, FL 32615. [email protected].
From wet-milling to nanometer-scale particle delivery, development of novel pharmaceuticals, cosmetics, and over-the-counter 'nano'products is growing in interest. Nanoparticle engineering technologies, initially focused on improving dissolution of insolubles, has been proven to: 1) improve the oral bioavailability of insoluble and poorly absorbed drugs that currently require injection (antibiotics, antivirals, anti-inflammatories, and nutritionals). 2) enable oral delivery of macromolecules (peptide/protein) and 3) provide pharmaceutical life-cycle management opportunities. This presentation will present examples and attempt to compare several companies and technologies, making the landscape of oral nanoparticle therapeutics clearer for different applications of interest.
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A4 – Invited Paper DRUG-LOADED PROTEIN AND DNA NANO-MESO-MICROSPHERES 144 FOR NON-SYSTEMIC INTRATUMORAL CHEMOTHERAPY Eugene P. Goldberg, Iris V. Enriquez, Shema Freeman, Amanda York Biomaterials Center, Department of Materials Science & Engineering, University of Florida, P.O. Box 116400, Gainesville, FL 32611, USA; [email protected]
The therapeutic activity of most cancer drugs is highly dose-dependant and systemic toxicity such as myelosuppression, mucosistis, and cardiomyalgia usually prevents the use of aggressive high dose chemotherapy. There is a consequent need for more efficient and localized non-systemic drug delivery. We have therefore explored the synthesis of drug loaded nano-meso-microspheres (meso-MS) and their application for intratumoral (IT) chemotherapy. Recent research has been aimed at the development of clinically practical compositions derived mitoxantrone (MXN) with biodegradable proteins (i.e. albumin, collagen, casein) or with deoxyribonucleic acid (DNA) as the meso-MS matrix. The synthesis developed in our laboratory involves steric stabilization of the aqueous biopolymer solution dispersions in an organic polymer solution continuous phase (i.e. cellulose acetate in tetrachloroethane) with crosslinking of the meso-MS uniquely via the organic phase using glutaraldehyde or heavy metal ions in some cases for DNA. Some emphasis has been on meso-MS compositions having 2-10 um particle size because of favorable injectability of dispersions, good tumor tissue permeation and fixation, and desirable surface area for efficient intratumoral drug release by diffussion and bio-degradation mechanisms. Although in vivo evaluations have demonstrated excellent tumor regression properties in murine models of ovarian and lung cancer, more recent pre-clinical animal studies have been conducted in a breast cancer model, a subcutaneous 16/C murine mammary adenocarcinoma using female C3H/HeJ mice. Intratumoral therapy with MXN loaded albumin meso-MS demonstrated greatly prolonged survival (>300%) with no systemic toxicity at MS drug dose levels more than 7x the toxic dose for conventional intravenous drug administration (48mg/Kg with meso-MS vs. 6.6mg/Kg for i.v. drug). Results of these studies suggest promising opportunities for the clinical use of intratumoral chemotherapy using drug loaded meso-MS to afford prolonged high drug concentrations in tumors and tumor regression with minimal general toxicity; especially for preoperative (neoadjuvant) chemotherapy to enhance outcomes for tissue-conserving breast and lung cancer surgery.
A4 – Invited Paper PREPARATION OF MAGNETIC POLYMERIC MICROSPHERES FOR 145 APPLICATION IN BIOMEDICAL FIELDS Changchun Wang, Jianhua Hu, Wuli Yang and Shoukuang Fu, Department of Macromolecular Science, Fudan University, and the Key Laboratory of Molecular Engineering of Polymers, Ministry of Education, Shanghai 200433, China; [email protected]
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Because of the wide applications in the area of biomedical and biotechnological fields, a great effort has been done to fabricate different kinds of microspheres with tailored structural and surface properties over the last decade. Among them, stimuli responsive microspheres are microspheres that show ability to change their physical-chemical properties and colloidal properties in response to environmental stimuli such as changes of temperature, pH, chemicals, light, electrical field, magnetic field or mechanic stress, etc. These microspheres have been under intensive study for their high potential applications in biomedical and biotechnological fields such as controlled drug delivery, biosensor, chemical isolation, cell culture substrates, enzyme immobilization, bioelectrocatalysis, and magnetically controlled electrochemical reaction. However, most of the reported stimuli responsive microspheres only show response to one specific stimulus. Up to now, to the best of our knowledge, only a few works have been directed to the fabrication of microspheres which show response to more than one stimulus. In this paper, a series of FITC-labeled magnetic PNIPAM microspheres with multi-resposive properties were prepared. The FITC-labeled magnetic PNIPAM microspheres suspended in saline water (0.9wt %) were introduced to the bodies of white rabbits. For comparison, a button magnet (1000Gs) was posited at the liver of one of the test rabbits. The main visceral organs including liver, spleen, lung, kidney and heart were taken out and sliced on freezing-microtome, and the slices of every visceral organ were observed in fluorescent microscopy to obtain the distribution of the FITC-labeled magnetic PNIPAM microspheres. The experimental results showed that the FITC-labeled magnetic PNIPAM microspheres have good magnetic target effect.
A4 – Contributed Paper FABRICATION OF BIODEGRADABLE POLYMER MICRODEVICES 146 FOR CONTROLLED DRUG DELIVERY SYSTEMS Aravind Chakrapani1, Nicholas J. Ferrell1, Derek J. Hansford1,2,3 1Biomedical Engineering Center, Ohio State University, 2Department of Materials Science and Engineering, Ohio State University, 3Ohio MicroMD Laboratory, Columbus, OH 43212, USA; [email protected]
We report a novel soft lithography based technique to fabricate non-spherical biodegradable polymeric microparticles of different sizes and shapes as drug delivery systems. Geometrical control over the shape and size of these microparticles renders them different aerodynamic and fluidic dynamic properties when compared to conventional spherical microparticles and may prove to be beneficial in certain drug delivery strategies, such as pulmonary and intravenous routes. The surface morphology of the particles was studied using a scanning electron microscope and size distribution of the particles was determined using a Coulter counter. The process is reproducible and millions of uniform biodegradable particles of various sizes and shapes with dimensions ranging from 2-50 µm have been fabricated. In addition, we demonstrated encapsulation of a model drug (FITC) and drug distributions within the particles were studied by confocal microscopy. Particle size is a critical parameter for several aspects of controlled drug delivery including control of drug-release kinetics, passive targeting to specific cell or tissue types,
128 Oral Abstracts biodistribution upon administration and available routes of administration. In conclusion, the uniform, biodegradable polymeric microparticles produced have potential to be used in a variety of drug delivery applications and polymer-based microfabrication technology holds promise to produce sophisticated, multi-functional drug delivery devices.
B2 – Invited Paper W/O/W MULTIPLE EMULSIONS CONTAINING NITROIMIDAZOLE 147 DERIVATES FOR VAGINAL APPLICATION Özgen ÖZER, Mine ÖZYAZICI, Ege University, Faculty of Pharmacy, Department of Pharmaceutical Technology, 35100 Bornova-Izmir/Turkiye; [email protected]
Vaginal trichomoniasisis a common problem frequently seen in clinical practice. While the vaginal flora of healty women is dominated by a low pH (3.5-4.5), for women suffering from vaginal infections an increase of the pH values is observed (>5). W/O/W multiple emulsions have two layers and due to the double layers of the emulsifier, the multiple emulsion facilitated transcutaneous passege of the water soluble substance compared to a classic emulsion and it was pleasent to apply. The aim of this study was to formulate a stable multiple emulsions containing two nitroimidazole derivates, metronidazole (MT) and ornidazole (OR) for vaginal therapy. MT and OR were located different phases of multiple emulsion and the in vitro release studies were realized in phosphate (pH 7) and lactate buffer (pH 4.5) solutions to investigate better the effect of pH and location of active substance on the release. The imaging studies were realized in rabbits following labeling MT and OR with Technethium-99m (99mTc) to evaluate the in vivo absorption characteristics. The percentage of MT and OR released from the multiple emulsions in alkaline media were 3.2 and 2.8 fold greater than that observed in acidic media, respectively, when they were introduced internal phase of the multiple emulsions. In this case, it was also possible to protect them against an eventual degradation besides having a prolonged release. 63.51 % of the administered OR dose remained in the rabbit’s vagina after 2.5 hours. This value was calculated as 54.59 % for MT, according to the activity detected from multiple emulsions and surrounding tissues. It was observed that especially in alkaline medium a high release was found which was convenient for the vaginal infections seen in the alkaline pH. It was concluded that W/O/W multiple emulsions were locally effective in vagina and they could be introduced as a new drug carrier system for vaginal delivery.
B2 – Invited Paper MECHANISMS OF EMULSION STABILISATION BY COLLOIDAL 148 PARTICLES Tommy S. Horozov and Bernard P. Binks, Surfactant & Colloid Group, Department of Chemistry, University of Hull, Hull, HU6 7RX, UK; [email protected]
It is known that certain solid colloidal particles can attach to oil-water interfaces and act as excellent emulsion stabilisers by forming a coherent particle layer around the emulsion
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droplets. It is accepted that the dense particle layers serve as a steric (mechanical) barrier keeping droplets apart and making particle-stabilised emulsions completely stable for years. There are, however, some rather puzzling results for stable emulsions with droplets just sparsely covered with particles, which call under question the accepted mechanism of stabilisation. Therefore the relation between the structure of particle monolayers, their behaviour in isolated thin liquid films and the mechanism of stabilisation in particle-stabilised emulsions is still unclear. We address this fundamental issue and present experimental results demonstrating the link between particle behaviour and interactions in planar monolayers, thin liquid films and the mechanisms of emulsion stabilisation. We show why strongly repulsive colloidal particles, which give well ordered dilute planar monolayers, can act as very effective emulsion stabilisers. This is due to the spontaneous particle accumulation in a dense monolayer bridging the emulsion droplets sparsely covered with particles, thus preventing coalescence, as revealed by direct in situ microscope observations. Our results imply that two general mechanisms of emulsion stabilisation by solid particles thus exist: (i) steric stabilisation of emulsion droplets covered by coherent particle layers and (ii) stabilisation by particle bridges when droplets are sparsely covered. This is very important for understanding the fundamentals of solid-stabilised emulsions and designing more efficient particle stabilisers.
B2 – Invited Paper PREPARATION OF BIODEGRADABLE NANOPARTICLES BY A 149 MEMBRANE BASED PROCESS K. Ebert, A. Ahmad, R. Just GKSS Research Centre Geesthacht GmbH, Max-Planck-Str. 1, 21502 Geesthacht/ K. Lützow, M. Scholz GKSS Research Centre Geesthacht GmbH Kantstr. 55, 14513 Teltow, Germany; [email protected]
Nanoparticles and nanocapsules are of special interest since they are effective carriers for different substances such as pharmaceutical agents, genes, proteins and peptides. Nanoprecipitation is a method capable for the lab-scale preparation of such particles with low mean particle size and low polydispersity. Briefly, a solution consisting of a polymer and a solvent is poured into a non-solvent bath containing a surfactant under agitation. Phase inversion occurs due to diffusion of solvent into the non-solvent bath. As a consequence, polymer precipitation occurs. The nanoparticles are stabilized by a simultaneously formed shell of surfactant molecules on the surface of the nascent particles. The direct scale-up of this method, however, results in particles with significantly less uniform particle size distribution. The aim of our work was the development of a process allowing large-scale manufacture of nanoparticles and nanocapsules with uniform particle size distribution. The new process utilizes porous membranes through which the polymer solution is homogeneously permeated into a circulating aqueous phase. So far, experiments with flat porous membranes of poly(acrylonitrile) (PAN) revealed that under the chosen conditions the polymer concentration has the strongest influence on the mean particle size while the concentration of the surfactant in the aqueous phase is determining the polydispersity of the nanoparticles.
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B2 – Invited Paper NEW FUNCTIONS OF GRAFTED-POLYMER LAYERS OF 150 CORE/CORONA POLYMER NANOSPHERES IN BIOSEPARATION Hidekazu Yoshizawa, Shintaro Abe and Tsutomu Ono, Graduate School of Environmental Science, Okayama University, Okayama 700-8530, Japan; [email protected]
Aqueous two-phase system is known for extraction technique for protein purification. Poly(ethyreneoxide)/dextran (PEO/Dex) system and poly(N-isopropyl acrylamide)/dextran (PNIPAAm/Dex) system are, for example, representative systems of aqueous two-phase system. The difference of hydrophobicity between two kinds of water-soluble polymer is mainly responsible for the partitioning of proteins. Whereas, functional polymeric nanospheres have large specific surface area and provide the noble separation field. We prepared core/corona polymeric nanospheres and examined the mechanism of protein adsorption on functional polymeric nanosphere. In this study, we report the behavior of protein partitioning between the grafted polymer layer on polymer nanospheres and Dex solution. Polymer nanospheres with PEO corona layer were prepared by dispersion copolymerization of PEO macromonomer and styrene. Whereas, PNIPAAm grafted polymer nanospheres were prepared by seed dispersion polymerzation. The prepared polymer nanospheres were dispersed in Dex solution and we observed the formation of aqueous two-phase partitioning system on the surface of polymer nanospheres. Proteins, phycocyanin and hemoglobin, were partitioned to PEO-rich phase and Dex-rich phase in liquid-liquid system, respectively. In nanosphere system, the similar tendency of protein partition was observed, that is, phycocyanin partitioned to PEO-core on nanosphere and hemoglobin partitioned to Dex solution. Untill now, phase separation in PNIPAAm/Dex system has been reported. However, we newly observed this system showed reversible thermosensitibily below the LCST of PNIPAAm. This phenomenon can be expected to realize thermo-sensitive purification system of proteins. Therefore, we carried out the protein partitioning in this system. Consequently, hemoglobin partitioned in Dex solution in liquid-liquid system was concetrated in solution phase of nanosphere system and lysozyme partitioned in PNIPAAm solution in liquid-liquid system was tranfered in PNIPAAm-gratfted layer in nanosphere system. This partitioning tendency in PNIAAm/Dex system was the same as the obtained result in PEO/Dex system.
B2 – Invited Paper RAPID STABILITY TESTING AND SHELF LIFE DETERMINATION OF 151 CONCENTRATED EMULSIONS Dietmar Lerche, Stefan Küchler, Titus Sobisch, Torsten Detloff and Dean Abadzic°, L.U.M. GmbH, Rudower Chaussee 29, 12489 Berlin, Germany; [email protected], °LUM Corp., 200 Boston Ave., Suite 2900, Medford, MA 02155, [email protected]
Many dispersions in the daily life are colloidal systems based on emulsions, partly containing additionally solid particles to improve their performance. The long term stability of the “quality” of emulsion based products is crucial, as between the production
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date and the time of their use or consumption often weeks and even month are passed by. Shelf life depends directly on physical stability besides other phenomena like degradation phenomena by light or alterations of emulsion’s texture. Centrifugation is the only method truly to accelerate physically sedimentation or creaming processes of emulsions and dispersions. Furthermore, analytical photocentrifugation is a very efficient tool to determine the demixing velocity. In this paper we demonstrate that from these velocities the shelf life may be directly determined. We present a SOP, how the influence of the elevated g-forces may be evaluated based on the analysis of a velocity versus g-force (centrifugal acceleration) dependency. Shelf life at gravity (1 xg) can be confidently calculated based on the linear or non-linear regression lines. Besides shelf life these studies reveal additional information regarding the structural (rheological) stability of emulsion based products.
B2 – Contributed Paper CERAMISPHERES: CONTROLLED RELEASE FROM SOL-GEL MICRO 152 AND NANOPARTICLES Christophe J. Barbé, Kim Finnie and Linggen Kong, CeramiSphere, ANSTO, PMB1, Menai NSW 2234, Australia; [email protected]
We present a generic approach to the synthesis of sol-gel silica matrices for encapsulating bioactive species, and controlling their subsequent release over periods ranging from hours to months. The bioactive species are incorporated into the matrix during gelation at, or near, ambient temperature, and remain entrapped until the gels are immersed in solution. The release profile can be tailored by controlling the internal structure of the gels (pore volume, size and tortuosity, and surface chemistry). In turn, the gel structure can be precisely tailored by varying such sol-gel processing parameters as the water-to-alkoxide ratio, pH, alkoxide concentration, ageing, drying time and temperature. Hence, the release rate of the encapsulated species is controlled by adapting the structure of the internal pore network to the physico-chemical properties of the active molecule. Combining emulsion synthesis with sol-gel technology enables the carrier to be produced in the form of mono-dispersed spherical particles, with an average size that can be varied from 50 nm to 50 µm. The particle diameter is determined by the size of the reverse micelles, which is controlled by the hydrophile-lipophile balance between the surfactant, aqueous phase and non-polar solvent. The release rate of the encapsulated species is controlled by the internal nanostructure of the spheres, which can be tailored (as in bulk gels) by varying the sol-gel chemical parameters. Another key feature of this process is its versatility, which allows the encapsulation of a wide range of actives, from simple hydrophilic dyes to larger biomolecules or nanocrystals. The combined versatility and independent control over the release rate and particle size gives this technology considerable potential as a delivery solution in the food, homecare, chemical, biocide, pesticide, cosmetic and pharmaceutical markets.
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B2 – Invited Paper CATIONIC ALBUMIN CONJUGATED PEGYLATED NANOPARTICLE 153 AS NOVEL DRUG CARRIER FOR BRAIN DELIVERY Wei Lu, Xinguo Jiang. PO Box 130, Department of Pharmaceutics, School of Pharmacy, Fudan University (Fenglin Campus), 138 Yi Xue Yuan Rd, Shanghai 200032, P.R. China; [email protected]
A novel drug carrier for brain delivery, cationic bovine serum albumin (CBSA) conjugated with poly(ethyleneglycol)-poly(lactide) (PEG-PLA) nanoparticle (CBSA-NP), was developed and evaluated. Copolymers of methoxy-PEG-PLA and maleimide-PEG-PLA were synthesized by ring opening polymerization of D,L-lactide initiated by methoxy-PEG and maleimide-PEG, respectively, which were applied to prepare pegylated nanoparticles by means of double-emulsion and solvent evaporation procedure. Native bovine serum albumin (BSA) was cationized and thiolated, followed by conjugation through the maleimide function located at the distal end of PEG surrounding the nanoparticle’s surface. To evaluate its blood-brain barrier (BBB) transcytosis and toxicity against BBB endothelial tight junction, we have explored a method of coculture with rat brain capillary endothelial cells on the top of micro-porous membrane of cell culture insert and rat astrocytes on the bottom side. BSA conjugated with pegylated nanoparticles (BSA-NP) was used as control group and 6-coumarin was incorporated into the nanoparticles as a fluorescent probe. Transmission electron micrograph showed CBSA-NP had a round and regular shape with a mean diameter around 100 nm. The unchanged permeability of 14C-labeled sucrose comparing to that of the sucrose in the appearance of 200 μg/ml of CBSA-NP proved that CBSA-NP did not impact the integrity of BBB endothelial tight junction. Permeability of CBSA-NP was about 7.76 times higher than that of BSA-NP, while the transcytosis was inhibited in the excess of free CBSA, indicating that CBSA-NP underwent absorptive-mediated transcytosis. Thirty minutes after a dose of 60 mg/kg CBSA-NP or BSA-NP injection in mice caudal vein, fluorescent microscopy of brain coronal sections showed a higher accumulation of CBSA-NP around the lateral ventricle, third ventricle and periventricular region than that of BSA-NP. This indicated CBSA-NP preferentially transported across BBB with little toxicity, which offered the possibility to deliver therapeutic agents to brain.
B2 – Invited Paper COLLOIDAL EMULSION APHRONS: STRUCTURE, PROPERTIES, 154 PRINCIPLES AND APPLICATIONS Yujie Dai, Tianjin Key Laboratory of Industrial Microbiology, College of Biotechnology, Tianjin University of Science and Technology, Dagu Nanlu 1038, Tianjin, 300222, China; Tong Deng, Institute of Process Engineering, Chinese Academy of Sciences, P.O. Box 353, Beijing, 100080, China; [email protected]
Colloidal emulsion aphrons (CEAs) can be considered as the micron-sized water-in-oil emulsion-cores encapsulated by a “soapy shell” consisting of multi-layer surfactant molecules. In this paper, their structure and some properties are summarized. The
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principles and affecting factors on CEA extraction are introduced. In addition, the CEA applications to drug over dose, detoxification and fine particle preparation were also investigated. It is similar to emulsion liquid membrane system. The mechanism for CEA separation can be approximately divided in two types. Type I (physical separation) and type II (carrier-facilitated diffusion). For type I mechanism, the solute transport across the soapy shell and the membrane is driven by the ability of the desired species to partition into the membrane phase and its diffusion rate through the membrane and the soapy shell. But for the solute which is not soluble in membrane, a carrier has to be used in the intervening membrane phase, which binds and releases the solute at the external and internal interfaces successively, allowing diffusion of the solute-carrier complex through the liquid membrane. This approach constitutes type-II carrier-facilitation mechanism. For type I separation, the CEA application to drug overdose treatment was studied using salicylic acid as the model drug, paraffin oil as the membrane phase, PEG-30 dipolyhydroxystearate (P135) as the hydrophobic surfactant, nonylphenol ethoxylate-10 (NP10) as the hydrophilic surfactant and NaOH solution as the receptor phase. When the pH value of the donor phase was lower than 2.0 and the suitable NaOH concentration of the receptor phase was higher than 0.02mol/L, more than 98.7% of salicylic acid was transported into receptor phase in half a minute. For type II separation, the heavy metal removal from the donor phase was studied with Cu2+ as a model ion, di-(2-ethylhexyl) phosphoric acid (D2EHPA) as a carrier, paraffin oil as the membrane phase, PEG-30 dipolyhydroxystearate (P135) as the hydrophobic surfactant, nonylphenol ethoxylate-10 (NP10) as the hydrophilic surfactant and EDTA solution as the receptor phase. The result showed that it only needed 3 minutes when 99.3% was removed from the outer donor phase with CEAs whereas it needed nearly 60 minutes for ELM extraction. The examples above exhibit the quick separation ability of CEA dispersion. Colloidal emulsion aphrons can also be used as a microreactor to synthesize fine powder materials. With ascorbic acid as reductant in inner phase, Ag+ was transferred into inner phase of CEAs and reduced to form fine silver particles. “Nano porous microsphere” of silver was synthesized by adjusting concentrations of stripping regent, solute and carrier, and also characterized by field emission scanning electron microscope(SEM).
C2 – Keynote Pape ENCAPSULATION OF DRUG NANOPARTICLES IN SELF-ASSEMBLED 155 MACROMOLECULAR NANOSHELLS Michael Pishko, Department of Chemical Engineering, 204 Fenske Laboratory, The Pennsylvania State University, University Park, PA 16802-4400. [email protected]
A layer-by-layer (LbL) self-assembly technique was used to encapsulate core charged drug particles in a polymeric nanoshell. This approach provides a new strategy in the development of polymeric vehicles for controlled release and targeting to diseased tissues and cells specific to human illness, such as cancer. A nanoshell composed of two biopolymers, poly-L-lysine and heparin sulfate, were assembled stepwise onto core charged drug nanoparticles. The exterior surface of the nanoshell was functionalized with biocompatible and targeting functional moieties, poly(ethylene glycol) (PEG) and folic
134 Oral Abstracts acid, respectively. Drug nanoparticles of dexamethasone, taxol, and 5-fluorouracil were fabricated using a modified solvent evaporation technique, producing particles within a range of 314.0 to 154.7 nm. Surface morphology of the encapsulated drug nanoparticles were viewed by TEM and SEM. TEM images indicated that the nanoshell was approximately 5 nm, and composed of two polyelectrolyte layers. Characterization of surface chemistry and charge of the nanoshell required the use of XPS and zeta potential, respectively. XPS data collected for PEG modified drug nanoparticles confirmed that the peak at 286 eV represented the repeat unit in a PEG molecule. Zeta potential results re-confirmed PEG’s presence at the surface. Chemisorption of PEG molecules neutralizes the surface of the nanoshell and this was illustrated by the measured neutral zeta potential of the drug particles. Preliminary biocompatibility studies to study phagocytosis of PEG modified drug particles were performed using a flow cytometric assay and suggested that the neutral charge of the nanoshell results in decreased phagocytosis after 24 hours of incubation.
C2 – Invited Paper EFFECT OF DRUG CHARACTERISTICS ON THE DEGRADATION 156 MECHANISM AND DRUG RELEASE RATE FROM BIODEGRADABLE POLYMER MATRICES Nily Dan, Department of Chemical and Biological Engineering, Drexel University, Philadelphia PA 19104; [email protected]
We compare the degradation and release of six different drugs from 50:50 poly lactic-co-glycolic acid: Thiothixene, Haloperidol, Hydrochlorothiozide, Corticosterone, Ibuprofen, and Aspirin. Despite using the same polymer matrix and drug loading (20% by weight), we find that the rate of polymer degradation and the drug release profile differ significantly between the drugs. We conclude that the design of biodegradeable polymeric drug carriers with high drug loadings must account for the effect of the drug on the polymer degradation and drug release rate.
C2 – Contributed Paper INVESTIGATION INTO THE INFLUENCE OF FATTY ACID PURITY ON 157 THE DRUG RELEASE BEHAVIOR OF TASTE-MASKING FATTY ACID MICROSPHERES Sheng Qi, Duncan Q.M. Craig, School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich NR4 7TJ, UK; and David Deutsch, GlaxoSmithKline Research and Development, Ware, Herts SG12 0DP, UK; [email protected]
Stearic acid has been used in pharmaceutical manufacturing for decades. However, commercial grade stearic acid contains a range of further fatty acids, including up to 50%w/w palmitic acid. The presence of "foreign" saturated fatty acids may significantly alter the performance of a fatty acid based formulation. In this study, taste-masking microspheres were prepared by a spray chilling technique using pure stearic acid, pure
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palmitic acid, and mixed stearic and palmitic acids (50:50 %w/w and 30:70 %w/w). The mixed fatty acid formulations were found to have the fastest drug release in alkaline media. DSC and XRPD were utilized for characterizing the fatty acid microspheres before and after exposure to alkaline media. Additional peaks associated with the formation of fatty acid-soaps were observed in the DSC and XRPD results of the microspheres after exposure to the alkaline buffer. The results of both techniques indicated that a lower amount of fatty acid-soap was formed in the single fatty acid systems than in the mixed systems. In order to gain a fuller understanding of this phenomenon, the behavior of fatty acid molecules at the interface of air/alkaline media were investigated using surface pressure-area isotherm measurements. It was noted that under a constant surface pressure (30mN/m) the surface area per molecule of the mixed systems decreased as a function of time. This reduction in film stability of mixed monolayers is a result of disrupted molecular packing in the monolayer. Neutron reflection experiments revealed that palmitic acid is partially soluble in the buffer and contributes to the instability of the mixed fatty acid monolayers. The combined effects of the dissolution of palmitic acid and the formation of fatty acid soaps may be responsible for the observed behaviour of mixed fatty acid microspheres and the difference in behaviour between "pure" and mixed systems.
C2 – Contributed Paper FUNCTIONAL MICRO-ENCAPSULATION USING HYBRID 158 MATERIALS Jacco Eversdijk and Sabine Fischer, TNO Science and Industry, De Rondom 1, Eindhoven 5612AP, The Netherlands; [email protected]
Micro-encapsulation is a method to enclose active ingredients in a shell material on a micron-sized scale. The main purpose of this technology is to protect the core material from environmental effects that could diminish or even destroy the functionality of the active compound. In most existing encapsulation technologies polymers are used as the encapsulating shell material. The performance of the encapsulation is therefore limited by the inherent properties of the applied polymeric material. At TNO Science and Industry, expertise has been obtained in hybrid materials consisting of (modified) inorganic nano-particles in polymeric matrices. The introduction of these in-organics can change the physical properties of polymers significantly. We are using these hybrid materials in functional encapsulating in order to increase or optimize desirable properties and functionalities such as barrier and controlled release. Although intelligent choice in polymeric shell-material can result in more or less satisfactory properties, a true selective optimization is mostly difficult to achieve. The introduction of (modified) nano-scaled inorganic particles in micro-encapsulation can offer an additional tool to the technology. The resulting hybrid material combines the standard polymeric encapsulation materials with modifiable inorganic nano-particles (either synthetically made or natural occurring). Through parameter such as: type of modification, shape and size of the mineral structure, an extensive toolbox is available to tune desired properties of the shell material such as: (oxygen) barrier, slow release and possibly release on demand. Results from ongoing research will be presented. Furthermore, a newly developed method of measuring the oxygen permeability of encapsulating materials will be presented. Said method uses the
136 Oral Abstracts non invasive OxySense technology, based upon the fluorescence quenching, in the presence of oxygen, of a metal organic fluorescent dye immobilized in a gas permeable polymer, in combination with coated films of the encapsulation material.
C2 – Invited Paper ACRYLATE-BASED NANOGELS AS MICROCAPSULAR MEMBRANE 159 COMPONENTS FOR PEPTIDE DELIVERY Hideki Ichikawa, Faculty of Pharmaceutical Sciences and Cooperative Research Center of Life Sciences, Kobe Gakuin University, Arise 518, Ikawadani-cho, Nishi-ku, Kobe 651-2180, Japan, [email protected]
Significant advances in biotechnology and biochemistry have led to the discovery of a large number of biologically active peptides for therapeutic use. In general these peptides have relatively short half-lives, requiring appropriate controlled release systems for better therapy. Controlled release of peptides is, however, not as easy as that of conventional drugs because their large molecular size is much more dramatic in hindering the diffusion and release from polymeric devices. From this perspective, we have been developing two types of microcapsular devices containing new acrylate-based nanogels with a specific solute-permeability for delayed- or thermosensitive-release of peptide drugs. The microcapsule preparation was accomplished by an air suspension coating process. A nanogel particle of acrylic terpolymer, ethyl acrylate-methyl methacrylate-2-hydroxyethyl methacrylate, was newly synthesized by emulsion polymerization to construct delayed-release microcapsules. By spray-coating the insulin-loaded lactose particles with the nanogels of acrylic terpolymers, microcapsules showing a pH-independent delayed-release profile can be obtained. The lag time of insulin release was adjustable by the membrane thickness. Oral administration of the microcapsules with the lag time of 6 hours to beagle dogs resulted in significantly reduced blood glucose concentration, leading to colon-specific insulin delivery with pharmacological availability of 5%. Meanwhile, nanogel particles of poly(N-isopropylcarylamide) (p(NIPAAm)) with a reversible temperature-dependent swelling property were prepared by dispersion polymerization to fabricate a microcapsular membrane with thermosensitively changeable permeability. The microcapsules constructed by coating of drug-loaded CaCO3 particles with blend mixture of the p(NIPAAm) nanogels and ethylcellulose pseudo-latex exhibited an ‘on-off’ positively thermosensitive drug-release, i.e., the release rate was remarkably enhanced at temperatures above a lower gel collapse point (temperature for complete deswelling) of 32˚C possibly due to the formation of voids through the shrinkage of p(NIPAAm) nanogels in the membrane. A possible application of this type of microcapsules can be found in externally temperature-activated pulsatile delivery of peptide drugs.
C2 – Invited Paper THERMOSENSITIVE MAGNETIC NANO- AND MICROCARRIERS AS 160 CONTACTLESS CONTROLLABLE DRUG CARRIERS AND TOOLS D. Müller-Schulte, MagnaMedics GmbH, Martelenberger Weg 8, D-52066
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Aachen, Germany, P.Borm, Hogeschool Zuyd, NL-6400 Heerlen, The Netherlands; [email protected]
By encapsulating magnetic colloids into a thermosensitive polymer matrices such as N-isopropylacrylamide and hydroxypropylcellulose, for the first time contactless controllable and manipulable nano- as well as micro carrier systems (stimulus-response carriers) can be prepared. The unique feature of these polymers is that a phase transition accompanied by a drastic shrink process occurs at temperatures above 30°C. By encapsulating ferro(i)magnetic colloids into the polymer matrix and exposing these magnetic beads to an external high frequency magnetic field (induction coil),the subsequent shrink process can be contactlessly induced. This process is reversible, i.e. the shape of the polymer goes back to its original form once the initial temperature has been reached again. The fundamental principle can be exploited for the construction of controllable stimulus-response carriers or, alternatively, nano- and micro tools. This stimulus response principle is the basis for a novel type of drug carrier. For this purpose biologically active substances (e.g. anticancer drugs) are encapsulated together with the magnetic colloid into the polymer matrix. The subsequent inductive heating above the critical phase transition temperature which is simultaneously accompanied by the de-swelling of the polymer (shrink process) leads to an immediate release of the encapsulated drugs. Depending on the size of the polymer carrier, this drug release occurs over a period of 2 to 5 minutes, thus making these carriers an ideal controllable drug depot. The proof of principles of the novel therapy approach is demonstrated using cancer drug model and doxorubicine.
C2 – Invited Paper MICROPARTICLE FORMULATION DEVELOPMENT – 161 CONSIDERATIONS FROM BENCH-TOP TO CLINICAL MANUFACTURING Peter Markland, Brookwood Pharmaceuticals, 756 Tom Martin Drive, Birmingham, AL 35211; [email protected]
Successful results from initial feasibility (or proof-of-concept) studies can place considerable pressures on product development programs to advance forward as rapidly as possible through the formulation selection, optimization, and scale-up process and on towards clinical testing (or first-in-man trials). This is true even for non-traditional dosage forms such as long-acting implantable microparticle (or microsphere) formulations. In addition to the obvious need to identify the formulation or composition that gives the desired in vivo release profile, there are numerous other factors that must be considered to help facilitate an efficient and successful product development program. This presentation will briefly review issues affecting the scale-up of emulsion-based microparticle (microsphere) processes. Following this introduction, the discussion will then describe in more detail other factors that can impact the successful development of a microparticle formulation program as it moves from bench-top on forward towards manufacturing of first-in-man clinical trial supplies.
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C2 – Contributed Paper VIBRATING NOZZLE PROCESSING IN INDUSTRIAL 162 MICROENCAPSULATION Rick Rehrig, Harper International, West Drullard Avenue, Lancaster, NY 14086; [email protected]
The majority of commercially available raw materials is sold as grains, flakes, blocks or powders. This granulometries have many disadvantages. Particularly the handling of active agents becomes difficult. In addition to possible instabilities of the active agent in air, one might need expensive equipment to dose potent expensive agents or to handle oily ones. BRACE microspheres and microcapsules can solve these problems. Microspheres are solid spheres with a matrix-encapsulated active agent whereas Microcapsules consist of a solid shell and a liquid or solidified core. The major difference between these two types of microgranules is the different release profiles of microspheres and microcapsules. Microspheres usually have diffusion controlled release profiles with a permanent release rate that is controlled kinetically by the particle size, whereas microcapsules expel their content by a single high burst as the shell breaks or (with special shell materials) extremely slow releases. The patented BRACE-processes for the production of microspheres and microcapsules are basically vibrating nozzle processes. With these processes it is possible to produce particles with a monomodal grain size distribution with a single sharp maximum. dmax/dmin- values lower than 1.10, 1.05 or even 1.01 are customary for spherical granules produced with a microsphere unit designed by BRACE. With these units, it is possible to obtain microspheres or microcapsules with a diameter range of 50 to 6000 µm. For the production of particles smaller or larger than that, BRACE manufactures special nozzles. A wide range of shell materials can be used in this highly scalable process. Tests have shown that the vibrating nozzle processes (laminar flow break-up) scale very well with Newtonian liquids. For industrial applications, it is possible to use multi-nozzle systems as well as high throughput settings.
C2 – Contributed Paper ON THE SYNTHESIS AND CHARACTERIZATION OF 163 BIODEGRADABLE DEXTRAN NANOGELS WITH TUNABLE DEGRADATION PROPERTIES T.G. Van Thienen1, B. Lucas1, J. Demeester and S.C. De Smedt1 Laboratory of General Biochemistry and Physical Pharmacy, Department of Pharmaceutics, University Ghent, Harelbekestraat 72, B-9000 Ghent, Belgium; [email protected]
Hydrogels are widely investigated as carriers in drug delivery. To be suitable carriers for intracellular drug delivery the hydrogels should be small enough to be able to enter cells. This presentation reports on the synthesis and characterization of both lipid coated and ‘naked’ biodegradable hydroxyethylmethacrylated dextran (dex-HEMA) nanogels. Dynamic Light Scattering (DLS), atomic force microscopy and transmission electron microscopy showed that lipid coated nanogels could be obtained by polymerization of an aqueous dex-HEMA solution entrapped in SOPC:DOTAP liposomes (SOPC and DOTAP
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respectively being 1-stearoyl-sn-glycero-3-phosphocholine and 1,2-dioleolyl-3-trimethylamonium propane chloride). Naked dex-HEMA nanogels were prepared by removing the lipid coating by Triton X 100. DLS measurements on dex-HEMA nanogels stored in buffer at 37 °C revealed that the degradation time depended on the cross-link density of the nanogels: dex-HEMA nanogel prepared from dextran lowly substituted with HEMA degraded fast while it took days to weeks for nanogels prepared from highly substituted dextran. Furthermore, confocal laser scanning microscopy showed that SOPC:DOTAP coated dex-HEMA nanogels can be taken up by VERO-1 cells.
D4 – Invited Paper LUIDIC FORCE DISCRIMINATION ASSAYS IN COMPLEX MEDIA 164h awn P. Mulvaney, Alison A. Glaser, Matthew D. Kniller, Michael Malito, Jack fe, Monica W. Stanton, Cy R. Tamanaha, and Lloyd Whitman, Naval Research aboratory, Code 6177: Surface Nanoscience and Sensor Technology, Washington C. 20375, USA; [email protected]
The Naval Research Laboratory’s compact Bead Array Sensor System (cBASS™) is under advanced development for multiplexed detection of pathogens and toxins in environmental and clinical samples. The portable cBASS™ prototype includes a suite of patented and patent-pending technologies for microfluidic, microbead-based microarray assays performed on a Bead ARray Counter (BARC®) sensor chip. There are two core aspects of the system: a Fluidic Force Discrimination™ (FFD) assay, whereby biomolecular targets are captured onto a microarray, labeled with microbeads, and nonspecifically bound bead labels are removed by controlled fluidic forces; and a detection system, whereby the number of microbead labels on each microarray element is counted. In cBASS™, detection of captured labels is performed directly by a BARC® sensor chip in a disposable cartridge. For higher throughput applications, labels can be counted optically in a multi-channel, slide-based system that mounts on a standard upright microscope. Highly sensitive DNA assays (<10 fM) and immunoassays (<1 ng/mL) have been demonstrated in less than 30 minutes at room temperature, without amplification or concentration steps (i.e. PCR). Successful assays have also been run in serum, plasma, whole blood, and complex environmental matrices. The technology is being commercialized in collaboration with Seahawk Biosystems (www.seahawkbio.com). S.P.M., A.A.G., M.D.K. M.M, and M.W.S. are employees of Nova Research Inc., Alexandria, Va.
D4 – Invited Paper MAGNETIC BEADS USED IN SAMPLE PREPARATION FOR MASS 165 SPECTROMETRY Geir Fonnum, Thuy Diem Thi Tran, Kristine Evensen, David Gillooly, Elizabeth Breivold, Marie Bosnes. Dynal, Invitrogen Corporation. Ullernchausèen 52, N-309 Oslo, Norway; [email protected]
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The use of magnetic beads in sample preparation is now a standard technique that enables the use of highly automated systems and opens up the possibilities of high throughput. The demand for both beads with higher protein/peptide capacity and shorter separations times lead to the development of new one micron magnetic beads. This bead type was characterized by magnetic measurements and compared to Dynabeads® of different diameters showing the relation between maghemite content, size and separation time. Identification of disease-specific protein and/or peptide biomarkers in body fluids is used to detect and diagnose disease and allow the assessment of disease severity, progression, and the effectiveness of treatments. Mass spectrometry (MS) is the analytical method of choice for biomarker discovery whereby protein and/or peptide profiles need to be obtained from large sample numbers. The complex nature of biological fluids, dynamic range constraints associated with MS, and the sensitivity of MS to salts and detergents means that reproducible sample preparation procedures reducing sample complexity and removing contaminants are required. We have developed a set of ion exchange and reversed phase magnetic beads based on one micron magnetic beads for this purpose. Weak and strong ion exchange magnetic beads can be used to fractionate complex mixtures of proteins, consequently increasing the amount of information that can be obtained from these samples. Reversed phase chromatography (RPC) magnetic beads have been developed for the fractionation, desalting and concentration of protein and peptide mixtures. We have data that shows that samples containing salts and contaminants that prevented the attainment of MS spectra could be used to obtain excellent MS spectra after using simple automatable experimental protocols involving RPC magnetic beads. Studies of different types of hydrophobic groups on the surface of RPC beads will be discussed.
D4 – Contributed Paper MAGNETO-RESISTIVE IMMUNOSENSORS: SENSITIVITY 166 ENHANCEMENT BY ACTIVE GUIDING OF SUPERPARAMAGNETIC PARTICLES R. Wirix-Speetjens, G. Reekmans, R. De Palma, C. Liu, W. Laureyn and G. Borghs, Interuniversity MicroElectronics Center (IMEC), MCP-ART, Kapeldreef 75, B-3001 Heverlee, Belgium; [email protected]
Microelectronics-based biosensors are increasingly gaining importance since they offer a number of advantages such as small size, fast in use, simplicity in operation, and eventually low cost. However, increasing the device sensitivity remains a challenging task in biosensor development. Magnetic biosensors are a suitable candidate for highly sensitive bio-sensing. In this approach, the classical label (e.g. fluorescent) is replaced by a super-paramagnetic particle, which can be detected using e.g. magneto-resistive sensors. The pioneering work in the field of magneto-resistive biosensors was performed by the Naval Research Laboratory followed by others which are mainly aiming at the detection of DNA hybridization. Previously, we have demonstrated the controlled manipulation of magnetic particles using integrated current conductors. In this paper, we report on the development of magneto-resistive biosensors (i.e. spin-valves) with the aim to increase the sensitivity by combining detection and manipulation of magnetic particles. For
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magneto-resistive sensors, the position of bound magnetic particles relative to the sensor location shows a clear dependence on the signal. Based on this phenomenon, we are exploring the ability to enhance the sensitivity and specificity via active guiding of magnetic particles using on-chip generated magnetic forces. First, a sandwich assay is build up on the device surface, followed by labeling with magnetic particles. Then, the bound magnetic particles are released and transported to that position that theoretically gives rise to the maximal signal, ensuring the most sensitive detection. Next to the development of this alternative detection mechanism, the impact of the surface chemistry, assay, type of particles and release mechanism has been fully evaluated in order to enhance the sensitivity, specificity and dynamic range for the detection of proteins. Currently, the detection of S100ββ, a marker for ischemic stroke (and brain damage in general), is being targeted.
D4 – Contributed Paper ENCAPSULATED MICROCRYSTALLINE PARTICLES AS LABEL 167 SYSTEMS FOR DIAGNOSTIS Wing Cheung Mak 1, Kwan Yee Ceung 1, Reinhard Renneberg 2 and Dieter Trau 1, 1Division of Bioengineering, NanoBioanalytics Laboratory, National University of Singapore, 9 Engineering Drive 1, Singapore 117576; 2Department of Chemistry and Sino-German Nano-Analytical Laboratory, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong; [email protected]
A novel class of biolabel system based on encapsulated organic microcrystalline particles was introduced. The significant of organic microcrystal biolabels compared with other particulate label systems is the dissolution property of the organic microcrystals upon exposure to a desired solvent which leads to the release of a large number of signal-generating molecules (~ 107 to 109 molecules per microcrystal); termed as the “Supernova Effect”. We have demonstrated various applications of encapsulated organic microcrystalline particle biolabels in diagnostics by utilizing organic microcrystalline particles with different properties such as electrochemical-active organic microparticles for silver enhancement or electrochemical immunoassay; fluorogenic precursor organic microparticles for fluorescence immunoassay and silole microparticles for aggregation-induction emission assay. Our biolabel technology provides a new strategy for biolabelling over the traditional enzyme-based, fluorescence-based and particle-based biolabel system. The technology for preparing organic microcrystalline particles biolabels and different strategies for utilizing encapsulated microcrystalline particles for diagnostics will be presented.
D4 – Invited Paper CHIP-BASED MOLECULAR DIAGNOSTICS USING METAL 168 NANOPARTICLES Wolfgang Fritzsche, Robert Möller, Grit Festag, Thomas Schüler, Andrea Steinbrück, Andrea Csáki, Institute for Physical High Technology (IPHT),
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Photonic Chip Systems Dep. A.-Einstein-Str. 9, 07745 Jena, [email protected]
Metal nanoparticles represent an interesting alternative as marker in molecular diagnostics. High stability and straightforward signal detection are possible when such particle labels are combined with enhancement techniques based on specific metal deposition. Light scattering at the (enhanced) particles is a simple and robust approach for microarray detection, and examples for easy readout as well as detection at the single particle level by standard means will be presented. The formation of metal layer around the original particle leads to a metal film at the substrate surface. This film could be detected by measurements of the electrical resistance along the surface. A system based on microstructured electrodes and a dedicated electrical chip reader will be presented. Problems related to the precise positioning of the capture DNA on such chips as well as to corresponding alternative, cost-efficient substrates were studied. Another field for investigations is the utilization of alternative approaches for metal enhancement. The performance of this enhancement is a key factor for the sensitivity of the method because the non-specific deposition of metal seeds determines the background level. An enzymatic system was studied that allows for a high performance electrical DNA chip detection and was successfully demonstrated for the identification of microorganisms from PCR samples.
D4 – Contributed Paper HYBRID PARTICLES AS SUBSTRATES FOR BIOTARGETS AND 169 METALIC IONS L. B. R. Castro, A.M. Carmona-Ribeiro and D.F.S. Petri. Instituto de Química, Universidade de São Paulo, Brazil; [email protected]
The synthesis of hybrid particles was carried out by emulsion polymerization of styrene in complexes formed by carboxymethyl cellulose (CMC), a polyanion, and a cationic surfactant, cetyltrimethylammonium bromide (CTAB). CMC chains with variable molecular weight and degree of substitution were tested. The polymerization condition chosen was that corresponding to CMC chains fully saturated with CTAB and to the onset of pure surfactant micelles formation, namely at the critical aggregation concentration. The hybrid particles were characterized by zeta potential and light scattering measurements. The period of colloidal stability stable in the ionic strength of 2.0 mol L-1 NaCl was observed visually. Upon increasing the CMC chain length, the particle characteristics remained practically unchanged, but the colloid stability was increased. The increase in the CMC degree of substitution led to particles with more negative zeta potential values. The adsorption of biotargets as concanavalinA (ConA) and hexokinase (HK), as well as of copper ions (Cu2+) on the surface of hybrid particles could be well described by the adsorption models. These results evidenced the potential of such hybrid particles for biotechnological applications.
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D4 – Invited Paper ASSEMBLY AND IN-VIVO TRACKING OF NANOPARTICULATE 170 CARRIERS FOR NEO-VASCULAR TARGETING Heinrich Haas, John Nieland, MediGene AG., Lochhamer Str. 11, D-82152 Martinsried, Germany, Leide Cavalcanti, Oleg Konovalov, Stephanie Corde, Thierry Brochart, Geraldine Le Duc, ESRF, Grenoble; [email protected]
In a new approach for targeted drug delivery, cationic nanoparticulate carriers are used for delivery of pharmaceutical compounds to activated (angiogenic) endothelial cells. Because angiogenesis is a key issue in a variety of pathological situations, like tumor growth or in inflammatory diseases, cationic vascular targeting provides new, alternative, options for treatment in those cases. In this context Medigene AG (www.medigene.com) is developing cationic liposome products (EndoTAG) for tumor therapy and diagnostics. For rational development of such novel drug delivery systems, parameters like molecular composition, drug load and organization on the molecular and mesoscopic scale must be correlated with the targeting characteristics in vivo. Here, results from physico-chemical characterization and from in vivo tracking of lipid-based carriers are given. X-ray scattering and spectroscopic techniques have been used to study drug loading to the lipid carrier matrix. For tracking of the distribution of the colloidal carriers in vivo, Monochromatic Quantitative Computed Tomography (MQCT) at the European Synchrotron Radiation Facility (www.esrf.fr) has been used. The method provides in vivo absolute concentration maps of contrast agents with good temporal (few seconds) and spatial resolution (350 μm), and it has been highlighted as a complementary tool to other imaging techniques in the field of preclinical and contrast medium research. After injection of several types of Gd-based liposomes in the rats bearing a 9L glioma, the Gd concentrations in the brain were mapped as a function of time. Different concentration profiles in the blood were found for Gd-loaded liposomes and free Gd. Specific patterns for tumor accumulation of cationic liposomes were found. The correlation between the molecular setup of the liposomes and the targeting characteristics was revealed. The data demonstrate, that in vivo tracking, such as by MQCT, can be a useful tool for screening and optimizing of colloidal drug delivery systems.
D4 – Contributed Paper TUNING OF LUMINESCENCE INTENSITY OF MANGANESE DOPED 171 CADMIUM SULFIDE QUANTUM DOTS BY CONTROLLING SURFACE PASSIVATION Subir K. Sabui, Swadeshmukul Santra, Nanoscience Technology Center, Department of Chemistry and Biomolecular Science Center, University of Central Florida, 12424 Research Parkway, Suite 400, Research Pavilion, Orlando, FL 32826; [email protected]
Brightness of manganese doped cadmium sulfide (CdS:Mn) quantum dot nanocryatals (~3.0 nm) depends on how well the nanocrystal surface is passivated. A coating of 1-2 atomic layer thick ZnS (a wide bandgap material) has shown to be effective to passivate
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CdS:Mn surface completely and minimize surface defects. As a result, highly bright yellow emission of CdS:Mn/ZnS is obtained and the emission remains insensitive to the environment. In this study, we have developed a unique synthesis protocol that allows synthesis of CdS:Mn nanocrystals at varoius extent of surface passivation with ZnS layer. As a result of partial surface passivation, this material behaves as if the emission is internally quenched. The present study showed that partially passivated Cd:Mn/ZnS nanocrystals are highly sensitive to the external environment. The solvatochromism and spectroscopic characteristics of partially passivated CdS:Mn/ZnS nanocrystals are thoroughly described. We believe that this study will initiate research in using quantum dots as optical based sensors.
D4 – Contributed Paper SINGLE-PARTICLE BIOAEROSOL MASS SPECTROMETRY FOR 172 MEDICAL AND BIOCHEMICAL DIAGNOSTIC AND ANALYSIS Matthias Frank, Michael Bogan, Keith R. Coffee, George Farquar, David P. Fergenson, Eric E. Gard, , Sue I. Martin, , Vincent J. Riot, Urs Rohner, Paul T. Steele, Herb J. Tobias, Lawrence Livermore National Laboratory, Livermore CA 94551, USA; [email protected]; Erica L. McJimpsey, Carlito Lebrilla, University of California Davis, Davis CA 95616, USA
We are developing a single-particle bioaerosol mass spectrometry (BAMS) system that can rapidly analyze individual biological particles in the size range from 500nm to 10 μm (e.g. bacteria, bacterial spores, clumps of viruses, liposomes) that are sampled from a nebulizer or directly from air into a time-of-flight mass spectrometer. An individial incoming particle is tracked and sized by laser light scattering and probed by UV-fluorescence before a short energetic pulse from a desorption/ionization laser vaporizes the particles and creates characteristic molecular ions characteristic for the biochemical composition of the particle. Mass spectra from both positive and negative ions created by matrix-free or matrix-assisted laser desorption and ionization are recorded simultaneously and can be analyzed and classified in real-time. At present, tens of particles can be analyzed per second this way. Using the characteristic mass spectra from individual biological particles our system is capable of discriminating, particle by particle and in real time, between bacterial spores, vegetative cells and other biological and non-biological background materials using the mass fingerprints obtained from those particles. In addition, selected species of bacteria can be discriminated from each other with this method. Here we will present the first results from exploring applying this technique to the rapid analysis and screening of human effluents, such as exhaled aerosol or re-aerosolized breath condensate, for the detection and diagnosis of respiratory disease. Other applications of this BAMS technique may include the high throughput characterization and quality control of aerosol particles that are used for drug delivery. This work was performed under the auspices of U.S. DOE by University of California Lawrence Livermore National Laboratory under contract No. W-7405-ENG-48.
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I2 – Keynote Paper DENDRIMERS, DENDRONS, DENDRISOMES AND DENDRIPLEXES 173 AS CARRIERS IN DRUG AND GENE DELIVERY A. T. Florence, K. Al-Jamal, C. Ramaswamy, P. Ruenraroengsak, S. Ribeiro, B. Singh, The School of Pharmacy, University of London, 29/39 Brunswick Square, London WC1N 1AX, United Kingdom; [email protected]
Dendrimers are the irreducibly small units that have been studied increasing widely as carriers for drugs, genes and other macromolecules. Irreducibly small because the molecular radius of many therapeutic agents is greater or similar to that of generations that can be produced in monodisperse form. However, the small diameter of dendrimers allows them certain advantages in diffusion and transport in tissue to deliver their load. There are at least two reasons why dendrimers can be useful in spite of the low carrying capacity for many molecules. The first is that multiple dendrimers associate with macromolecules such as DNA and heparin, a process often electrostatically driven, so that compact complexes are formed which have the ability to be taken up into cells. Secondly dendrimers with appropriate disposition of hydrophobic and hydrophilic geometry can associate to form larger structures, dendrisomes (vesicle-like forms) or into nanoparticles, two of the systems we have studied. Dendrimers which have been complexed with DNA to form dendriplexes, can be incorporated into PLGA nanoparticles to provide a slow release system which would not be possible with the dendrimer construct itself. Dendrimers are thus versatile building blocks for the exploration of means of enhancing drug, gene and vaccine delivery. Although products are beginning to reach the clinic, there are still obstacles to overcome. These include control of drug release often rapid due to the large dendrimer surface area, the ability of these colloidal particles to flocculate and aggregate; nonetheless the ability of dendrimers to diffuse into cells and experience lesser obstruction in the crowded cell interiors than larger nanoparticles must be an advantage. Data on dendron (partial dendrimer) and dendron behaviour in vitro and in vivo will be discussed in this survey of where we are and how dendrimers are shaping up to be a superior alternative to nanoparticulate delivery vectors.
I2 – Invited Paper DENDRIMERS: A PLATFORM FOR CUSTOM-DESIGNED DRUG 174 DELIVERY Sonke Svenson, A.S. Chauhan, L.A. Reyna, D.A., Tomalia, Dendritic NanoTechnologies, Inc., Mount Pleasant, MI, 48858, USA; [email protected]
Many potential drugs and launched products suffer from low bioavailability due to poor water solubility or membrane permeability. Drug carriers can improve solubility and permeability, and can provide the option of targeted delivery, i.e., passive targeting through size exclusion (EPR Effect) or active targeting through ligands that interact with receptors over-expressed on tumor cell surfaces (e.g. folic acid interacting with the high affinity folate receptor). Successful drug carriers should also reduce the cytotoxicity of an
146 Oral Abstracts encapsulated drug. Poly(amidoamine) (PAMAM) STARBURST® dendrimers are a class of core-shell nanostructures with precise architecture and low polydispersity, which are synthesized in a layer-by-layer fashion around a core unit, resulting in a high level of control over size, branching points and surface functionality. This control over dendrimer architecture, and the resulting ability to tailor dendrimers to the needs of a drug, makes them ideal carriers for drug delivery applications, as exemplarily shown using the anti-cancer drug cisplatin and the non-steroidal anti-inflammatory drug (NSAID) indomethacin. Both drugs have been encapsulated into STARBURST® dendrimers, and their encapsulation efficiency and release profiles in DI water and PBS at pH 7.2 have been studied. Cisplatin encapsulation was successful using dendrimers with carboxylate surface. Dendrimer size (generations 3.5 or 4.5) and size of the core molecule (C2, C4, or C12) had little effect on the encapsulation efficiency. The release profiles in DI water and PBS revealed a two-step process, burst release over 30 minutes, followed by sustained release over several hours. The release in PBS was shifted to slightly higher percentages due to its higher ionic strength. Indomethatcin encapsulation was successful in dendrimers - - with amino (NH2), hydroxy (OH) and carboxylate (COO ) surfaces, with NH2>OH>COO . The encapsulation efficiency increased with dendrimer size; however, the core size (C2 vs. C12) had little effect. The release profiles were dependent on surface groups - (NH2
I2 – Invited Paper WHAT CONSTITUTES A PARTICLE? ASSEMBLY OF 30-500 NM 175 DENDRITIC (FRACTAL) HYPERPOLYMERS VIA SEQUENCE RECOGNITION ENABLED POLYMERS Thor W. Nilsen, Genisphere, Hatfield, PA 19440, USA; [email protected]
Dendritic “particles” have the characteristic of presenting a “surface” with a maximum number of reactive moieties with a minimum number of structural elements. Sequence recognition, such as that exhibited by nucleic acids and nucleic acid like polymers, is an optimal starting point for the assembly of molecular networks. The ability of a linear polymer to “recognize” a cognate polymer sequence is the underlying principle. A polymer with linear recognition elements, such as DNA, can be used to assemble 3-D structures with defined characteristics. We present the assembly and characterization of molecules of 300Å to 4200 Å (MW 70,000 Æ 12,000,000). We further present the application of these molecular constructs for amplification of signal to the single molecule detection limit and assembly of molecular capture networks in chromatography applications.
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I2 – Invited Paper FUNCTIONALIZED MESOPOROUS SILICA NANOPARTICLES FOR 176 GENE TRANSFER, INTRACELLULAR CONTROLLED RELEASE, AND BIOSENSOR APPLICATIONS Victor S.-Y. Lin, Department of Chemistry, US DOE Ames Laboratory, Iowa State University, Ames, IA 50011-3111, USA; [email protected]
We have recently synthesized a series of multi-functionalized, MCM-41 type mesoporous silica nanosphere (MSN) materials. The mesopore surface of these materials was derivatized with fluorescence sensor groups that could recognize and react with amino acid-based neurotransmitters. The exterior surface of the MSN materials was covalently coated with polylactides or polypeptides. By utilizing the polylactide (PLA) or polypeptide layer as gatekeepers to regulate the rates of diffusion of several structurally similar neurotransmitters, such as dopamine, tyrosine, and glutamic acid, into the sensor mesopores, a highly selective fluorescence biosensor that could distinguish dopamine from glutamic acid under physiological condition was synthesized. These MSN materials were also designed as a stimuli-responsive controlled release delivery system. Several pharmaceutical drugs and neurotransmitters were encapsulated inside the mesopores of MSN by capping the openings of the mesopores with various chemically removable caps, such as surface-derivatized cadmium sulfide (CdS) nanocrystals, superparamagnetic iron oxide nanoparticles (Fe3O4), and cell membrane permeable dendrimers, e.g. polyamidoamine (PAMAM) dendrimer, to block the molecules of interest from leaching out. We studied the stimuli-responsive release profiles of several drug/neurotransmitter-loaded MSN systems by using various non-cytotoxic chemicals as release triggers. Furthermore, the gene transfection efficacy, uptake mechanism, and biocompatibility of the capped-MSN system with various cell types, such as neural glia (astrocytes), human cervical cancer (HeLa), and Chinese hamster ovarian (CHO) cells were investigated. The mesoporous structure of the MSN material allows membrane impermeable molecules, such as pharmaceutical drugs and fluorescent dyes, to be encapsulated inside the MSN channels. We envision that these MSNs can serve as a universal transmembrane carrier for intracellular drug/gene delivery and imaging applications.
I2 – Invited Paper A STANDARDIZED PROCEDURE FOR DERIVATIZATION OF 177 NANOPARTICLES WITH FUNCTIONALLY ACTIVE PROTEINS Marina V. Backer*, Achintya K. Bandyopadhyaya† , B.T.S. Thirumamagal† , Werner Tjarks† , Rolf F. Barth**, and Joseph M. Backer*, *SibTech, Inc., Newington, CT 06111, † College of Pharmacy, The Ohio State University, Columbus, OH, 43210, and **Department of Pathology, The Ohio State University, Columbus, OH, 43210, USA. [email protected]
We have developed a robust platform technology for facile and uniform derivatization of nanoparticles with functionally active proteins. This technology is based on a novel 15-aa humanized, cysteine-containing tag, named C-tag, that contains a unique cysteine residue for site-specific conjugation via thiol-directed chemistries. We found that in a variety of
148 Oral Abstracts proteins expressed with C-tag, a cysteine residue in C-tag can be modified with different payloads without affecting protein functional activity. To validate this platform, C-tag was fused to either N- or C-termini of several proteins, expressed in bacteria, refolded under red-ox conditions, purified, and site-specifically conjugated to various payloads, such as dendrimers, quantum dots, liposomes. In all cases, functional activities of loaded proteins tested in vitro and in vivo were similar to that of free proteins. In a specific example, C-tagged vascular endothelial growth factor (VEGF) was conjugated to nanoparticles that combine therapeutic and diagnostic components for targeted treatment and imaging of tumor vasculature. These “theranostic” nanoparticles were constructed on the basis of 5th generation of PAMAM dendrimers that were derivatized with 105-110 decaborate (Na(CH3)3NB10H8NCO) for boron neutron capture therapy and with Cy5-dye for near-infrared fluorescent imaging in vivo. Derivatized nanoparticles were conjugated site-specifically to C-tagged VEGF via a bi-functional crosslinker sulfo-LC-SPDP. VEGF on the surface of the theranostic nanoparticle retained functional activity in tissue culture assays and provided for imageable accumulation of nanoparticles in endothelial cells of tumor vasculature in mouse models. We expect that the use of C-tagged proteins would provide for rapid, facile and reliable method for coupling functional proteins to nanoparticles.
I2 – Invited Paper BRANCHED NANOPARTICLES FOR DRUG DELIVERY ACROSS THE 178 BLOOD BRAIN BARRIER Eun S. Gil,1 Young S. Kim,2 Annie Mitsak,3 Jianshu Li,3 Huining Xiao3 and Tao L. Lowe1,2,3, Departments of Surgery,1 Bioengineering,2 Biomaterials and Bionanotechnology Summer Institute,3 and Materials Science and Engineering,4 Pennsylvania State University, 500 University Drive, Hershey, Pennsylvania 17033, U.S.A.; 3Department of Chemical Engineering, University of New Brunswick, Fredericton, NB, Canada E3B 5A3; [email protected]
The blood-brain barrier (BBB) is a dynamic and complex structure composed principally of specialized capillary endothelial cells held together by highly restrictive tight junctions. It allows the passage of nutrients into the brain, while preventing the passage of many substances, posing a problem for cerebral drug delivery from the blood stream. In this work, we have developed branched nanoparticles including thermo-responsive and biodegradable dendrimers and beta-cyclodextrin for controlled drug delivery across the BBB. The effects of the nanoparticles on the integrity of bovine blood brain endothelial cell (BREC) and bovine retinal brain endothelial cell monolayers, in vitro BBB models, were studied. The permeability of the nanoparticles with/without model protein drugs, bovine serum albumin (BSA) and nerve growth factor (NGF), through the BBB models, is also investigated. Completion of this project will have significant impact on the treatments of neurological disorders in the brain.
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N1 – Keynote Paper NANOSUSPENSIONS FOR DELIVERY OF POORLY SOLUBLE DRUGS: 179 STATE OF THE ART AND LATEST DEVELOPMENTS Rainer H. Müller 1, 2 and Jan Möschwitzer1; 1Free University of Berlin, Department of Pharmacy, Kelchstrasse 31, 12169 Berlin, Germany; 2PharmaSol GmbH, Blohmstrasse 66a, 12307 Berlin, Germany; [email protected]
The number of drugs being poorly soluble is steadingly increasing. About 10% of the drugs in the development pipelines are poorly soluble, even up to 60% of compounds coming directly from chemical synthesis. Very often, the poor solubility is correlated with a poor oral bioavailability. Intravenous injection as alternative is not possible because of the too large injection volumes required due to the poor solubility. Therefore there is a definite need for smart technological formulation approaches to overcome the problem of poor oral bioavailability and/or provide formulations suitable for intravenous injections. Ideally such a formulation approach should be universally applicable to any drug. Such an universal formulation approach applicable to almost any drug is “nanonization”. The drug powder is transferred to drug nanocrystals, typically by wet milling processes leading to drug nanocrystals suspended in a liquid dispersion medium, the so called nanosuspension. The “makeability” of drug nanocrystals for pharmaceutical formulations has been proven by the first products introduced to the pharmaceutical marked. Drug nanocrystals can be produced by “bottom up” or “top down” technologies. Industrially most relevant are the top down technologies, that means production of drug nanosuspensions by pearl milling or by high pressure homogenization. The high pressure homogenization can be performed either having the drug particles dispersed in water or alternatively using non-aqueous or water-reduced media. Production of drug nanocrystals in liquid or solid polyethylene glycols (PEGs) has the advantage that these suspensions can directly be filled into capsules for oral delivery. It is also possible to combine bottom up and top down technology, that means preparing a fine drug suspension by precipitation (adding solvent to non-solvent) and subsequently applying a high energy step such as high pressure homogenisation (NANOEDGE technology). A clear disadvantage of using precipitation is the need for solvent removal (at least in most cases) and the problem of solvent residues, in addition making the process costly. The special feature of the drug nanocrystals is an increased surface area but also an increased saturation solubility of the poorly soluble drugs. This leads to a distinctly enhanced dissolution velocity, thus reaching a sufficiently high oral bio availability in case the dissolution velocity is the absorption limiting step. In addition, nanosuspensions can be injected intravenously as highly concentrated suspensions. Saturation solubility and dissolution velocity increase with decreasing particle size. Therefore to obtain extremely fast dissolving particles with a very high saturation solubility, it would be desirable to have particles with a size of about 100nm or even distinctly less. Therefore one present trend is to produce drug nanocrystals of 100nm or below. In addition, there is a clear demand for shortening the production time. Pearl milling requires hours to days, high pressure homogenisation typically 10 to 20 homogenization cycles. Drug nanocrystals dissolve fast, however for certain oral applications it is desirable to have a delayed release or a prolonged / sustained release. Therefore technologies are required to
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modify drug dissolution from drug nanocrystals during transit of the gastrointestinal tract. In high pressure homogenization, the number of homogenization cycles could be reduced by 50% or more by applying a special pre-treatment step in drug synthesis before the obtained powder is processed as suspension by high pressure homogenisation (so called “H42 technology”). In some cases drug nanosuspensions can be obtained with only a few homogenisation cycles. Nanocrystals below 100nm could be produced applying a precipitation method under special conditions (H69 technology) or alternatively again by high pressure homogenization using an especially pre-treated drug powder (H96 technology). To protect the produced drug nanocrystals against the acidic environment of the stomach, a coating process was developed for the coating of the single nanocrystals without the need of organic solvents [6]. For controlled release in the gut, various Eudragit polymers were applied for the production of different types of pellet systems. This allows to adapt the release and dissolution profile of the nanocrystals to obtain optimal blood levels for the treatment.
N1 – Invited Paper NANOSUSPENSIONS OF POORLY SOLUBLE ACTIVE INGREDIENTS 180 Ingrid Fischbach, Jens Rieger, BASG-AG, GVC/F – J542; Ludwigshafen 67056, Germany; [email protected]
Many organic active ingredients exhibit poor solubilities in both aqueous media and organic solvents. This represents a severe problem to the formulator, since low solubilities make it very difficult to produce formulations with high bioavailabilities. Nanoparticulate formulations are of particular interest for such actives, since particle sizes in the range of 50 – 500 nm go along with a dramatic increase in solubility, enhancement of the biological resorption and the modification of optical, electrooptical and other properties. Such ultrafine dispersions can be obtained from a solution of the active via controlled precipitation. Interfacial activity and colloidal stability is achieved by additional excipients (surfactants, polymers). The mode of action of polymeric excipients for the control of crystallization and for the stabilization of ultrafine dispersions will be discussed in detail considering some selected examples.
N1 – Contributed Paper SIZE CONTROL OF NANOPARTICLES IN CONTINUOUS 181 CRYSTALLIZATIONS: SOLUBILITY EFFECT Ingo H. Leubner, Crystallization Consulting, 35 Hillcrest Drive, Penfield, NY 14526-2411, USA, [email protected]
Continuous crystallization is an attractive method for large scale preparation of crystalline materials. To control the crystal size of the product it is desirable to have a theory based on fundamental science that allows simple modeling and accurate predictions. The balanced nucleation and growth (BNG) theory predicts the size dependence as a function of experimentally controlled parameters. In the present work, the predicted size dependence on solubility is experimentally supported for continuous crystallization in the CSTR
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(MSMPR) crystallizers. As a model system, silver chloride was chosen where the solubility was varied from 0.81 to 8.3E-04 mole/l (60 C and 3.0 min residence time). The average crystal size varied from 380 to 580 nm. The model and experimental results allow to determine all parameters to predict crystal sizes, and to obtain fundamental understanding of the critical processes. Combining model and experiments, the maximum growth rate of the crystals at steady state, Gm, was determined to 0.96 nm/s, and the ratio of critical to average crystal size to 0.79. The supersaturation at steady state was determined to 1.0029 (+/-0.0004). The prediction, that the average crystal size is independent of reactant addition rate and of suspension density, was experimentally confirmed. Since the model is based on fundamental science, it is generally applicable to all crystalline systems.
N1 – Invited Paper APPLICATIONS OF NANOTECHNOLOGY IN THE PHARMA 182 INDUSTRY – AN OVERVIEW Hemant N. Joshi, Spectrum Pharmaceuticals, Inc., 157, Technology Drive, Irvine, CA 92618, USA; [email protected]
Nanotechnology is the latest buzz-word in our lives. As the word indicates, nano is less than micro and nanoparticles can not be seen by naked eyes. Nanotechnolgy has been part of the pharma industry for the last couple of decades. However, the real applications are being studied in now in more recent times. Nanotechnology is playing a prominent role in other industries too. Terms such as nanocrystals, nanotubes, nanoparticles, etc. are becoming common terms in the pharma industry. However, the word nano is still rather loosely used in the pharma industry. It is important to take into consideration the “effective” particle size rather than the individual particle size in ascertaining the particles in the system as nanoparticles. This paper will define the term and set the stage for presentations to follow. Nanoparticles can increase the surface area for solubilization, but true nanoparticles (less than 200 nm) can also form very stable suspensions which can be delivered intravenously. The real achievement for nanotechnology is the targetted delivery of drugs to cell components where the drug treatment is needed. Nanotechnology provides potential promises, but it is certainly not without problems. Nanoparticles can enter our body via the GI tract, skin and lungs. Key applications of the technology and potential problems are discussed. Nanoparticles may pose risks to the professionals manufacturing the products, and those who are handling the formulations.
N1 –Contributed Paper ELECTROSTATIC INTERACTIONS BETWEEN LYSOZYME AND 183 POLY(ACRYLIC ACID) MICROGEL PARTICLES Christian Johansson, Martin Malmsten, Department of Pharmacy, Uppsala University, Sweden; [email protected]
Microgel particles are polymer networks that swell/deswell as a response to changes in the environment. As a model for studying electrostatic interactions between protein drugs and
152 Oral Abstracts polyelectrolyte microgels, we study the interactions between lysozyme (positively charged) and negatively charged microgel particles of loosely cross-linked poly(acrylic acid). When lysozyme is incorporated into the microgel, the composite system deswells osmotically. We study how ionic strength, pH and lysozyme concentration affect the interactions between protein and microgel. Binding isotherm studies show the effect on protein loading. Confocal microscopy and micropipette-assisted light microscopy provide information on the time-dependency in protein distribution and microgel deswelling, respectively. At low ionic strength, protein incorporation is found to occur at lower protein concentration. Lysozyme incorporation increases strongly with decreasing pH, a pH-dependence which is less pronounced at lower ionic strength. Binding isotherms show that lysozyme incorporation mostly is governed by protein charge, rather than by the combined charge contrast. At some conditions a “lysozyme shell” forms in the microgel, with much higher lysozyme concentration in the outer part than in the centre. Ionic strength, pH and lysozyme concentration are found to determine whether shell formation occur or not. High lysozyme concentration, low ionic strength and low pH all promote shell formation. Shell formation strongly increases lysozyme uptake. It also affects the binding kinetics of lysozyme to the microgels, resulting in a lag time, often in the order of minutes, before any lysozyme can diffuse from the shell towards the microgel centre.
N1 – Invited Paper USE OF PARTICLES AS EMULSION AND FOAM STABILIZERS 184 María I. Briceño, María T. Celis, Jean-Louis Salager, Laboratory FIRP, School of Chemical Engineering, University of Los Andes, 5101 Mérida, Venezuela, [email protected]
Although the stabilizing effect of colloidal-size nanoparticles on emulsions and foams has been known since the early twentieth century, it has been the last 10 years that industry and academia have acknowledged the vast potential of using particles as surfactants or co-surfactants in the preparation and stabilization of dispersed systems. In fact, particles are the main stabilizers of naturally occurring emulsions and foams, such as the ones encountered in water-in-crude oil emulsions and froths in fermentation processes. In the first part of the lecture, the mechanisms by which particles may stabilize a surface or an interface are reviewed, as well as the effect of particle wettability, size and concentration on stability. Some applications in the pharmaceutical and food industry are presented in the second part, e.g., ice cream and multiple emulsions for controlled drug delivery.
Plenary 3 SUPERPARAMAGNETIC PARTICLES FOR DIAGNOSTIC AND 185 THERAPEUTICS, Heinrich Hofmann, Ecole Polytechnique Fédéral Lausanne,EPFL, Institute of Materials, Powder Technology Laboratory, Station 12, CH-1015 Lausanne, Switzerland; [email protected]
Nanoparticles for biomedical applications such as target drug delivery, non-viral vectors for gene delivery, contrast agent for in vivo imaging (MRI) or in biosensors have to fulfill
153 Oral Abstracts very different requirements simultaneous. To achieve the physical requirements, such as fast transport in a magnetic field gradient, local influence of the magnetic field strengths or photoluminescence the particles has to be so small that properties like superparamagnetism and quantum confinement determine the properties. From the biological point of view, the size plays also an important role regarding up take mechanism by cells or to control the colloidal behaviour in contact with blood. Recent results show that even more important are the nature of the b In this presentation, methods for the synthesis of biocompatible particles with properties which fulfill the physical as well as the biological needs will be presented. Especially superparamagnetic particles for target drug delivery or for gene therapy are difficult to synthesis, because several coatings of inorganic and/or organic nature are necessary. Additionally derivatisations with biological molecules, which are often larger then the core particle are needed. Th colloidal stability of the suspension during fabrication and application is one of the major challenges during synthesis of the particulate systems. In a second part a review medical application of such particles will be given. Especially the relationship between particle properties, including surface properties, and cells will be discussed. Additionally to the particle properties, external magnetic field could have a interesting effect of transfection rate, drug targeting efficacy and the imaging process.
A5 – Keynote Paper EXPLOITING EMERGING TECHNIQUES IN IMPRINT LITHOGRAPHY 186 TO MAKE HIGHLY UNIFORM, SHAPE-SPECIFIC DRUGS, IMAGING AGENTS, AND NANO-CARRIERS Joseph M. DeSimone and Larken E. Euliss, University of North Carolina at Chapel Hill, Department of Chemistry, CB #3290, Venable and Kenan Laboratories, Chapel Hill, NC 27599; [email protected]
The delivery of therapeutic, detection and imaging agents for the diagnosis and treatment of cancer patients has improved dramatically over the years with the development of nano-carriers such as liposomes, micelles, dendrimers, biomolecules, polymer particles, and colloidal precipitates. While many of these carriers have been used with great success in vitro and in vivo, each suffers from serious drawbacks with regard to stability, flexibility, polydispersity, or functionality. To date, there has been no general particle fabrication method available that afforded rigorous control over particle size, shape, composition, cargo and chemical structure. Recently, we have pioneered a contemporary breakthrough in the materials useful for imprint lithography—an emerging technology adapted from the microelectronics industry—that enables an extremely versatile and flexible method for the direct fabrication and harvesting of monodisperse, shape-specific nano-biomaterials. By utilizing the method we has designed referred to as Particle Replication In Non-wetting Templates, or PRINT, we can fabricate monodisperse particles with simultaneous control over structure (i.e. shape, size, composition) and function (i.e. cargo, surface structure). By utilizing this method, we can impart control over the cross-link density of the polymeric matrix. Unlike other particle fabrication techniques, PRINT is delicate and general enough to be compatible with a variety of important next-generation cancer therapeutic, detection and imaging agents, including various cargos (e.g. DNA, proteins, chemotherapy drugs,
154 Oral Abstracts biosensor dyes, radio-markers, contrast agents), targeting ligands (e.g. antibodies, cell targeting peptides) and functional matrix materials (e.g. bioabsorbable polymers or stimuli responsive matrices). PRINT makes this possible by utilizing low-surface energy, chemically resistant fluoropolymers as molding materials and patterned substrates to produce functional, harvestable, monodisperse polymeric particles.
A5 – Invited Paper MOLECULAR SPRING ASSEMBLIES OF NANOPARTICLES AND 187 NANOWIRES Nicholas A. Kotov, Jim Lee, Departments of Chemical Engineering, University of Michigan, Ann Arbor, MI; and A. Govorov, Department of Physics, Ohio University, Athens, OH; [email protected]
The development of micro- and nano-fluidics devices demands in-situ measurements of many parameters in the stream of liquids. Local gradients of solvent concentration, temperature, pH and other parameters have tremendous importance for the development of lab-on-a-chip applications and similar analytical tools. The same problem also appears in biomedical fields that require evaluation of local concentrations and other parameters for intricate geometries of 3D cell scaffolds, which affect the development of cells residing on the scaffolds. Both tasks are difficult to accomplish using traditional sensor deployment schemes, which often require substrate and wires. In this presentation, we address these problems by designing a nanoscale sensing device from different types of nanoparticles and nanowires connected by molecular springs made from flexible PEG oligomers. A library of assemblies of nanoparticles and nanowires was created using PEG tethers. The polymeric linkers afford continuous and dynamic change of conformations in such structures leading to the variations of the distance between the nanoscale colloids reversibly changes depending on conditions or analyte concentration and can be evaluated by fluorescence measurements. Plasmon-exciton interactions result in tremendous enhancement of luminescence and for some systems in characteristic wavelength shift depending on the analyte concentration. Understanding plasmon-exciton interactions will contribute to the photonics and nanoscale optics and create a knowledge base for other technologies such as lasing in nanomaterials, energy conversion, and nanoscale electronic.
A5 – Contributed Paper DEVELOPMENT OF NANOENGINEERED CALCIUM PHOSPHATE 188 -BASED PARTICULATES FOR GENE DELIVERY Hyunbin Kim, Renato P. Camata, Rakesh Kapoor, Dept of Physics, Univ. of Alabama at Birmingham, Birmingham, AL, USA; and Selvarangan Ponnazhagan, Dept of Pathology, Univ. of Alabama at Birmingham, Birmingham, AL, USA; [email protected]
Particulate dispersions formed during co-precipitation of plasmid DNA and calcium phosphate are widely used in gene transfection protocols. Despite its simplicity these methods exhibit variable and often irreproducible efficiency. Basic investigations on the
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mechanisms of this versatile non-viral gene transfer methodology have been limited, however, because of the heterogeneous nature of the calcium phosphate nanoparticles commonly employed in these studies. Moreover, there are currently no efficient probes capable of tracking the intracellular kinetics of gene transfer using calcium phosphate, making detailed studies of gene delivery processes virtually impossible. Rare-earth doped particles are well known for efficient infrared-to-visible upconversion. In this work, we focus on the development of custom-designed calcium phosphate nanoparticles of well-controlled size, structure and chemical composition that are doped with Er3+ or Yb3+. The upconverted fluorescence of these doped calcium phosphate nanoparticles may allow tracking of different intracellular processes involved in transfection. The nanoparticles are produced using hybrid laser/aerosol method known as nanoparticle beam pulsed laser deposition, in which a hydroxyapatite target containing ~0.01-0.1 wt.% of the rare-earth species is ablated in inert gas. Nanoparticles formed are size selected, and dispersed in balance solutions for subsequent mixing with plasmid DNA and exposure to cells for gene transfer experiments. Nanoparticle sizes can be tuned in the 3-20 nm range while nanoparticle concentrations may be varied between 105 and 109 cm-3 in solution. We have targeted the control over crystal phase make-up of calcium phosphate nanoparticles, which determines its dissolution behavior, and the rare-earth dopant concentration. If the dissolution kinetics of these DNA-carrying nanoparticles can be matched to the temporal sequence of specific cell cycles, there is a clear opportunity to exert control over the gene transfer process. In-vitro measurements of gene transfer efficiencies as a function of calcium phosphate nanoparticle size, composition, and chemical environment will be discussed.
A5 – Contributed Paper PRECISION ASSEMBLY OF MULTICOMPONENT 189 NANOPARTICULATE COLLOIDS Joseph F. Bringley, Eastman Kodak Company, Research & Development Laboratories, Rochester, NY 14250, [email protected]
We describe a general methodology whereby colloidal electrostatic forces can be utilized to prepare a wide array of core-shell nanomaterials. The assembly process can be described by simple geometrical considerations, and as such, the experimental method may be adapted to design core-shell assemblies from a simple knowledge of the geometrical relationship between the core and the shell. We show the precision-directed assembly of particles upon particles, and polymers and molecules upon particles. We further show that the method can be used to design nanoparticle colloids having complex and multiple functionalities. The method is demonstrated in detail for polyelectrolytes assembled onto silica nanoparticles. 1H NMR spectroscopy is used to evaluate the adsorption isotherm and the saturation adsorption for polyethyleneimine assembled onto a 15 nm silica colloid. We describe a method for crosslinking the polyelectrolyte shell and permanently fixing it to the colloid surface, and demonstrate its effect on colloid stability. The colloid and surface properties of the materials will be discussed in detail.
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A5 – Contributed Paper THE SELF-ASSEMBLY BEHAVIOR OF THE CHOLESTERYL 190 DERIVATIVES OF ACYCLOVIR AND THE PREPARATION OF SELF-ASSEMBLED NANOPARTICLES Yiguang Jin, Rui Xin, Department of Pharmaceutical Chemistry, Beijing Institute of Radiation Medicine, Beijing 100850, PR China; [email protected]
We had prepared the self-assembled nanoparticles (SAN) from the long-chained glyceride derivative of acyclovir in our previous works, and the SAN showed targeted delivery in vivo and sustained drug release. We named the SAN-like aggregates prepared from amphiphilic prodrugs as self-assembled drug delivery systems (SADDS). In this study, we synthesized a new lipid prodrug of acyclovir – cholesteryl-succinyl-acyclovir (CSA), and prepared the self-assembled nanoparticles. CSA was obtained through condensing succinyl acyclovir with cholesterol. Also sodium salt of it (Na-CSA) was prepared. CSA was free soluble in tetrahydrofuran (THF), a water-miscible solvent, and Na-CSA was soluble in methanol. They are amphiphiles with cholesterol moieties and guanosine moieties. Self-aggregates of the amphiphiles were tried to prepare using the injection method or the film method, and CSA solutions in THF and Na-CSA solutions in methanol were used. A homogeneous opalescent suspension containing about 2 mg/ml CSA and 50% THF was obtained after CSA solution was injected into agitating water. After being kept at room temperature for a few hours, the suspension would like to change to a white gel (organogel). A lot of fibers were found in the gel with a negative-stained transmission electron microscope (TEM). Contrastively, CSA could form vesicle-like nanoparticles (ca. 30-nm size) in the suspension without organic solvents using the film method. Na-CSA always formed vesicle-like nanoparticles (ca. 50-nm size) using both the injection method and the film method. The hydrophobic interaction between cholesterol moieties could drive CSA and Na-CSA to self-assemble into bilayers and further formed vesicles. However, in the THF-water media, the hydrogen bonds between guanosine moieties of CSA would likely improve molecules aggregating head-to-head and extending into fibers. No hydrogen bonds would form among Na-CSA due to the ionization of guanosine. The self-assembled nanoparticles of CSA or Na-CSA might become a novel delivery system of acyclovir.
A5 – Invited Paper 191 TARGETED NANOPARTICLES FOR BONE THERAPIES Neal K. Vail; Southwest Research Institute, Microencapsulation and Nanomaterials Department, 6220 Culebra Road, San Antonio, Texas 78238, USA; [email protected]
Maintenance of skeletal health is one of the more challenging current public health issues. We are developing nanoparticles designed to target the mineralized phase of bone to selectively deliver therapeutics to treat bone maladies and improve bone health. Our current focus is the treatment of multiple myeloma with proteasome inhibitors, which are
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not selective to bone and their therapeutic-toxic window may be narrow. We previously identified several bone-binding ligands and selected methylene bisphosphonate (MBP) as a model targeting ligand. Amino-MBP was synthesized and its structure confirmed by 1H NMR. This and other identified ligands were covalently linked via sulfhydryl conversion of the amine to maleimide-terminated PEGylated phospholipids or polymers. Functionalized PEG-b-PLA copolymers were synthesized and their composition confirmed by NMR. Ligand attachment was confirmed by UV-Vis, NMR, and MALDI-TOF/MS. Liposomes were prepared by controlled hydration and sized by extrusion to typically 120nm. ± 20 nm. Lipid-ligand conjugates were inserted into the preformed liposomes by post-insertion. Polymer nanoparticles were prepared by nanoprecipitation and typically were 75nm to 150nm, depending on the nanoparticle formulation. Particle size decreased with increasing PEG content. Similarly, zeta-potential decreased with increasing PEG content, probably due to shielding of the PLGA surface by the surface PEG groups. Ligand content of the various nanoparticle systems was quantified by complexing the MBP with 99mTc and performing gamma counting. MBP ligand contents varied between 0 and 5 mol % of the particle formulation. In vitro studies showed that MBP ligands conjugated to the surfaces of nanocapsules convey preferential targeting accumulating significantly on hydroxyapatite substrates, compared to non-ligand-containing nanoparticles, which had no significant affinity for the HAp substrate. We demonstrated the formulation and characterization of bone-targeting nanoparticles. These nanoparticles preferentially adhere to bone-like surfaces in vitro. We proceeding with in vivo studies to examine the biodistribution and uptake of these nanoparticles.
A5 – Invited Paper IRON NANOPARTICLES AS MAGNETIC CARRIERS 192 Shannon A. Morrison, Michael D. Shultz, Melissa A. Michaels, Sweta H. Naik, Farrah N. Radwan, Everett E. Carpenter, Virginia Commonwealth University, Chemistry Department, 1001 W. Main Street, Richmond, VA 23298, USA; [email protected], [email protected]
Magnetic carriers based on iron oxides are used almost exclusively due to biological and chemical inertness. Because of the relatively weak magnetic flux density of iron oxides, the nanoparticles need to be in the micron size range to withstand the flow conditions required of a magnetic carrier. The inherent sizes of these particles have potential problems however, such as prompting immunological responses and difficulty traversing the cell membrane. We propose to use a multi-layer magnetic carrier based on iron metal nanoparticles. This core-shell structure allows for smaller carriers, 10-20 nm in size, while still retaining the necessary magnetic properties. Core-shell nanoparticles are synthesized using the reverse micelle technique. Iron is first precipitated within the aqueous core using sodium borohydride. The iron core then acts as a nucleation source for the subsequent iron oxide shell formation. A description of the magnetic properties of these superparamagnetic particles will be presented along with a comparison to other commercially available magnetic nanoparticles.
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A5 – Contributed Paper BROAD-BAND EFFICACY DETERMINATION FOR DISCRETE SILVER 193 NANOPARTICLES Stan Farnsworth, Nanotechnologies, Inc.1908 Kramer Ln, Building B, Austin, TX 78727, [email protected]
Silver has long been known to demonstrate anti-microbial efficacy with an ionic interaction mechanism. Now, discrete non-aggregated silver powders with average particle size 25 nm produced by Nanotechnologies, Inc have been established to have efficacy with bacteria, virus, and fungus, including vegetative as well as dormant spore forms. Bacterial forms tested include gram-positive and gram-negative, while viral forms include both enveloped and non-enveloped, as well as DNA and RNA cores. Notably, work has demonstrated that the particles have a direct interaction with the pathogens, beyond the traditional ionic mechanisms. Electron imaging shows that bacteria walls are lysed, and virus capsids have silver attached corresponding with specific protein sites. Work is now shifting to determining the efficacy of the silver nanoparticles when used as simple surface coatings, incorporated into polymers, and suspended in creams and ointments. Results from these tests could indicate likely best-commercial application candidates, including medical textiles, hospital and food-preparation surfaces, wound dressings, and veterinary products.
A5 – Invited Paper NANOCLUSTERS AS A UNIQUE DRUG DELIVERY PLATFORM 194 Lianjun Shi1 and Cory Berkland1,2; 1Department of Chemical and Petroleum Engineering, University of Kansas; 2Department of Pharmaceutical Chemistry, University of Kansas; [email protected]
Clusters of nanoparticles, also referred to as agglomerates, colloidal clusters or colloidal crystals, are a class of assemblies at the micrometer or sub-micrometer scale consisting of a large number of closed-packed nanoparticles. Materials such as these offer some intriguing properties not observed in the bulk forms of corresponding materials. For instance, three dimensional assemblies of uniform nanoparticles can be designed to exhibit a full photonic band gap not seen in dispersions of the same. New techniques to synthesize organic or inorganic nanoparticles demonstrating control of size, size distribution, and surface chemistry, continue to fuel emerging nanocluster applications. Typically, nanoclusters are not able to disperse into individual nanoparticles, even when applying intense sonication. The proximity of nanoparticles leads to irreversible agglomerates held together by strong van der Waals interactions. Here we report nanoclusters with pharmaceutically relevant properties such as pH-sensitive dispersion and biodegradability, using solvent-extraction and self-assembly fabrication techniques.
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G2 – Keynote Paper THE CELLULAR AND MOLECULAR TOXICITY OF LOW SOLUBLITY 195 NANOPARTICLES Vicki Stone, Centre for Health and Environment, School of Life Sciences, Napier University, Edinburgh EH10 5DT, UK; [email protected]
The number and diversity of newly engineered nanoparticles is steadily increasing due to their applications in medicine but also in food, clothing, cosmetics and IT applications. Due to the wide variety of applications for which these are developed, including, the potential for human exposure is inevitable and so it is important to consider the hazard of these materials. Although new nanoparticles for medical applications are usually thoroughly tested prior to clinical use, the mechanism of toxicity is often poorly understood. Improved understanding of such mechanisms could help in the improved design of new nanoparticles. The toxicology of bulk manufactured nanoparticles such as carbon black, TiO2 and polystyrene beads hax been investigated for several years, often in relation to investigating the mechanisms driving the adverse health effects of ambient air pollution. The results from these studies are useful in providing a platform from which an effective strategy for nanoparticle toxicity testing can be established. We have previously reported that carbon (14 nm diameter) or polystyrene (54 nm diameter) nanoparticles generate more inflammation in the rat lung than larger particles, and this inflammation is correlated well with the surface area of the particle dose. The smaller particles have been demonstrated in a number of assays to generate more reactive oxygen species (ROS) than larger particles leading to oxidative stress in the lung epithelial cell lines and in macrophages. In addition non-lethal doses of these particles induced a significant increase in cytosolic calcium leading to the production of the pro-inflammatory cytokine tumour necrosis factor alpha (TNFα). In conclusion, the relatively small size of nanoparticles enhances their potency in a number of in vivo and in vitro systems resulting in the production of oxidants and the generation of pro-inflammatory mediators. Such test systems would be useful in investigating interactions between medical nanoparticles and the body. Funding: The Colt Foundation and BP.
G2 – Invited Paper FORMULATION PROCESSES AND CHARACTERIZATION OF DNA 196 AND siRNA NANOPARTICLES R. Knowle, A. Werner, M. Fisher, and Rob DeLong, Louisburg College, 501 N. Main Street, Louisburg, North Carolina 27549, USA; [email protected]
There are a wide variety of potential applications for nucleic acid-peptide nanoparticles in research and development ranging from molecular medicine to diagnostics. My research interests are in formulation processes and characterization chemistry for nanoparticles formed of DNA and RNA. In previously published work we have studied nanoparticles formed when plasmid DNAs or antisense oligomers were complexed with cationic polymers, protamine, polyarginine or oligo-ARG, and the relationship between their
160 Oral Abstracts physico-chemical characteristics and chemical, biochemical or biological activity. More recently we have been studying sedimentation and partitioning methods for preparing these types of nanoparticles and have extended them to the preparation of siRNA and DNA vaccines. These types of particles are limited by relatively inefficient and heterogenous loading or coating capacities and by their tendency to aggregate and to precipitate in physiological fluid and serum containing media. Further their bioprocessing, down-stream, sterilization and storeage steps can often lead to the chemical degradation of the bio-molecules even before any enzymatic based mechanisms in serum or their delivery to cells and tissues. We are using a combinatorial and design of experiment approach towards developing more effective means of producing nanoparticles with better loading and stability properties. Taking into account and using to our advantage in some cases, some of these properties to better prepare nanoparticles on this basis, using a high throughput luciferase splice-site correction model to rapidly screen nanoparticles produced under a variety of design of experiments screening for their biological activity.
G2 – Contributed Paper PHYSICOCHEMICAL ANALYSIS AND EFFECTS ON YEAST CELLS OF A 197 PARTICULATE TRADITIONAL INDIAN MEDICINE, JASADA BHASMA. Tridib K. Bhowmick, A. K. Suresh, S.G. Kane, Jayesh R. Bellare, Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Maharashtra, Mumbai-400076, India; and Ajit. C. Joshi, 22, Shukrawar Peth. Pune, Maharashtra, India; [email protected]
Herbo-mineral preparations called Bhasma are unique to Ayurveda and Siddha system of Indian traditional medicine. These preparations are particulate powders and have long been used and claimed to be an effective and potent medicine. However, there are only a few analytical scientific studies carried out on these products, and even these suffer from incomplete analysis. Use of a wide variety of materials (e.g. minerals, metals, herbal juices) during manufacture also makes these kinds of medicines extremely difficult to analyze. In this study, different analytical techniques have been employed for complete elemental analysis as well as the physical characteristics of Jasada Bhasma which is made from zinc metal. When analyzed with the help of XRD, it shows the presence of hexagonal ZnO as a major phase in the sample. For ICP analysis the sample completely dissolved by a two step process. In the first step sample was dissolved in (1:1) mixture of conc. HNO3 and HClO4. The bulk of the sample dissolves in such a solution. The residue which is still left behind is dissolved in acid after digesting with LiBO2. The analysis result shows that the sample contains eleven elements (Na, Mg, Al, Si, S, Ca, Mn, Fe, Cu, Zn and Sn) among which Zn is present in maximum amount (78.57 wt %). EDAX has also been done to confirm the elemental composition. It detects the same eleven elements plus oxygen (11.70 wt%) and this result is in good agreement with the ICP result. An interesting finding is that oxygen (32.63 atom %) present in the material is not stoichiometrically sufficient to satisfy all the Zn (54.07 atom %) as a ZnO, nor with the other metals present. Combining the XRD, ICP and EDAX result it can be said approximately equal amount of amorphous Zn present in the sample. The size and size distribution of the particles have been determined by employing DLS and TEM. Particle size distribution has been studied after suspending the materials in aqueous dispersing phase at pH 3.0 and filtering through 0.22 micron membrane filter. TEM photographs of the filtrate show
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considerable amount of particles present in the nanoparticle (10-15 nm) range. The biological activity of Jasada Bhasma medicine has been studied by growing Saccharomyces cerevisiae cells in shaker flask in presence of 300ppm Bhasma sample. The result revealed that cell cycle of Saccharomyces cell changed dramatically when they are treated with small size (below 500nm) particles.
G2 – Invited Paper PHYSICAL PROPERTIES AND CYTOTOXICITY OF INORGANIC 198 NANOPARTICLES FOR BIOMEDICAL APPLICATIONS Gan-Moog Chow, Department of Materials Science and Engineering, National University of Singapore, Kent Ridge, Singapore 119260. [email protected]
Inorganic nanoparticles find potential applications in targeted delivery of drugs and biomolecules, separation, sensing and imaging. The properties of nanoparticles depend not only on the size but also the structure, microstructure and surface coating. In this talk, examples of some of our work on inorganic nanoparticles for biomedical applications are presented. Near-infrared-sensitive Au-Au2S, magnetic γ-Fe2O3 and NiFe2O4, and
IR-to-visible upconversion fluorescent Yb-Er, Yb-Ho and Yb-Tm co-doped LaF3 nanoparticles were chemically synthesized. The physical and chemical properties were optimized for the application in question. For drug delivery, surface modification of particles using suitable surfactants was tailored for adsorption and release of anti-cancer drugs (cis-platin or carboplatin). For bio-probes and displays, visible emission of colloidal up-conversion nanocrystals under IR excitation was studied and compared with the NaYF4:Yb,Er nanocrystals. The cytotoxicity of nanoparticles depends not only on the particle size but also the surface coating, both of which are controlled by the synthesis and processing conditions. For example, the dependence of cytotoxicity on particle size and on the presence of oleic acid as surfactant on nickel ferrite particles were investigated in vitro using the Neuro-2A cell line as a model. For nickel ferrite particles without oleic acid prepared by ball milling, cytotoxicity was independent of particle size within the given mass concentrations and surface areas accessible to the cells. For nickel ferrite particles coated with oleic acid prepared by the polyol method, the cytotoxicity significantly increased when 1 layer or 2 layers of oleic acid were deposited. Large particles showed a higher cytotoxicity than smaller particles. The effects of surface energy and effective interaction areas of adsorbed functional groups on cytotoxicity were suggested.
G2 – Invited Paper GREEN NANOTECHNOLOGY THROUGH COLLABORATIVE 199 RESEARCH ON MECHANISMS OF NANOMATERIAL TOXICITY Agnes B. Kane and Robert Hurt, Department of Pathology and Laboratory Medicine and Division of Engineering, Brown University, Providence, RI 02912; [email protected], [email protected]
It is now recognized that the recent world-wide investment in new nanomaterials should be
162 Oral Abstracts accompanied by parallel efforts to explore and understand their potential impacts on human health and the environment. By virtue of their small size, nanomaterials may penetrate biological membranes, enter cells, carry high concentrations of adsorbed species, or show elevated surface reactivity relative to their macroscopic counterparts. This talk introduces the emerging field of nanotoxicology and presents results from our collaborative research on carbon nanomaterials. It will be shown that iron nanoparticles that are residues of the growth catalyst can be redox active, produce DNA damage, and are toxic to macrophages. A unique feature of this effort is a close collaboration between toxicologists and materials scientists with the joint goal of identifying the specific material features (size, shape, surface chemistry, purity) that contribute to toxicity. The long-term promise of this approach is to learn how to modify synthesis or purification procedures to fabricate “green” nanomaterials – those co-optimized for performance and minimal health impact. This research was supported by the grants from US-EPA, NSF, NIEHS, and the Superfund Basic Research Program.
G2 – Invited Paper NANOPARTICLE INTERACTIONS WITH THE CYTOSKELETON 200 James McGrath and Morton Ehrenberg, Department of Biomedical Engineering, University of Rochester, Rochester NY 14642, USA; [email protected]
The use of nanometer particles as intracellular mechanical probes requires the design of particles with controlled and specific interactions with cytoskeletal filaments. Also, the demonstrated toxicity of some nanoparticles particles is of increasing concern as nanoengineered materials become more commonplace in industry and the environment. With these issues in mind, we characterized the natural capacity of various particles to bind to the proteins contained in fibroblast cytoplasm and bovine brain extracts. We found that all particles tested (polystyrene, TiO2, and SiO2 with various levels of surface carboxylation) bound primarily to cytoskeletal proteins in both types of extracts, although the profile of bound proteins do vary with particle surface chemistry and extract type. In the fibroblast extracts, we found that carboxylated polystyrene beads bind most prominantly to actin and the intermediate filament protein vimentin, and through surface modification we could eliminate the binding of actin. These biochemical studies were supported with microrheological measurements in living fibroblast cytoplasm. Here we tracked microinjected particles with high temporal and spatial resolution inside of cells and found that the untreated particles made random movements of diminshed amplitude compared to the particles treated to block actin binding. We also found that the random movements of the two nanoparticles chemistries were indistinguishable when the particles were endocytosed rather than microinjected, and that the amplitude of the motion of endocytosed particles was significantly higher microinjected particles. There is considerable evidence in the literature that small nanoparticles can circumvent the membrane trafficking pathways of cells to become free in cytoplasm. Our data suggests that the such particles will strongly interact with the elements of the cytoskeleton.
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O1 – Keynote Paper NANOPARTICULATE VACCINE DESIGN: THE VESIVAX SYSTEM 201 Gary Fujii, Molecular Express, Inc., 13310 South Figueroa Street, Los Angeles, CA 90061; [email protected]
The VesiVax® system is based upon the concept that highly potent vaccines can be designed by engineering proteins that are capable of stably inserting themselves into nanoparticulate liposomes. Such a nanoscale liposomal particle could then serve as an immunogen for vaccine development. This approach requires a fundamental understanding of the interactions of proteins and/or peptides with lipid bilayers and an in-depth knowledge of the processes used in the commercial preparation of liposomes. Structural and functional studies on the interactions of proteins and peptides with lipid bilayers previously identified structural amphiphilicity and hydrophobicity as important parameters that control the nature of the interaction between proteins and membranes. The VesiVax® vaccine technology platform takes advantage of these parameters and features a flexible and easily modified gene cassette designed to rapidly engineer and produce, using standard recombinant manufacturing processes, antigen fusion proteins that are compatible with liposomal bilayer membranes. The recombinant antigen proteins are fused to an aqueous soluble hydrophobic domain that makes purification simple and allows stable insertion of the immunogen within the lipid membrane. This technology eliminates the issues associated with hydrophobic protein domains that have previously made large-scale vaccine development difficult. Vaccines based on the VesiVax®system have been developed against the influenza virus and herpes simplex type 2 virus, the causative agent of genital herpes. Both vaccines have been tested in animal models and have demonstrated significant protective efficacy from challenge with lethal doses of virus. Assays of the immunological parameters suggest that both T and B cell responses can be elicited by VesiVax® vaccines. The safety profile of the VesiVax® vaccines is expected to be much better than that of vaccines prepared by conventional techniques.
O1 – Invited Paper CLINICAL DEVELOPMENT OF A THERAPEUTIC MICROPARTICLE 202 FORMULATION Thomas M. Luby and Mary Lynne Hedley, MGI PHARMA Biologics. 44 Hartwell Ave. Lexington, Ma. 02421; [email protected]
Injection of microparticle encapsulated DNA has been shown to elicit immune responses to the plasmid-encoded antigens, and the use of this technology in a therapeutic cancer vaccine is described. The tumor-associated antigen Cytochrome P450 1B1 (CYP1B1) is overexpressed in many tumor types, but has a relatively restricted normal tissue expression profile, making tumors that express it excellent targets for immunotherapy. Despite the fact that it is a self protein, T cells specific for CYP1B1 epitopes exist in the human repertoire, and these are cytotoxic for tumor cells expressing the antigen. Microparticle formulations containing plasmid DNA are phagocytosed by antigen presenting cells resulting in the expression of the antigen-encoding DNA in cell types well suited for
164 Oral Abstracts inducing an immune response. Preclinical studies in mice have demonstrated that a formulation made of PLGA microparticles encapsulating a CYP1B1 encoding plasmid DNA are able to induce immune response to CYP1B1. Additionally, it appears that the microparticle formulation has natural adjuvant activity and is amenable to alterations that result in enhanced immunity. This formulation has been tested in a phase I clinical trial of 17 patients with various cancers types, and was shown to be safe and well tolerated. Interestingly, clinical efficacy appeared to correlate with immune responses induced by the drug, leading to strategies to further potentiate the immune response in subsequent trials.
O1 – Contributed Paper BIOMIMETIC PARTICLES FOR VACCINE DESIGN 203 Nilton Lincopan, Noeli M. Espíndola, Adelaide J. Vaz, Ana M. Carmona-Ribeiro, Instituto de Química, Dept. Bioquímica, Universidade de São Paulo, P.O.Box 26077, 05513-970 São Paulo, SP, Brazil; Faculdade de Ciências Farmacêuticas, Laboratório de Imunologia, Universidade de São Paulo, São Paulo, SP, Brazil; [email protected]
Electrostatically driven bilayer coverage from bilayer fragments onto oppositely charged latex produces a highly homodisperse particulate for antigen presentation. Polystyrene sulfate (PSS) particles were covered with single cationic dioctadecyldimethylammonium bromide (DDA) bilayers and used for antigen adsorption and presentation. The antigen was a mixture of recombinant 18/14 Taenia crassiceps proteins (18/14-Tcra). Firstly, the DDA/PSS assembly was characterized at 5 x 109 PSS particles/mL over a range of DDA concentrations (0.01-1 mM) by means of dynamic light scattering for particle sizing and zeta-potential analysis. 0.01 mM DDA is enough to produce perfectly homodisperse and cationic bilayer-covered particles. Secondly, under these experimental conditions, 18/14-Tcra adsorption isotherms onto biomimetic particles or onto aluminum hydroxide (Freund`s adjuvant) yield limiting adsorption of 0.36 and 1.32 mg protein adsorbed per mg biomimetic particles or Al(OH)3, respectively. Finally, the foot-pad swelling test for delayed type hypersensitivity, a marker for cellular immunoresponse, was performed in mice to prove the higher efficiency of antigen presenting biomimetic particles in comparison to Freund´s adjuvant under equivalent experimental conditions and antigen concentration. Thereby, this work represents a proof-of-concept that cationic bilayer-covered particles represent a novel, highly organized and general immunoadjuvant for antigen presentation and subunit vaccine design. Financial support: FAPESP and CNPq
O1 – Invited Paper DESIGN OF SYNTHETIC COLLOIDAL ANTIGEN CARRIERS FOR 204 VACCINE PURPOSES Thierry Delair, Unité-Mixte CNRS-bioMérieux, ENS-Lyon, 46 allée d’Italie 69007 LYON; [email protected]
Vaccine have long been based on the use of attenuated or denaturated pathogens injected to patients. To improve the safety of new vaccines, instead of the whole pahtogen, sub-units
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can be used, i.e. one or several proteins cons tituting the pathogen (or the genes coding for them). But to have an efficient immune response, antigen carriers have to be used with the antigen either within the matrix of the polymer or adsorbed onto the surface of the particles. The physico-chemical properties of the particles have to be tuned to the applications. In this presentation we will adress various methods to obtain colloidal carriers for either the delivery of DNA or proteins and we will describe some results obtained in various animal samples. Particles for the delivery of DNA need to be positively charged in order to interact via electrostatic interactions. Various cationicpolymers have been immobilized onto the surface of the colloid and we will show that not only the chemical nature of the polymer but the elaboration process as well impact the adsorption process of the DNA. For protein delivery, we will show that surfactant free particle can be efficient antigen carriers that can ellicit both cellular and humoral responses in various animal models such as mice, rabbit and non-human primate.
O1 – Invited Paper NOVEL INJECTABLE BIOMATERIALS FOR PROTEIN AND VACCINE 205 DELIVERY Balaji Narasimhan, Department of Chemical and Biological Engineering, Iowa State University, 2035 Sweeney Hall, Ames, IA 50011-2230, USA; [email protected]
Polyanhydrides are a class of hydrophobic biomaterials that degrade by hydrolytic cleavage of the anhydride bond and their hydrophobicity confers a “surface erosion” mechanism upon them. Our overall objective is to engineer polyanhydrides with tailored chemistry and nanostructure to regulate drug/protein distribution and to design delivery systems with physiologically meaningful release profiles. Two examples of controlled delivery systems based on these materials will be discussed. First, proteins can be stabilized within and released from polyanhydride microspheres. The effect of polymer chemistry, and microsphere fabrication method on the structure and activity of several proteins was investigated. It was found that the mechanism of denaturation of the protein and its interactions with the polymer carrier play a significant role in the design of the delivery system. A systematic approach was developed for selecting compatible polymer/protein systems prior to encapsulation. Second, single dose vaccine carriers based on polyanhydrides were designed with the unique capability to modulate immune response mechanisms. Our studies have shown that by altering the chemistry of the polyanhydride, a TH2 response can be selectively diminished without reducing the overall antibody titer. This leads to a balanced TH1/TH2 response, which has important implications for developing vaccines to intracellular pathogens. The fundamental treatment of the mechanistic aspects of release and immune response will ultimately facilitate rational means of designing single-dose vaccines.
D5 – Keynote Paper COLLOIDAL ASSEMBLY EXTENDED INTO THE BIOMATERIALS 206 DOMAIN: COMPOSITE COATINGS AND MEMBRANES FROM LIVE
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CELLS Orlin D. Velev, Shalini Gupta, Lindsey B. Jerrim, Rossitza G. Alargova and Peter K. Kilpatrick; Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695-7905, USA; [email protected]
The co-assembly of live cells and synthetic nanoparticles could be a route to making new biomaterials, in which the functionality of the cells is complemented by the functionality of the particles. We will demonstrate a few classes of such biocomposite materials made by using techniques for controlled colloidal assembly into 2D and 3D structures that we have developed earlier. We will first demonstrate how on-chip dielectrophoresis can be used to co-assemble arrays of latex spheres and yeast cells or NIH/3T3 mouse fibroblasts. Depending on the frequency of the field and relative polarizability of the cells and particles, one and two dimensional assemblies can be obtained. These arrays can be bound into permanent biocomposites by using lectin-conjugated spheres as biocolloidal binders. Particles with magnetic cores added an additional functionality to the live cell 2-D matrix and we were able to manipulate these materials with external magnetic fields. Such cell-nanoparticle chains and membranes can form the basis of sensors, microscopic bioreactors and artificial tissue. We also present a method for assembling and immobilizing large-scale coatings from yeast cells. The coating method is based on convective assembly and deposition in a moving meniscus to make close packed two-dimensional arrays. A robust technique for rapid deposition of monolayer cell coatings was designed on the basis of this method. One immediate application of these cell coatings is in biosensors and test beds for toxicity and drug action.
D5 – Invited Paper GENETIC CONTROL OF NANOPARTICLE ASSEMBLY AND ITS 207 APPLICATION IN COLORIMETRIC SENSING Yi Lu,* Juewen Liu and Daryl P. Wernette, Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA; [email protected]
Genetic control of synthesis and properties of nanoparticles in response to chemical stimuli under ambient conditions has been one of the hallmarks in biology. Employing such a process in nanopartcle research can result in novel nanomaterials that rival or even exceed the structural and functional properties of naturally occurring materials. Toward this goal, we have taken advantage of recent advance in biology where catalytic DNA molecules (DNAzymes) or aptamers can be selected from a large (up to 1015 different) DNA library with specific binding selectivity toward an analyte of choice. By using DNAzyme, aptamer or aptazymes for directed assembly gold nanoparticles, genetic control of such assembly in response to chemical stimuli such as metal ions or organic molecules have been demonstrated. Because of the highly intense color change from the blue assembled gold nanoparticles to the disassembled red particles, this work has resulted in simple, sensitive and selective colorimetric sensors for a number of molecules. A careful investigation of the DNAzyme biologyhas resulted in a new derivative of DNAzyme that allows a tunable
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assembly of nanoparticle and thus tunable dynamic range of detection. In addition, a detailed understanding of DNAzyme and nanoparticle interactions resulted in imporvemnet of the sensor performace, allowing room tempeature and fast operation for on-site, real-time sensing, much the same as pH indicators. In addition to controlled assembly of materials, biology also controls the disassembly of materials in responsive to chemical stimuli so that useful materials can be released or harmful processes can be prevented. We have recently succeeded in employing such a process in disassemble gold nanoparticle aggregates in responsive to metal ions such as Pb2+. This endeavor has resulted in even further improvement of the colorimetric sensor by transforming a “light-down” sensor into a “light-up” sensor. Recent progress in this work will be presented.
D5 – Contributed Paper DEVELOPMENT OF A MULTIPLEX PEPTIDE NANOTUBE-BASED 208 PATHOGEN ASSAY Robert I. MacCuspie, Hiroshi Matsui, Department of Chemistry, Hunter College and The Graduate Center of the City University of New York, 695 Park Ave, New York, NY 10021, [email protected]; Ipsita A. Banerjee, Department of Chemistry, Fordham University, Bronx, NY; Andrea Bertke, Sanjay Gummalla, Howard Mostowski, Phil Krause, U.S. Food & Drug Administration, Center for Biologics Evaluation and Research, 29 Lincoln Drive, Bethesda, MD 20892.
Rapid detection of pathogens is of key importance in both the fight against bioterrorism and in the screening of regulated products such as vaccines and blood supplies. Here, a novel multiplexed peptide-nanotube based assay is presented that detects in-tact, whole viruses in solution both rapidly and efficiently. Using multifunctional peptide nanotubes coated at the ends with detecting antibody and along the sidewalls with fluorescent dye signaling molecules, the nanotubes bind to viruses to form three-dimensional networks whose size and fluorescence intensity increase proportionally to the virus concentration. Antibodies bind the virus creating an aggregate with a central core containing nanotube fingers radiating outward similar in shape to a sea urchin. As the concentration of virus increases, additional viruses tether these sea-urchin structures together into 3-D fluorescent scaffold-like networks. In this system, fluorescent intensity is directly proportional to the size of each network. Flow cytometry allows collection of scattering data concurrently with fluorescent data, confirming the correlation between the 3-D network size and fluorescent intensity. When the ratio of nanotubes to virus was very high formation of several smaller networks was favored as opposed to fewer larger networks. This method allows determination of an unknown virus concentration level down to trace levels as low as 100 virus particles per mL in a 0.5mL sample. Standard curves of fluorescent intensity vs. concentration of virus were created for several different viruses. By assigning different antibodies to unique fluorophores, detection of several fluorescence intensities simultaneously is possible. Proof of principle experiments were performed to quantify a mixture of two viruses. The multiplex ability of the system is only limited by the ability to resolve each unique fluorophore and the number of photodetectors on the flow cytometer.
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D5 – Contributed Paper SURFACE MODIFIED LANTHANIDE DOPED NANOPARTICLES AS 209 NEW ROBUST BIOLABELS Frank van Veggel, University of Victoria, PO Box 3065, Victoria, British Columbia, V8W 3V6 Canada. [email protected]
Trivalent lanthanide (Ln3+) ions have unique optical transitions in the sense that they have quite sharp, almost line-like, non-overlapping absorption and emission bands from the visible to the near-infrared (which could be adavantageous in multiplexing). In addition, they can have several long-lived excited states, from micro- to millisecond, allowing easy removal of autofluorescence from, for instance, biological moieties through gated spectroscopy. Moreover, they have efficient upconverting processes that convert low-energy photon into high-energy photons. These characterstics make these ions potentially very useful as biolables. In particular, absorption and emission in the near-infrared allow for deeper tissue penetration and their photocycles are extremely 3+ robust. We have developed routes to stabilize Ln doped LaF3 nanaoparticles with ligands such as citrate and 2-aminoethylphosphate to make them water soluble, with retention of the emissive properties of the dopant Ln3+ ions. The primary amine of the 2-aminoethyl- phosphate ligands could be reacted with activated esters to impart biological function, i.e. introduction of specific ligands such as biotin. Binding studies with immobilized avidin on beads showed that specific binding had been achieved. The second route we developed is based on the coating of these nanoparticles with a thin layer of SiO2 in order to have a material that can easily be functionalized and is benign to animals and human. Primary amino or thiol groups can be introduced by standard silane chemistry and reacted further to introduce a biological function (i.e. biotin). Binding studies with the avidin that was labeled with fluoresceine as marker showed that specific binding had been obtained. This approach led to very effcient emission from the visible, Eu3+ and Tb3+, to the near-infrared (Nd3+ and Er3+). Moreover, effcient upconversion was achieved though the Yb3+ sensitized emission of Er3+ and Tm3+. The latter is particularly attractive because the excitation at 980 and the emitted upconverted light at 800 are both in the water window.
D5 – Contributed Paper NANOPARTICLE QUANTUM DOTS FOR TARGETED 210 NEUROMODULATION Tania Q. Vu, Department of Biomedical Engineering, Oregon Health & Sciences University, 20000 N.W. Walker Road, Beaverton, OR 97006-8921; [email protected]
Increased interest in nanocrystal quantum dot (QD) technology as fluorescent probes possessing unprecedented brightness, photostability, and multi-color capability has made apparent their promising potential for live tracking of single cell and in vivo processes. Presently, QD biorecognition is largely achieved through attachment of antibodies to the QD surface, resulting in a bio–inorganic complex combining biomolecular-specificity with fluorescence. A less explored approach is to use small ligands or synthetic analogs that
169 Oral Abstracts when conjugated to the QD, would bind not only to receptor proteins, but also serve to activate signaling cascades that regulate cellular phenotype and behavior. Here we discuss the development of QDs complexed with nerve growth factor, a peptide hormone belonging to the family of neurotrophic factors that has generated therapeutic interest due to the ability of these factors to promote the survival of central and peripheral neurons after neuronal damage and during development. Surface modification and composition of complexed QD nanostructures were confirmed and bioactivity of ligands and ligand-QD complexes were tested using functional imaging based assays. Despite known structural and chemical stringency of ligand-receptor activation, evidence supports activation and modulation of cell function and behavior. Three aspects of this work will be discussed: 1) design and assembly of drug-bound QDs, 2) visualization and chemical analysis of biomolecule-coupled quantum dot complexes, and 3) anatomical and physiological targeting in neural tissue. Development of this QD-based capability bears significance for development of tools for studying inter-molecular interactions, assessing effectiveness of drug compounds, and visualization of cellular function for a variety of biological studies.
D5 – Invited Paper TARGETED AND FUNCTIONAL NANOPARTICLES FOR BIOMEDICAL 211 DIAGNOSTICS Yvon Durant, Romuald Couronne University of New Hampshire, Materials Science Program, G101 Parsons Hall, durham NH 03824, USA; and Ebo de Muinck, Angiogenesis Research Center, Dartmouth Medical School, Borwell Research Building 734E, HB 7700, 1 Medical Center Drive, Lebanon, NH 03756, USA; [email protected]
Medical diagnostic can be facilitated by the use of magnetic nanoparticles tagged with specific homing peptides. The synthesis of such particles is done in 3 steps. The first one is the synthesis of the magnetites (the core of the particles), the second step concerns their encapsulation into a protective polymer shell, and the third step is the functionalization with a homing peptide and optionally a fluorescent tag. The synthesis is done from Iron II and Iron III salts in basic conditions in the presence of oleic acid. This process create primary magnetite particle in the size range or 8-25nm. Upon liquid extraction of the aqueous dispersion with styrene we obtain an organic dispersion of magnetite at solid content ranging up to 20%. To this organic solution is added hexadecane and acrylic acid, which is in term sonicated with an aqueous solution of sodium dodecyl sulfate. Upon polymerization with potassium persulfate, a unimodal miniemulsion is obtained with an average size of 130nm. This magnetic latex is then diluted to 5% solid content and ultrafiltrated on 100KD cellulose filters to eliminate residual monomers with Potassium buffer system (PBS). The latex is then functionalized with an adhesion promoter amino functional peptide such as cNRG and tagged with a fluorescent label, such Alexa Fluor 647 Cadaverine in the presence of a carbodiimide coupling agent such as EDCI. The coupling yield is 50%. The particles are again ultrafiltrated with PBS to eliminated uncoupled molecules. The resulting particles have a ferromagnetic behavior and have a saturating magnetic moment of 50 emu/g at 0.1 T. These diagnostic nanoparticles can be used both in confocal fluorescence microscopes, fluoro-endoscopes and Magnetic Resonance Imaging units.
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D5 – Invited Paper DETECTION OF DRUGS OF ABUSE USING A MAGNETIC BIOSENSOR 212 F. de Theije, T. van der Wijk, W. Hardeman, H. Brans, A. Immink; Philips Research and Philips Applied Technologies, High Tech Campus 4 (WAG02), 5656AE, Eindhoven, The Netherlands, and S. Marlin, A. Jehanli, D. Baldwin, G. Jowett, Cozart plc, 92 Milton Park, Abingdon, Oxfordshire, UK OX14 4RY; [email protected]
We are developing assays for fast detection of drugs of abuse in saliva, based on giant magneto-resistance (GMR) biosensors packaged in a compact disposable microfluidics cartridge. The possibility of magnetic actuation, which rapidly concentrates the magnetically labeled bio-molecules at the sensor surface, is a unique feature of the magneto-electronic device that allows a fast assay by speeding up reaction time and making time-consuming fluid wash steps obsolete. The high signal to background ratio due to the fact that body fluids are non-magnetic makes the GMR the device of choice for drugs of abuse testing. Additionally, electronic callibration techniques make the system very robust in diverse environments among which the road-side environment. Drugs of abuse generally are small molecules that only possess a single epitope for antibody binding, thus requiring a competitive assay format. Our assays are based on the capture of biologically functionalized superparamagnetic beads on a sensor surface. Antibodies raised against the drug of interest are linked to magnetic beads. Drug molecules bound to the sensor surface compete with drugs from the sample for the antibody binding sites. Using morphine as a model system, different assay protocols were investigated using both well-plate and cartridge experiments, aiming for an assay at minute time scale. We will present several competitive assay formats tuned to magnetic nanoparticle labels, compare assays performed on magnetic biochips and well-plates, and show high-speed drug immunoassay dose-response data.
D5 – Contributed Paper SENSING SUBSTRATES MODIFIED WITH GOLD NANOPARTICLE 213 FILMS K. Bonroy, F. Frederix, G. Reekmans, H. Jans, R. De Palma, K. Jans, G. Borghs, IMEC, Kapeldreef 75 Heverlee 3001, Belgium; [email protected]
A wide range of different materials is used as sensing area of biosensor devices. Most of these materials form relative flat sensor substrates. Besides the transduction principle, the sensitivity of affinity-based biosensors is, however, also partially dictated by the amount of immobilized receptor molecules on the sensor substrate. Three-dimensional (3D) sensor substrates, created by assemblies of gold nanoparticles, would allow for a larger amount of immobilized receptor molecules per surface area and could therefore increase the sensor signal significantly in comparison to standard 2D substrates. In addition, according to numerous studies, nanoparticle films are very promising for the development of novel electrical and optical sensors. In this paper, we report on the formation of 3D substrates of gold nanoparticles on plane gold, quartz and SiO2 substrates using mercapto-silane chemistry.
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In a first series of experiments, the surface roughness of the resulting 3D gold surfaces was characterized using SEM, CV and AFM, while the Localized Surface Plasmon Resonance (LSPR) properties were evaluated using UV/Vis absorption spectroscopy. The sensing properties of the obtained 3D gold nanoparticles films were further explored on Quartz Crystal Microbalance (QCM) crystals, SPR slides, interdigitated electrodes (IDE) and LSPR sensors. Hereby, the sensitivity and the enhancement qualities of the nanoparticles films were demonstrated for different protein systems such as antibody/antigen and bacterial S-layer proteins.
D5 – Invited Paper RADIOLABELED LIPID NANOPARTICLES FOR IMAGING AND 214 RADIONUCLIDE THERAPY Beth Goins, University of Texas Health Science Center at San Antonio, Radiology Dept., San Antonio, TX 78229-3900; [email protected]
The goal of this paper is to discuss the uses of radiolabeled lipid nanoparticles, or liposomes, for scintigraphic imaging and radionuclide therapy. Use of radiolabeled liposomes as a tool in the development of liposome-based therapeutic agents will also be discussed. Methods for radiolabeling preformed lipid nanoparticles with single photon emitting radionuclides for both diagnostic applications (technetium-99m) and radionuclide therapy (rhenium-186) will be described. Examples of preclinical testing of radiolabeled liposomes using state-of-the-art dedicated small animal single photon emission computed tomography (SPECT) and computed tomography (CT) scanner will be given. These examples include non-invasive tracking and quantitation of the distribution of radiolabeled liposomes in rat tumor models following injection by intravenous, intratumoral and intraperitoneal routes.
E3 – Keynote Paper A NEW PARADIGM FOR LOCAL DRUG DELIVERY: TEMPERATURE 215 TRIGGERED RELEASE LIPOSOMES FOR ANTI-TUMOR APPLICATIONS David Needham, Duke University, CIEMAS, Science Drive, Durham, NC, 27708, USA; [email protected]
Invented in 1996, the temperature-sensitive liposome, is now in phase 1 human clinical trials. This presentation will recap earlier work, and present new data that shows unexpected mechanistic features that make this triggered release system an emerging new paradigm for local drug delivery, i.e., drug release in the blood stream, where drug gets access to tumor endothelia as well as to the neoplasm. Drug release from the liposomes occurs via grain boundary permeabilization when it is heated at its phase transition temperature, Tm, of 41C. This rate of release of an encapsulated drug like doxorubicin is enhanced 10X over that of the pure DPPC bilayer by the inclusion of 10mol% lysolipid. The lysolipid appears to not desorb from the membranes as initially postulated, but remains in the membranes stabilizing long lasting pores (when held at or close to Tm) through
172 Oral Abstracts which small molecules and drugs can freely diffuse. This is the basis for the temperature-triggered nanotechnology for drug release. As for drug action a series of preclinical studies in window chamber and flank tumors have established that the mechanism of action is one of vascular shut down because of the unique ability of Hyperthermia (HT) to release Doxorubicin from the liposomes intravascularly as they pass through the blood vessels of the mildly heated tumor. Thus, a new target for Doxorubicin delivered in this unique way appears now to be vascular endothelia, in addition to its cytotoxicity for cancer cells. We have established that the action of this drug-releasing liposome in combination with HT has both anti-vascular effects that reduce blood flow and eventually shut down the blood vessels, as well as anti-cancer cytotoxicity (due in part to 50% of drug delivered to the tumor being bound to DNA in the first hr after administration).
E3 – Invited Paper RETROGRADE DELIVERY OF BACTERIAL TOXINS PASSES 216 THROUGH THE RECYCLING ENDOSOME OF MAMMALIAN CELLS David Sheff, Department of Pharmacology, Carver College of Medicine, University of Iowa, 2-570 BSB, Iowa City, IA 52317, USA; [email protected]
All mammalian cells continuously internalize surface receptors and membranes through a process of constitutive endocytosis. Bacterial toxins, such as shigella toxin and cholera toxin exploit this process to gain entry into the cytoplasm. Shigella toxin B subunit binds to plasma membrane globoside and is rapidly delivered to early endosomes. Similarly, cholera toxin B subunit binds to GM1 gangliosides. Both are delivered in a non-clathrin dependent pathway to the early endosomes. Both are then targeted to the Golgi apparatus before reaching the ER. However, the pathway to the Golgi apparatus remains unclear. Here we find that both toxins pass from early endosomes, through the recycling endosome - en route to the Golgi apparatus. AlF4 treatment unexpectedly results in blockade of retrograde traffic from the recycling endosome thus trapping bacterial toxins prior to Golgi entry. Furthermore, in cytoplasts lacking a Golgi apparatus, shigella toxin is capable of accessing the cytoplasm directly from the recycling endosome without passage through Golgi apparatus or ER. The ability of the bacterial toxin to bypass the Golgi suggests that passage through the Golgi may in fact be a result of a host defense mechanism against bacterial toxins removing toxin from the endosomes before it can exit into the cytoplasm. These findings also suggest that in cases where Golgi entry is blocked, direct entry into the cytoplasm, from endosomal comparments, may be enhanced.
E3 – Contributed Paper POLYELECTROLYTE SELF-ASSEMBLY TO MODIFY SURFACE 217 PROPERTIES OF PROMAXX INSULIN MICROSPHERES Ramin Darvari1, Julia Rashba-Step1, Quinmin Lin1, Joan Kelly1,Tatsiana Shutava2, Yuri Lvov2, Terrence Scott1; 1Epic Therapeutics, Inc., A Wholly Owned Subsidiary of Baxter Healthcare Corporation, Norwood, MA, USA
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02062; 2Institute for Micromanufacturing, Louisiana Tech University, Ruston, LA 71272, USA; [email protected]
PROMAXX insulin microspheres are the product of a process developed at Epic Therapeutics, Inc., in which the microspheres are formed by controlled phase separation of the active agent from aqueous solutions of solubility reducing agents such as polyethylene glycol. These microspheres contain over 90% of the active agent with a narrow particle size distribution. This study aimed at developing a process to coat PROMAXX protein microspheres by means of polyelectrolyte deposition to modify the surface charge, and dissolution properties of the microspheres. Due to highly soluble nature of the uncoated microspheres, the polyelectrolyte deposition was performed in aqueous solutions of solubility reducing agents. The reaction medium consisted of 16% polyethylene glycol (PEG), 0.7% NaCl, and the polyelectrolyte at 0.15 to 1.5 mg/ml concentration range. The pH of the medium was ranging between 5.7 to 7.0. The microspheres were incubated for 1 hour in the reaction medium, and the unreacted polyelectrolyte was washed out by medium replacement. Surface charge modification of the microspheres was shown by comparing the zeta potential of uncoated PROMAXX insulin mirospheres (-18.0+6.0 mV) with the coated microspheres using protamine sulfate (19.1+9.1 mV), poly-l-lysine (35.3+3.5 mV), and poly-l-arginine (37.3+10.2 mV). Confocal microscopy of the microspheres coated with FITC-labeled polyelectrolytes also confirmed the success of the deposition process. The in vitro release study of the protamine-coated PROMAXX insulin microspheres showed a significant reduction of the particles solubility under simulated physiologic conditions. In vivo performance of the same microspheres was studied in rats receiving 1 IU/kg of the uncoated and coated formulations. The results revealed significant effect of the coating on the pharmacokinetic parameters of insulin, which in turn extended the duration of serum glucose depression by over 2.5 fold in comparison with the results obtained from the uncoated formulation.
E3 – Contributed Paper POLYMERSOME ENCAPSULATED HEMOGLOBIN (PEH): A NOVEL 218 TYPE OF OXYGEN CARRIER Dian R. Arifin, Department of Mechanical Engineering. Monash University, Clayton, VIC 3800, Australia, [email protected]; and Andre F. Palmer, Department of Chemical and Biomolecular Engineering, University of Notre Dame, Notre Dame, IN 46556, USA; [email protected], [email protected]
Bovine hemoglobin (Hb) was encapsulated inside polymer vesicles (polymersomes) to form polymersome encapsulated Hb (PEH) dispersions. PEHs are 100% surface PEGylated with longer PEG chains and possess thick hydrophobic membranes as compared to conventional liposomes. Polymersomes were self-assembled from poly(butadiene)-poly(ethylene glycol) (PBD-PEO) amphiphilic diblock copolymers with PBD-PEO molecular weights of 22-12.6 kDa, 5-2.3 kDa, 2.5-1.3 kDa and 1.8-0.9 kDa. The first two diblock copolymers possessed linear hydrophobic PBD blocks, while the later possessed branched PBD blocks. PEH dispersions were extruded through 100 and 200 nm
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pore radii membranes. The size-distribution, Hb encapsulation efficiency, P50, cooperativity coefficient and methemoglobin (metHb) level of PEH dispersions are consistent with the values required for efficient oxygen delivery in the systemic circulation. The influence of different molecular weight diblock copolymers on the physical properties of PEH dispersions was analyzed. PBD-PEO copolymers with molecular weights of 22-12.6 kDa and 2.5-1.3 kDa completely dissolved in aqueous solution to form polymersomes, while the other two copolymers formed a mixture of solid copolymer precipitates and polymersomes. PEHs self-assembled from 22-12.6 kDa and 2.5-1.3 kDa PBD-PEO copolymers possessed Hb loading capacities greater than PEG-LEHs, PEGylated actin-containing LEHs and non-modified LEHs, although their sizes were smaller and their hydrophobic membranes were thicker. The Hb loading capacities of these polymersomes were also higher than those of lipogel encapsulated hemoglobin particles and nanoscale hydrogel encapsulated hemoglobin particles. PEH dispersions exhibited average radii larger than 50 nm and exhibited oxygen affinities comparable to human erythrocytes. Polymersomes did not induce Hb oxidation. The interaction between Hb and the membrane of 2.5-1.3 kDa PBD-PEO polymersomes improved the monodispersity of these particular PEH dispersions. These results suggest that PEHs could serve as efficient oxygen therapeutics.
E3 – Invited Paper LIPOSOMAL DELIVERY OF ALL-TRANS-RETINOIC ACID: 219 CHALLENGES AND RESULTS Krzysztof Dziewiszek, Cristina Musselli, David O’Donnell, Lavesh Gwalani, Daniel Levey, Roman M. Chicz, Antigenics Inc., 3 Forbes Rd., Lexington, MA 02421 [email protected]
Tretinoin (all-trans Retinoic acid) is a known anticancer agent used in the treatment of Acute Promyelocytic Leukemia (APL) to cause maturation of promyelocytic cells, triggering a repopulation of bone marrow and peripheral blood with normal hematopoietic cells. It is currently supplied as Vesanoid® in gel capsules for oral administration. However, its oral administration is hampered by erratic plasma concentrations and in continuous treatment, by a progressive decrease in plasma concentration. Due to potentially severe side effects (RA-APL syndrome), Vesanoid’s use has to be monitored and it should be administered in a facility with available supportive services to manage the cases with severe intolerance. This greatly diminishes the benefits of oral administration. On the other hand, parenteral formulation, which would circumvent the plasma concentration variability, is difficult to develop due to minimal water solubility of tretinoin. Liposomes are well known vehicles suitable for lipophilic drugs that do not adversely affect the activity of Active Pharmaceutical Ingredient (API) and this technology was proven in the cases of doxorubicin and amphotericin B. Liposomal formulation of tretinoin, which contains Dimyristoylphosphatidylcholine (DMPC) and small amounts of soybeen oil has been developed. Administration of this formulation circumvents problems resulting from the oral drug’s availability and its toxicity, which is associated with the presence of free tretinoin. Biological activity of two tretinoin formulations will be discussed.
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E3 – Invited Paper TRANSFERRIN-LOADED NIDO-CARBORANE LIPOSOMES: 220 SYNTHESIS AND INTRACELLULAR TARGETING TO SOLID TUMORS FOR BORON NEUTRON CAPTURE THERAPY Hiroyuki Nakamura, Yusuke Miyajima, Department of Chemistry, Faculty of Science, Gakushuin University, Tokyo 171-8588, Japan; Shinichiro Masunaga, Koji Ono, Research Reactor Institute, Kyoto University, Osaka, 590-0494, Japan; Yasuhiro Kuwata, and Kazuo Maruyama, Biopharmaceutics, School of Pharmaceutical Science, Teikyo University, Kanagawa 199-0195, Japan; [email protected]
The nido-carborane lipid 2 which has a double-tailed alkyl moiety was synthesized from heptadecanol by 5 steps. Analysis in a transdmission electron microscope by negative staining with uranyl acetate showed that the lipid 2 formed a stable vesicle in which calcein was encapsulated. Furtheremore, the lipid 2 formed a stable boron liposome at 25% molar ratio toward DSPC with cholesterol after sizing by a extruder. Transferrin was able to be introduced on the surface of the boron liposome (Tf-PEG-CL-liposome) by coupling of transferrin to PEG-CO2H moieties of PEG-CL liposomes. The biodistribution of Tf-PEG-CL-liposome, in which 125I-thylaminyl inulines were encapsulated, exhibited that Tf-PEG-CL-liposome was accumulated into tumor tissue and stayed for a prolonged time enough to increase a ratio of the tumor/blood concentration, although PEG-CL-liposome was gradually released from tumor tissue along with the time course. The high accumulation and long retention time in tumor tissue was due to a transferrin receptor-mediated endocytosis and 22 ppm of 10B concentration in tumor tissue was achieved by injection of 10B-enriched Tf-PEG-CL-liposome at 7.2 mg 10B/kg in tumor-bearing mice at 72 h after administration of the liposome. The mice were anesthetized with sodium pentobarbital solution, placed in an acrylic mouse holder, where whole bodies of mice except the tumor-implanted leg were shielded with the acrylic resin, and irradiated in the KUR atomic reactor for 37 min at a rate of 2 x 1012 neutrons/cm2. The average of survivals of mice without treated with the Tf-PEG-CL-liposome was 21 days, whereas that of mice treated with the Tf-PEG-CL-liposome was 31 days. The longer survivals were observed in mice treated with the Tf-PEG-CL-liposome and one of them survived for 52 days after BNCT.
J2 – Keynote Paper NUCLEIC ACID MICROSPHERES FOR TYPE 1 DIABETES 221 INTERVENTION Larry Brown1, Kimberly Gillis1, Joseph Worrall1, Nick Giannoukakis 2, Janet Knox1, Jennifer Machen2 1Epic Therapeutics, Inc., A Wholly Owned Subsidiary of Baxter Healthcare Corporation, Norwood, MA, 02062, 2University of Pittsburgh School of Medicine, Diabetes Institute, Rangos Research Center, Pittsburgh, PA, 15213, USA; [email protected]
Type 1 diabetes mellitus occurs as a result of the autoimmune destruction of the insulin producing beta cells of the pancreas. This is mediated by macrophages and T cell-mediated
176 Oral Abstracts destruction of the beta cells. CD40, CD80 and CD86 surface cell antigens expressed on the dendritic cells (DC) are crucial regulators of T cell activation. Our goal was to form antisense (AS)-oligonucleotide PROMAXX microspheres targeting the primary transcripts of CD40, CD80 and CD86 in order to prevent autoimmune insulin-dependent diabetes in the NOD mouse. Three phosphothioated AS-oligonucleotides were dissolved in an aqueous solution and combined with polylysine and water-soluble polymers such as polyethylene glycol and polyvinylpyrollidone. The solutions containing the polymer, the antisense oligonucleotides and the polycation were incubated at 58 to 70°C, and cooled to 23°C. Excess polymer was removed by washing with deionized water. The microspheres were about 1 to 3 microns in diameter by light scattering. In-vivo studies treated one group of NOD mice with DC that had been treated ex-vivo with AS-oligonucleotides directed against CD40, CD80 and CD86. Group 2 NOD mice were injected with AS-oligonucleotide PROMAXX microspheres. Untreated controls were injected with DC not exposed to AS-oligonucleotides. In vivo results showed that all of the untreated DC control mice developed diabetes by 23 weeks. 20% of the DC AS-oligonucleotide treated mice remained diabetes-free at 40 weeks. 71% of the AS-oligonucleotide PROMAXX microsphere treated group remained diabetes free at 40 weeks. This study showed that the PROMAXX delivery system enabled superior in-situ delivery of the AS-oligonucleotides and significantly enhanced the therapeutic efficacy of diabetes suppression. The microspheres provided effective uptake of AS-oligonucleotides by DC resulting in the prevention of diabetes in NOD mice by inducing immune tolerance. The PROMAXX AS-oligonucleotide microsphere approach is a promising new preventative treatment for Type 1 diabetes and other autoimmune diseases.
J2 – Invited Paper DEVELOPMENT OF NANOTRANSFECTION COMPLEXES FOR 222 SYSTEMIC TREATMENT OF CANCER Sean M. Sullivan, Department of Pharmaceutics, University of Florida College of Pharmacy, Gainesville, FL 32610-0494, USA; [email protected]
Binding of plasmid DNA to cationic amphiphiles converts the plasmid from a random coil with an approximate radius of gyration of 100nm to a toroid composed of multiple plasmid DNA molecules with a 80 nm radius. This condensed DNA form the basis for the nanoplex. Systemic administration of nanoplexes results in distribution of plasmid DNA into the major organs, including the lung, liver, spleen, heart, muscle and kidneys with the lungs yielding the highest level of gene expression followed by the heart, muscle and spleen. Using this strategy to treat vascularized tumors will require selective gene transfer to the tumor and avoid uptake or at least expression in the non-tumor tissue. In vivo studies have also shown that the nanoplexes can not extravasate out of the blood stream or if so, not very efficiently. Hence, the tumor vasculature is the primary target for treating the cancer that will lead to the destruction of the vasculature itself and/or secretion of cytotoxic proteins into the tumor from the tumor endothelial cells resulting in tumor cell killing. Coating the surface of the nanoplexes with peptides that bind to receptors on the tumor endothelial cells can achieve selective gene transfer to the tumor cells. Peptides that bind
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to CXCR4, a chemokine receptor for stromal derived factor-1 (SDF-1), and basic fibroblast growth factor receptor (bFGFR) with nanomolar binding affinity have been derivatized to phospholipid analog and incorporated into nanoplexes composed of a dilaurylspermine coformulated with a mixture of lyso-phosphatidylcholine, monoacylglycerol and fatty acid. CXCR4 is expressed on proliferating endothelial cells and the receptor density is dramatically increased in a positive autocrine feedback loop by vascular endothelial derived growth factor (VEGF). Receptor mediated transfection of both endothelium and brain cancer cells are demonstrated using a combination of tissue culture and blood vessel arteriograph organ cultures. These peptide targeted nanoplexes will deliver a cytotoxic gene and the expression is controlled by a proliferating endothelial cell promoter to further restrict expression to the tumor vasculature. A cytotoxic gene has been engineered to yield a secreted gene product that is composed of a cytotoxic domain and a membrane permeability domain. The cytotoxic domain is a 14 amino acid sequence derived from P19ARF that binds Mdm 2 and inhibits ubiquination of proteins, including P53, a tumor suppressor protein. The membrane permeability domain is also composed of a 14 amino acid sequence derived from HIV-Tat. Peptide modeling studies of the expressed gene product yielded an IC50 of 3 uM. Transfection of human brain cancer cells with an expression plasmid encoding the bifunctional cytotoxic protein resulted in 50% cell death with only 8% of the cells transfected. This could be increased to 90% cell killing when transfection efficiencies were increased to 18%. Furin, a protease secreted by both human and rat brain cancer cells was shown to hydrolyze the cytotoxic gene product into discrete bands thus being a potential inactivator. Addition of a recombinant furin inhibitor increased cell killing as a function of dose with the highest dose yielding a 3 fold increase in cell killing. These studies have identified and characterized a biochemical and molecular biological targeting system for treatment of vascularized tumors, specifically brain cancer. A synthetic gene has been created that kills cells through a bystander effect thus increasing the therapeutic effectiveness by impacting the neighboring untransfected cells surrounding the transfected endothelial cells.
J2 –Contributed Paper ENHANCEMENT OF THE EFFICIENCY OF NON-VIRAL GENE 223 DELIVERY BY APPLICATION OF PULSED MAGNETIC FIELD S.W. Kamau, P.O. Hassa, B. Steitz, A. Petri-Fink, H. Hofman, M. Hofman, B. von Rechenberg, and M.O. Hottiger, Institute of Veterinary Biochemistry and Molecular Biology, University of Zurich, Winterthurerstr. 190, CH-8057 Zurich, Switzerland; [email protected]
Non-viral gene vectors have several advantages, however, the transfection efficiencies remain lower than those with viral vectors and a need exists to enhance the efficiency of non-viral gene delivery. Superparamagnetic nanoparticles (SPIONs) based on iron oxide, have an advantage in gene delivery since their combination with magnets that can draw particles to a desired location and keep them at a specific site. We explore the use of smaller polyethylenimine (PEI) coated SPIONs, use of novel shorter DNA fragments (PCR products) instead of plasmids and application of a pulsating magnetic field, on enhancing green fluorescent protein (GFP) gene delivery. Our results show that smaller 50
178 Oral Abstracts nm PEI coated SPIONs were as effective as the larger 200-250 nm nanoparticles, however they required more time to sediment on the cells after exposure to a magnetic field. The transfection rates achieved with SPIONs within 5 min were significantly higher than those achieved with conventional transfection methods. This was also observed when novel PCR products containing only the sequence of the gene of interest, were used. The application of a pulsating magnetic field, after sedimentation of the particles with a static magnetic field increased the transfection efficiency significantly, over the magnetofection. In conclusion, we show that PCR products are effective gene vectors when coupled to SPIONS and are attractive alternatives to plasmids. The application of a pulsating magnetic field enhances the already high transfection rates in presence of static magnet. Smaller SPIONS are as effective as large particles and would be more suited in future in vivo studies.
J2 – Invited Paper SYNTHESIS, CHARACTERIZATION & TRIGGERED DETHREADING 224 OF CYCLODEXTRIN-POLYETHYLENE GLYCOL POLYROTAXANES BEARING CLEAVABLE ‘CLICK’ ENDCAPS David H. Thompson, Scott Loethen, Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, IN 47907, USA; Tooru Ooya and Nobuhiko Yui, Japan Advanced Institute of Science and Technology, School of Materials Science, 1-1 Asahidai, Nomi, Ishikawa, 923-1292, Japan; [email protected]
We report the synthesis, characterization and degradation kinetics of three α-cyclodextrin (α-CD)-polyethylene glycol (PEG) polyrotaxanes with endcapping groups that were installed using Cu (I)-catalyzed Huisgen cyclization (a.k.a., “click” reaction). PEG1500, aminated at the α- and ω-termini of the polymer with azidoacetic acid, was used to thread α-CD onto the polymer chain to form a polypseudorotaxane. The polypseudorotaxane was then endcapped in up to 82% yield with three different substituents to provide polyrotaxanes that were either acid-, base- or fluoride-sensitive. NMR, GPC, AFM, and XRD were used to characterize the polyrotaxanes. Turbidity analysis was used to monitor their dethreading rates upon exposure to mild deprotection conditions. Rates of solubilization triggered by endcap hydrolysis and polyrotaxane dethreading were found to be pH dependent. The vinyl ether endcapped polyrotaxane is stable at pH 7 for 16 h, but is solubilized at approximately 0.0211 min-1 at pH 4. The ester-endcapped polyrotaxane is rapidly solubilized at pH 12.1 with a rate of 0.0122 min-1. These results show that pH-triggerable polyrotaxanes can be readily and efficiently prepared from polypseudorotaxanes in high yield by Huisgen cyclization of azido- and alkynyl-modified precursors in the presence of Cu(I).
J2 –Contributed Paper TARGETED NEURONAL DELIVERY OF A SINGLE ANTIVIRAL SIRNA 225 PROTECTS MICE AGAINST ENCEPHALITIS CAUSED BY TWO DISTINCT FLAVIVIRUSES Sang-Kyung Lee, Department of Bioengineering, Hanyang University, 17
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Haengdang-dong Seongdong-gu, Seoul 133-791, Korea; Priti Kumar, Premlata Shankar and N. Manjunath Swamy, CBR Institute for Biomedical Research, Harvard Medical School, 800 Huntington Ave, Boston, Massachusetts 02115, USA; [email protected], [email protected]
Japanese encephalitis (JE) and West Nile (WN) viruses are neurotropic flaviviruses that can cause acute encephalitis with a high degree of fatality. Infections by diverse neurotropic flaviviruses are clinically indistinguishable and currently there are no effective drugs to treat these infections. Thus it is important to develop therapeutic approaches that are effective against multiple viruses within and across the flaviviral species. In this study, we employed RNA interference (RNAi) targeting a cross-species conserved sequence in the flaviviral genome as a broad-based intervention against flavivirus infection. By using a lentiviral vector pseudotyped with the rabies virus glycoprotein (RVG) that enabled specific and targeted delivery of antiviral siRNA to neurons we were able to completely abrogate flaviviral replication in the neuroblastoma cell line, Neuro 2a. Moreover, a single administration of vector-delivered short-hairpin RNA was able to completely protect mice against a lethal intracranial challenge with as high as 50LD50 of virus. RVG-mediated targeting also resulted in better levels of protection compared to the conventional methods of delivery using the Vesicular Stomatitis virus glycoprotein, possibly due to better retro-axonal spread attributed to RVG. As a further demonstration of the feasibility of an RNAi-based therapy for viral encephalitis, we used cationic lipid formulations that mediate effective brain delivery of short interfering (si)RNA. A single intracranial application of lipid-complexed siRNA targeting conserved viral sequences before viral challenge and more importantly, as late as 18 hours after viral challenge was sufficient for protection of mice against lethal encephalitis induced by both JEV and WNV. These results demonstrate the practicality of using lipid-based delivery of single siRNA targeting a conserved sequence as a broad-based therapeutic strategy against multiple related viruses. Efforts are currently underway towards developing a nanoparticle-mediated approach for targeted brain delivery following peripheral administration of this common siRNA.
J2 – Invited Paper RECEPTOR-MEDIATED GENE DELIVERY USING CHITOSAN 226 DERIVATIVES IN VITRO AND IN VIVO Chong Su Cho, Tae Hee Kim, School of Agricultural Biotechnology, Seoul National University, Seoul 151-742, South Korea; Hua Jin, Hyun Woo Kim, and Myung-Haing Cho, College of Veterinary Medicine, Seoul National University, Seoul 151-742, South Korea; [email protected]
Chitosan has been considered to be a good candidate for gene delivery system, since it is already known as a biocompatible, biodegradable, and low toxic material with high cationic potential. However, low specificity and low transfection efficiency of chitosan need to be overcome prior to clinical trial. In this study, we focused on the chemical modification of chitosan for enhancement of cell specificity and transfection efficiency. To
180 Oral Abstracts induce the receptor-mediated endocytosis into liver cells, chitosan was coupled with lactobionic acid (LA) bearing galactose group as the specific ligand to asialoglycoprotein receptor (ASGP-R). Also, mannosylated chitosan (MC) was prepared by coupling chitosan with mannopyranosylphenylisothiocyanate bearing mannose group to induce the receptor-mediated endocytosis into dendritic cells (DCs). And their physicochemical properties, morphology, cytotoxicity, and transfection efficiency of the complexes in vitro and in vivo were studied. The potential of antitumor effect in vivo by using MC for delivery of IL-12 genes was also evaluated. Transfection efficiency of GC/DNA complex into HepG2 which has ASGP-R was higher than that into HeLa without ASGP-R. MC had low cytotoxicity and exhibited much enhanced gene transfer efficiency on the macrophage cell lines compared with chitosan itself. Increased IL-12 production by DC induced increase of IFN-γ production. In vivo study shows that intratumoral delivery of IL-12 genes using MC is an effective method of generating antitumor immunity. In conclusion, it is expected that relatively non-toxic GC and MC is suitable for repeated administration to maintain sustained gene expression, thereby opening the possibility for cancer gene therapy.
J2 – Invited Paper NANOPARTICLE-MEDIATED DELIVERY OF TUMOR SUPPRESSOR 227 GENES Rajagopal Ramesh 1, Lin Ji 1, Began Gopalan 1, Jack A. Roth 1, Louis A. Zumstein2, 1University of Texas MD Anderson Cancer Center, Houston, TX, 2 Introgen Therapeutics, Houston, TX 77030, USA; [email protected]
Nanoparticle delivery of tumor suppressor genes is a promising approach for the treatment of cancer. Our nanoparticles comprise a therapeutic tumor suppressor gene surrounded by a lipid bilayer. This lipid bilayer allows for systemic delivery of gene therapies, without generating an immune response, as well as potential modification of the surface layer of the nanoparticle. We have developed a novel nanoparticle which delivers the Fus1 tumor suppressor gene, which has completed preclinical testing and is undergoing clinical testing in cancer patients. Fus1 was identified as a tumor suppressor gene in a region of chromosome 3p21 which is frequently deleted in human lung and breast cancers. FUS1 protein is frequently not expressed in lung cancer, and expression of FUS1 protein induces apoptotic cell death in cancer cells. Sequence analysis suggests that FUS1 has a protein kinase A domain, a protein-protein interaction domain, and a myristolyation domain. The lipid is a 1:1 (molar) mixture of DOTAP and cholesterol. The formulation steps, which include extrusion, result in a DNA-lipid nanoparticle with a unique bilamellar vase-like structure, which is taken up by cells by phagocytosis. In animal studies this nanoparticle allowed for high efficiency of delivery and a broad tissue distribution. The nanoparticle is also not immunogenic, a potential advantage over viral gene therapy vectors. In animal models of cancer, nanoparticle FUS1 demonstrated potent antitumor activity in solid primary tumors (delivered intratumorally) and metastatic lesions (delivered intravenously); antitumor activity was demonstrated by the complete inhibition of growth of solid tumors, a dramatic decrease in the number of metastatic lesions, and increased survival of the mice. A Phase 1 intravenous dose-escalating safety study is underway at University of Texas MD Anderson Cancer center with nanoparticle Fus1 in non-small cell
181 Oral Abstracts lung cancer patients; to date nanoparticle-Fus1 is well tolerated, and the dose-escalation is continuing.
A6 – Invited Paper GLUCOSE-SENSING MICROGELS 228 Todd R. Hoare and Robert H. Pelton, McMaster Centre for Pulp and Paper Research, Department of Chemical Engineering, McMaster University, 1280 Main St. W., Hamilton, Ontario, Canada L8S 4L7; [email protected]; [email protected]
Hydrogels which can reversibly respond to both physical and chemical stimuli have great potential as switchable chemoselective probes and feedback-controlled delivery vehicles for drug or macromolecule transport. Microgels with similar functionality are even more attractive since they respond much faster and more predictably to external stimuli and can be administered readily via flow processes. We have synthesized amphoteric poly(N-isopropylacrylamide) (PNIPAM)-based microgels with covalently tethered phenylboronic acid (PBA) functional groups. Phenylboronic acids form reversible covalent complexes with carbohydrates containing cis-diol groups, such as glucose; thus, PBA-microgels are tunably responsive to pH, temperature, ionic strength, and glucose concentration. The thermal phase transition of the PNIPAM backbone can be used to amplify the electrostatics-driven gel swelling induced by glucose binding, while the zwitterionic nature of the microgel surface maintains the colloidal stability and glucose responsiveness of the microgels in the high-temperature, high-salt physiological environment. The amphoteric nature of the microgels also permits the design of microgels which can both swell and shrink in response to increases in the glucose concentration according to the pH of the medium. The potential applications of such microgels as turbidimetric glucose sensors, “smart”, self-regulating insulin-delivery vehicles for diabetes treatment, and selective cell bioflocculants have been demonstrated.
A6 – Invited Paper PEPTIDE NANOPARTICLES FOR MEDICAL APPLICATIONS: NOVEL 229 DRUG TARGETING, DELIVERY AND VACCINATION STRATEGIES Alexandra Graff, Senthilkumar Raman, Gia Machaidze, Ariel Lustig, M.E. Müller Institute for Structural Biology, Biozentrum, University of Basel, Switzerland; and Peter Burkhard, The Institute of Materials Science, University of Connecticut, 97 North Eagleville Road, Storrs, CT 06269-3136, USA; [email protected]
Artificial particulate systems such as polymeric beads and liposomes are finding multiple applications in drug delivery, drug targeting, antigen display, vaccination, and other technologies. Here we describe the structure-based design of a novel type of nanoparticles with regular polyhedral symmetry and a diameter similar to small viruses, which self-assemble from single polypeptide chains. Each peptide chain is composed of two protein domains with trimeric and pentameric oligomerization states that are joined into
182 Oral Abstracts one single peptide chain by a short linker segment. E.g. a de-novo designed trimeric coiled-coil domain is joined to the pentameric coiled-coil domain of COMP to give a nanoparticle with a size of about 17 nm. These computer designs are verified by structural and biophysical analyses including electron microscopy, analytical ultracentrifugation, CD-spectroscopy, dynamic light scattering and many more. These nanoparticles represent a novel type of nanomaterial ideally suited as drug targeting and delivery systems. Since they are composed of peptides they are fully biocompatible and biodegradable. Furthermore, since they are built from protein domains this allows for a highly specific design in terms of shape, size and stability of the nanoparticles. Their modular composition enables an easy and rapid design and generation of different combinations of drugs and targeting entities. Hence these nanoparticles can be used as a drug targeting toolbox: different drugs (e.g. radioisotopes, doxorubicin, etc.) can be combined with different targeting entities (e.g. somatostatin, bombesin, etc.) in an easy and straight-forward manner. Furthermore, such nanoparticles with regular polyhedral symmetry represent an ideal repetitive antigen display system. Surface proteins of pathogens or fragments of such proteins can easily be engineered into the peptide sequence of the nanoparticle. Notably, the surface proteins of enveloped viruses contain a trimeric coiled-coil sequence essential for their cell penetration mechanism. For example, by extending the trimeric coiled-coil of the nanoparticle by the respective coiled-coil sequences of HIV, influenza, Ebola, or another enveloped virus, a subunit vaccine against these viruses can be designed. Finally, since they are composed of small protein oligomerization domains they are easy to synthesize by standard recombinant E. coli protein expression systems. The vaccines can be produced in one single manufacturing process without the need of any additional chemical modifications and, as opposed to most virus like particles, they are stable without the need of DNA packaging inside.
A6 – Contributed Paper PREPARATION OF MONOFUNCTIONAL GOLD 230 NANOPARTICLE-DENDRIMER (PAMAM) CONJUGATES BY COVALENT APPROACH Qiu Dai, James G. Worden, Xiong Liu, Jinhai Wang, Jianhua Zou, and Qun Huo, Nanoscience Technology Center and Department of Chemistry, University of Central Florida, 12424 Research Parkway Suit 422, Orlando, FL, USA; E-Mail: [email protected]
The synthesis and study of gold nanoparticles (NPs) is a major area of current nanomaterials research. Due to their unique optical and electrical properties, gold nanoparticles may potentially be applied in catalysis, sensing, environmental, Surface Enhanced Raman Scattering, nonlinear absorption processes, and optoelectronics. There are two significant challenges which first must be overcome; the first is the synthesis of the gold nanoparticles with the proper size and surface functionality. The second is how to assemble the gold nanoparticles together with desired properties. Recently we report our work on the preparation of monofunctional gold nanoparticles using a solid phase synthesis method. Compared to multifunctional gold nanoparticles, these nanoparticles offer greater versatility for the preparation of more complex nanostructures. Gold
183 Oral Abstracts nanoparticles with a single functional surface group may be treated like regular molecules and used in traditional organic chemistry reactions. Herein we report that gold nanoparticles-dendrimer conjugate clusters were prepared by the covalent coupling of monofunctional gold nanoparticles with a generation 5 PAMAM dendrimer. Monocarboxylic group modified gold nanoparticles with an average core diameter of 2.8 nm were covalently coupled with 5 PAMAM dendrimer which has 128 primary amine groups on its surface. TEM images show that most of the conjugates consisted of 5-10 nanoparticles with a diameter of 10-13 nm. This work demonstrates that the potential of convent bond chemistry in the bottom-up approach towards the nanomaterials development. This approach can be readily extended to the fabrication of other types of more sophisticated nanomaterials with well-controlled structures and properties for biomedical applications such as drug delivery and molecular imaging purposes.
A6 – Invited Paper CHLOROTOXIN-BOUND SUPERPARAMAGNETIC NANOPARTICLES 231 FOR BRAIN TUMOR DIAGNOSIS AND THERAPEUTICS Omid Veiseh, Conroy Sun, Jonathan Gunn, Nathan Kohler, Narayan Bhattarai, Donghoon Lee, Raymond Sze, Richard.Ellenbogen, Andrew Hallahan, Jim Olson, Miqin Zhang, Department of Materials Science and Engineering, University of Washington, Seattle, WA 98195, USA; [email protected]
A bifunctional nanoconjugate for targeting gliomas and inhibiting tumor migration was developed by coating iron oxide superparamagnetic nanoparticles with chlorotoxin (Cltx). The preferential binding of Cltx-conjugated nanoparticles to glioma cells, as compared to dextran-coated nanoparticles, was evaluated qualitatively by magnetic resonance imaging (MRI) and quantitatively by inductively coupled plasma atomic emission spectroscopy (ICP-AES). The specificity of glioma-targeting was evaluated through a comparison of the nanoparticle binding to glioma cells that express matrix metalloproteinase 2 (MMP-2) versus healthy cells that do not express detectable MMP-2. The ability of the nanoparticle conjugate to inhibit cell migration was demonstrated by the matrigel invasion test in which Cltx-conjugated nanoparticles showed similar efficacy to free Cltx at a concentration lower than that of free Cltx peptide. This indicates that the Cltx retained its bioactivity after conjugation to the nanoparticle and that the nanoparticles enhanced the cellular uptake and retention of Cltx in the target cells. The Cltx conjugated nanoparticles can be potentially used for highly sensitive detection of glioma tumors and targeted therapy in the central nervous system.
A6 – Invited Paper FUNCTIONALIZATION OF NANOPARTICLES FOR DISEASE 232 DETECTION AND TARGETED DRUG DELIVERY. Lucienne Juillerat-Jeanneret, Feride Cengelli, University Institute of Pathology, Bugnon 25, 1011, Lausanne, Switzerland; Heinrich Hofmann, Alke Petri-Fink, EPFL, 1015, Ecublens, Switzerland; Justyna Grzyb, Stephen
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Hanessian, University of Montreal, Canada; [email protected]
Super Paramagnetic Iron Oxide Nanoparticles (SPIONs) combined with MRI are under clinical evaluation to enhance disease detection. A major improvement would be to link therapeutic drugs or targeting agents to the SPIONs to achieve targeted drug delivery, either at the cell surface or intracellularly, together with active and selective disease detection, without inducing cell reaction. Our objectives are to define the characteristics of SPIONS able to achieve cell-specific interaction with tissue structures and to develop the necessary chemistry to achieve these goals. Our system consists in an ironoxide core (9-10 nm diameter) coated either with dextran or various functionalized polyvinylalcohol (PVA). We investigated the cellular uptake, the cytotoxicity and the interaction of these various nanoparticles with tumor cells in aggregate cultures, tumor-associated stromal cells and brain-derived endothelial cells, microglial cells and differentiating 3-dimensional aggregates. None of the nanoparticles coated with dextran or the various PVAs was cytotoxic or induced the production of the inflammatory mediator NO used as a reporter for cell activation. Only aminoPVA-SPIONs were taken up by the cells, but did not invade cell aggregates lower than the first cell layer or induced microglial cell activation and NO production in the aggregates. Fluorescent aminoPVA-SPIONs derivatized with a fluorescent reporter molecule and confocal microscopy demonstrated intracellular uptake by microglial cells. Therefore functionalized aminoPVA-SPIONs represent biocompatible potential vector systems for drug delivery which will not produce massive tissue invasion and can be combined with MRI detection of active lesions in diseases. In order to develop therapeutics-derivatized-SPIONs and to couple defined molecules for targeted detection and drug-delivery purposes, we have designed and synthesized a multivalent linker, to which drugs are covalently coupled via a biologically labile linkage before the preparation of derivatized SPIONs.
A6 – Contributed Paper PRECIPITATION OF AN AMORPHOUS PHARMACEUTICAL SOLID 233 Hsien-Hsin Tung, Elizabeth Kwong, Aaron Cote, Brian Johnson, Cindy Starbuck, Alex Chen, Chad Dalton, Sophie-Dorothee Clas, Wayne Mullett, Robert Papp, Bernard Cote, Xiaoyi Gong and Theresa Natishan, Merck Company, Rahway, NJ 07065; [email protected]
Amorphous solids (glasses) can play an important role in improving bioavailability, especially for water insoluble pharmaceutical compounds. A traditional method for the generation of amorphous solids is via precipitation. In this study, we investigate the confined impinging jet mixer for the precipitation of an amorphous pharmaceutical solid. Impinging jet provides scalable rapid micro-mixing between the batch solution (DMSO) and anti-solvent (water), which generates a controlled local high supersaturation environment for the precipitation. Conversion of the amorphous solid to crystalline solid during the precipitation was effectively minimized by operating the system near the freezing point of solvent mixture without adding additives. The process was scaled up successfully from the grams scale to the hundred grams scale. Physically, the resulting amorphous solid shows a 30-fold increase in surface area (amorphous: 37.9 m2/gram vs.
185 Oral Abstracts crystalline 1.3 m2/gram), a 15-fold increase of aqueous solubility (amorphous 0.5 μg/mL vs. crystalline 0.03 μg/mL) and a 75-fold increase of solubility in DMSO/water 1/1 solvent mixture (amorphous 0.23 mg/ml vs. crystalline 0.003 mg/ml). The amorphous impinging jet precipitate further shows a significant improvement of in-vivo performance with a 10-fold increase in bioavailability. The dry amorphous solid with a high glass transition temperature of 150°C, is physically and chemically stable under ambient conditions and was found to be acceptable for development into a solid oral dosage form to support Phase I studies.
A6 – Contributed Paper SEMICARBAZIDE FUNCTIONALIZED SILICATE NANOPARTICLES 234 FOR PEPTIDE LIGATION Monique Smaïhi, Tristan Doussineau, IEM, UMR 5635 CNRS-ENSCM-UM2, 1919 route de Mende, 34 193 Montpellier Cedex 5, France ; and Ouafâa El-Mahdi, Rémi Desmet, Christophe Olivier, Oleg Melnyk, Biological Institute of Lille, UMR 8525 CNRS/Université de Lille 2/Institut Pasteur de Lille, 1 rue du Pr Calmette 59021 Lille Cedex, France ; and Jean-Olivier Durand, CMOS, UMR 5637, case 007-Université Montpellier II, Place Eugène Bataillon, 34095 Montpellier Cedex 05 France; [email protected]
The versatility of silica in synthesis aspects as well as surface modifications offers a great advantage to the use of the material in bioanalysis. Therefore, we have used monodisperse silicate nanoparticles to develop a site-specific ligation method which enables the control of the biomolecule orientation at the solid–liquid interface. Two types of zeolite colloidal nanoparticles were used for peptide anchoring : zeolite beta which posses a Si/Al ratio of 25 and silicalite-1 which is a fully silicic material. Zeolite-type materials are microporous silicate with pores of a well-defined size ranging from 2 to 20 Å. This discrete pore system gives molecular sieve properties to these materials. Semicarbazide-functionalized (SC) silicate colloids have been prepared for the site-specific ligation of alpha oxo aldehyde (COCHO)-modified polypeptides. The obtained colloidal suspensions have been characterized by complementary techniques providing information on the size distribution, morphology, chemical nature of the grafting and porosity of the particles. The specificity of the particles surface for the SC group has been studied by fluorescence spectroscopy with two peptides labelled with rhodamine. The first peptide bares a COCHO functionality, which should bind covalently with the semicarbazide surface. The second peptide bares an amine end-group which should interact by non specific adsorption with the surface. The results demonstrated that the peptide/colloids reactivity is dramatically influenced by the chemical composition of the particles surface. Indeed, while zeolite beta (aluminosilicate) particles react indifferently with the two peptides, silicalite particles (pure silicate) anchor exclusively the peptide bearing the COCHO functionality. This particular physisorption phenomenon of zeolite beta, is attributed to the aluminum atoms present at the particles surface which have a specific affinity for peptides. Since fluorescent molecules have been confined in the microporous zeolite cavities, these nanoparticles could find applications in immunodiagnostics and drug delivery.
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A6 – Contributed Paper INTEGRATION OF MAGNETIC NANOPARTICLES INTO 235 TEMPERATURE RESPONSIVE HYDROGEL SYSTEMS Reynolds A. Frimpong and J. Zach Hilt Department of Chemical and Materials Engineering, University of Kentucky Lexington, KY, USA; [email protected]
Magnetic nanoparticles have been integrated with temperature responsive hydrogel systems to form composites expected to have wide applicability in various biomedical applications. These systems particularly show great promise as active components of micro- and nanoscale devices. Several N-isopropylacrylamide systems were synthesized by UV free radical polymerizations with varying crosslinking densities and lengths based on ethylene glycol dimethacrylate, tetraethyleneglycol dimethacrylate and polyethylene glycol 400 dimethacrylate. For the first composite system, magnetic nanoparticles were incorporated into a hydrogel matrix by mixing them with the monomer solutions prior to polymerization. Additionally, core-shell composites were prepared through surface initiated polymerization of hydrogels on core magnetic nanoparticles. The various composite hydrogel systems were characterized for their swelling response to environmental temperature. Also, the response of these composites to the application of an external oscillating magnetic field and the resultant heating was characterized as a function of the amount of particles present in the composite.
A6 – Keynote Paper ENGINEERED NANOPARTICULATE SYSTEMS FOR BIOMEDICAL 236 APPLICATIONS Brij Moudgil, P. Sharma, S. Brown, V. Krishna, D. Dutta and S. Santra*, Department of Materials Science & Engineering, and Particle Engineering Research Center, University of Florida, Gainesville, Florida 32611, USA; [email protected]; *University of Central Florida, Orlando, Florida, USA
The development of innovative nanoparticulate-based systems for next-generation processes and devices requires cross-disciplinary expertise. Through cross-disciplinary collaborations, advances have been made in manipulating particulate-based systems at the molecular/nanoscale for enhanced performance in biomedical applications. Novel techniques for nanoparticle synthesis and surface modification have led to progress in enhanced drug delivery, drug detoxification, and multi-faceted bio imaging. In this presentation, highlights of significant advances made in targeted areas such as photostable nanoparticle-based imaging systems, pulmonary therapeutics, nanoparticle toxicity, engineered nanocomposites for biohazard reduction, and microemulsion technology in detoxification of drugs in the human body will be discussed.
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K2 – Keynote Paper FACTORS TO CONSIDER IN DEVELOPING A DRY POWDER INHALER 237 David L. Gardner and Robert A. Casper, Respirics Inc.-6008 Triangle Dr. Suite 101 Raleigh, NC 27617, USA; [email protected]
Although metered dose inhalers (MDIs) are the mainstay of delivering drugs topically to the lungs for the treatment of asthma and COPD, DPIs are being actively pursued because of two primary factors, i.e., the ban imposed upon CFC-based MDI products and the emergence of GSK’s Advair Diskus® DPI combination product for the treatment of asthma and COPD. Although several CFC-based MDIs have been reformulated into HFA-MDIs, DPIs have certain attributes that make them a viable alternative to the MDI, i.e., the elimination of environmental concerns related to CFC- or HFA-based products and the common coordination issue associated with MDI use. One issue with the current marketed DPIs is their dose variability due to the inspiratory flow rate achieved by the patient and the flow resistance within the DPI. This dose variability implies that different amounts of the drug are reaching the respiratory tract, dependent upon the patient’s inspiratory effort. Thus, this variability will result in sub-optimal management and control of the patient’s disease. Respirics, Inc. is developing DPIs (trademarked Acu-Breathe™) that are designed to deliver medications to the respiratory tract, either on an acute or chronic basis, and which we believe will reduce dose variability. The dose variability issue is addressed through the I-point™ technology incorporated in the DPIs, i.e., a dose is released to the patient only after a predetermined inspiratory flow rate has been achieved by the patient. This paper will provide (1) a brief overview of the inhalation technologies available and some of the issues surrounding their use; (2) discuss the principle factors to consider in the development of a DPI product; and, (3) to present in vitro data of model drugs relating to dose delivery and respirable mass in support of the Acu-Breathe™ technology.
K2 – Invited Paper PARTICLE FABRICATION FOR AEROSOL DRUG DELIVERY 238 Andrew R. Martin, and Warren H. Finlay, Department of Mechanical Engineering, University of Alberta, Edmonton, AB, Canada, T6G 2G8; [email protected]; [email protected]
The site within the respiratory tract at which an inhaled aerosol particle will deposit is strongly influenced by the particle’s size and shape. For topical treatment of certain lung diseases, it is preferable to target deposition in the tracheo-bronchial airways, while for systemic delivery to the blood stream, deposition in the alveolar region is required. Delivery of therapeutic agents to non-targeted sites not only reduces the efficiency of treatment, but can lead to undesirable side effects. Current delivery systems are able to target specific regions of the lungs in a broad sense, primarily by tailoring aerodynamic particle sizes to encourage deposition in targeted areas. Particle size and shape also influence translocation of deposited particles by pulmonary clearance mechanisms. For example, industrial hygienists and toxicologists are familiar with the ability of inhaled nanopartilces to avoid mucociliary clearance, and of long, thin fibers to hinder clearance by
188 Oral Abstracts alveolar macrophages. In the context of drug delivery, such characteristics might be benificial in reducing clearance of particles away from targeted regions. Presently, we are exploring methods of delivering aerosols of unconventional size and shape to the lungs. These include delivery of nanoparticles as micron-sized clusters, as well as generation of fibrous aerosols from porous thin films. In the latter case, separation of columnar thin film microstructures from their substrate has yielded discrete particles. In the long term, we anticipate that the influence of particle size and shape on physical mechanisms involved in deposition and clearance of inhaled particles will play an important role in the development of next-generation respiratory drug delivery systems.
K2 – Invited Paper PARTICLE AGGREGATION IN THERAPEUTIC AEROSOLS 239 Philippe G. Rogueda, AstraZeneca R&D Charnwood, Bakewell Road, Loughborough, LE11 2HD, UK; [email protected]
The aim of this work was to test the theories of aggregation in aerosol droplets developed by Igor Gonda in the mid 80’s. In order to explain high dose limitations in suspension pMDIs (pressure metered dose inhalers), it has been suggested that solid drug particles form large aggregates in aerosol droplets. Gonda’s theory was developped to explain the aggregation, but has never been tested in pMDI HFA (Hydro fluoro alkanes) aerosols. Experiments were carried out on 2 model systems: micronised lactose (Dv50 = 1.4 μm) and a micronised inhalation drug (3-[2-(4-hydroxy-2-oxo-3H-1,3-benzothiazol-7-yl ) ethylamino]-N-[2-[2-(4-methylphenyl) ethoxy) ethyl] propansulphonamide, Dv50 = 1.5 μm). The drugs were suspended in HFA 227, with and without Triton X100 (TX100) as a stabiliser. 50 μl Valois valves were used. 20 suspensions of varying solid drug content were prepared for each system, concentrations ranged from 0.1 % to 10 %w/w. Aerosol sizing measurements were performed with the Malvern Spraytec (light scattering measurements yielding geometric diameter volume size distribution) and the TSI APS (time of flight measurements yielding aerodynamic diameter number size distribution). Droplet sizes measured by both techniques did not evidence the size increase predicted by the theory. It is suggested that this theory may not apply to HFA pMDI systems. As a consequence dosing limitation at high solid contents in pMDI may not be due to the formation of large aggregates.
K2 – Invited Paper AN OVERVIEW OF ALKERMES AIR® DRY POWDER PARTICLE 240 FORMULATION TECHNOLOGY FOR PULMONARY DELIVERY Michael M. Lipp, Alkermes Inc., 88 Sidney St., Cambridge, MA 02139, USA; [email protected]
Particle design and engineering has enabled several advances in the area of dry powder formulations for pulmonary drug delivery. Modern methods for creating aerosol particles include spray-drying, supercritical fluid-based methods and additional sophisticated liquid droplet production technologies. Porous particles for inhalation produced via the
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Alkermes AIR® technology are characterized by geometric diameters > 5 microns and low densities (< 0.4 g/cc), yielding aerodynamic diameters of approximately 1-5 microns for optimal lung deposition (Edwards et al., 1997; Dunbar et al., 2002). Increasing the geometric diameter increases the dispersibility of the powder, making it possible to efficiently deliver a wide range of doses using a simple, passive DPI. An overview is provided of the Alkermes AIR® technology with a focus on the impact of particle engineering on the resultant preferred properties of the particles.
K2 – Invited Paper ELECTROSTATIC MEASUREMENT METHODS IN 241 PHARMACEUTICAL AEROSOLS Matti Murtomaa, Laboratory of Industrial Physics, Department of Physics, FI-20014 University of Turku, Finland; [email protected]
The electrostatic charge carried by respirable particles has many effects on the function of a dry powder inhaler (DPI) and on the lung deposition. Used powders, both carries and active drugs, tend to be electrically insulating by nature, and therefore they will inevitably charge during the operation. The charge transfer process is very complicated due to the fact that the powder is usually a mixture consisting of two materials which differ considerably in particle size. The particles will acquire charge when the DPI is moved, the dose is measured, the dose is released, and when the drug and carrier particles separate. The key factors in the charging process are the materials, moisture and the nature of the contact. Unfortunately, the frictional charging (or tribo-charging) is a phenomenon which is not thoroughly understood and predictions about the magnitude or the even polarity of the charge are hard to make. Since the importance of the electrostatics in the pharmaceutical aerosols is widely accepted, laboratory measurements are needed to assess the phenomena. In this presentation, different measurement methods are presented. It is shown that by using various complementary methods, a comprehensive picture of the situation can be formed. For example, a simple net charge measurement can give information about the effect of device materials or particle morphology, when a time-resolved measurement gives information about the separation of the drug and carrier particles. The presentation focuses on the devices which have been designed and built at the University of Turku, Finland, but other measurement methods will also be covered. Examples of the results which have been obtained with the devices will be presented.
K2 – Invited Paper POWDER SURFACE IMAGE ANALYSIS IN SCREENING OF 242 FUNCTIONAL BULK PROPERTIES OF PHARMACEUTICAL PARTICLES Niklas Sandler, Solid State Group, School of Pharmacy, University of Otago, Dunedin New Zealand and Osmo Antikainen, Division of Pharmaceutical Technology, University of Helsinki, Finland; [email protected]
It is necessary to obtain particle size data of materials in various phases of the drug
190 Oral Abstracts development process including the preformulation stage of new chemical entities (NCE) and later stage drug product manufacturing. For instance, the particle size of a NCE has to be evaluated with respect to whether it has a significant effect on dissolution rates, bioavailability, stability, prosessability, or drug product content uniformity. Furthermore, the bulk properties and behaviour of a material in different unit operations used in drug manufacturing depend to a great extent on its particle size distribution. Various techniques for measuring the particle size distribution of powders exist and the choice of method has to be made carefully based on material characteristics and type information required. Common methods used with conventional pharmaceutical powders are sieving, laser diffraction and image analysis (IA). Recently, a new IA concept of characterizing physical material properties by examining texture features of monochromatic images of pharmaceutical powder surfaces instead of properties of single particles has been introduced. In controlled illumination conditions the particle size of a material can be determined fast and reliably from digital images of undispersed powder surfaces. The technique has been successfully used in at-line particle size monitoring during wet granulation and in granule growth kinetics research. By using different surface imaging setups screening of functional properties of bulk powders and granules can be possible. The purpose of this presentation is to demonstrate the use of the surface image analysis approach in particle size monitoring during fluidized bed granulation, in prediction further prosessability using multivariate visualisation and to show the potential of the technique in evaluation of particle size related segregation behaviour during tablet manufacture.
K2 – Contributed Paper A NOVEL POROUS HOLLOW SILICA NANOPARTICLES FOR 243 DRUG/PESTICIDE RELEASE CONTROL Li-Xiong Wen, Fan Liu, Zhu-Zhu Li, Jian-Feng Chen, Key Lab for Nanomaterials, Ministry of Education;Research Center of the Ministry of Education for High Gravity Engineering and Technology, Beijing University of Chemical Technology, Beijing 100029, PR China; and Jimmy Yun, NanoMaterials Technology Pte Ltd., Blk26 Ayer Rajah Cresent 07-02, Singapore 139944; [email protected]
Carrier materials play a key role in the controlled drug delivery applications. However, conventional carriers have various limits such as low loading capacity, synthesis complexity, high cost, environment maleficent, etc. Novel porous hollow silica nanoparticles (PHSNs) were, therefore, developed by our group through a novel sol-gel route using inexpensive inorganic nano-templates. These new environment-friendly materials had a core-shell structure with nearly uniform meso-pores and a high surface area. The simple synthesizing method enabled successful and readily repeatable preparation of PHSNs in both flask and pilot reactors without any significant negative scaling-up effect. The as-synthesized PHSNs were applied to encapsulate different water-soluble or oil-soluble drugs/pesticides, including brilliant blue F (BB), bentazone, ibuprofen, avermectin and validamycin, through either a simple immersing method or a supercritical fluid loading method. The active substances could all be loaded efficiently on PHSNs, with a loading capacity as high as 1.67:1 (avermectin:PHSNs) by weight. All of
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the entrapped active substances demonstrated a typical sustained release pattern from the carriers when immersed in some release medium and the PHSNs also showed a remarkable UV shielding ability for the entrapped active drugs. The drug release rate could be controlled by adjusting the pore size and shell thickness of PHSNs, and it was also influenced by the pH and temperature of the release medium. Using the avermectin-loaded PHSNs, an avermectin controlled release formulation was prepared and the biological tests on its killing effects were conducted. It was found that the new formulation could kill pest more efficiently and last for longer than conventional formulations, with lower toxicity and cost. It therefore illustrated that the novel PHSNs can be utilized as a promising carrier for the controlled drug/pesticide release for industrial applications.
K2 – Invited Paper Hans Leuenberger, President of the Swiss Society of Pharmaceutical 244 Scientists, Institute of Pharmaceutical Technology, Pharmacenter, Klingelbergstrasse 50, 4056 Basel, Switzerland; [email protected]
FDA‘s PAT (Process Analytical Technology) Initiative is a revolutionary paradigm change concerning the “Pharmaceutical cGMPs for the 21st century”. The actual performance of the pharmaceutical industry is ca. Two Sigma, i.e. ca. 4.5% defectives. The defectives are eliminated by a final and costly “testing-in the quality”. The champion is the chip industry with Six Sigma, i.e. with only 2ppb defectives. The new FDA requirements focus on “Design of Quality” and not to “test -in“ the quality. Ca. 80% of the pharmaceutical products on the market are solid dosage forms, which are developed based on pharmaceutical powder particle technology. Thus, the development of a rigorous scientific framework in this area is of highest priority. The paper addresses typical problems of critical processes and formulations and proposes a “Road Map” to achieve as fast as possible the top of the “Knowledge Pyramid” often cited by FDA. The road map proposes the extensive use of a multivariate design of experiments including artificial neural networks (ANN), and the use of percolation theory for a better understanding of complex systems. An important question is whether it is possible to “translate” the existing laws of physical chemistry into the area of powder particle technology, where we have the problem that the number of particles involved is much lower than the Avogadro number NA. For this purpose, the results of Nanoscience should be integrated, especially in the area of mathematical modeling of the fascinating special physical properties of nanoparticles, which again consist of a finite number N of “particles” in this case atoms, respectively molecules, with N << NA.
H2 – Invited Paper LIPOSOMAL FORMULATION OF A PHOTOSENSITIZER: GREEN 245 LIGHT FOR AN OLD SUBSTANCE Alfred Fahr, L. Restetzki, FSU Jena, Institute of Pharmacy, FSU Jena, Lessingstrasse 8, D-07743 Jena, Germany; D. Scheglmann and V. Albrecht, Biolitec AG, Winzerlaer Strasse, D-??, Jena, Germany; [email protected]
192 Oral Abstracts
Photodynamic therapy (PDT) is a promising new technique being explored for use in a variety of medical applications and is known as a well-recognized treatment for the destruction of tumors. Another possible application of PDT is the treatment of infectious diseases due to pathogenic micro organisms. The use of PDT for the treatment of various types of disease has been limited due to the inherent features of photosensitizers (PS). These have included their high cost, extended retention in the host organism, substantial skin photo toxicity, low solubility in physiological solutions (which also reduces its usefulness for intravascular administration as it can provoke thromboembolic accidents), and low targeting effectiveness. These disadvantages, had led to the administration of very high doses of a photosensitizer, which dramatically increase the possibility of accumulation of the photosensitizer in non-damaged tissues and the accompanying risk of affecting non-damaged sites. The usual formulation of these photosensitizers consists of an ethanolic solution, which causes the solubilized drug after i.v. injection to precipitate at the injection site. This induced very slow pharmacokinetics, more resembling an oral PK profile. Solubilization of the drug by liposomes changes this PK profile dramatically. It looks now like a typical i.v. profile, the drug does not stay for a long time in the blood circulation, but despite of the rapid elimination from blood the photosensitizer penetrates to a larger extent than the ethanolic formulation into tumour tissue, presumably as host inside the liposomal membrane. This is maybe due to the enhanced permeability and retention effect. As the drug sits because of its hydrophobicity in the liposomal bilayer, it is able to diffuse out of the membrane to e.g. surrounding tumour cells at longer times and may increase by this its pharmacodynamic activity in tumour tissue. The patient benefits from this rather physical formulation approach also by a reduced topical phototoxicity because of the rapid clearance from blood.
H2 – Invited Paper USE OF A SCATTERING ELEMENT AND DIRECT DE-IONIZED 246 WATER-COOLING IN HIGH POWER SEMICONDUCTOR DIODE LASERS TO ENHANCE THE BEAM DIVERGENCE AND PERFORMANCE FOR USE IN PHOTODYNAMIC THERAPY (PDT) Amaranath Premasiri, Gemunu Happawana, Mechanical Engineering Department, Southern Methodist University, Dallas, TX; Gary Evans, Electrical Engineering Department, Southern Methodist University, Dallas, TX; and Arye Rosen, Electrical and Biomedical Engineering Department, Drexel University, Philadelphia, PA; [email protected]
Use of high power semiconductor diode lasers in a light delivery system to be used in photodynamic therapy (PDT) of cancer treatment makes the system portable and inexpensive. Semiconductor diode lasers convert electrical power to optical power with higher efficiency than any other device, yet there are two main disadvantages associated with semiconductor laser when it is used in such systems. One is high temperature within the laser which contributes to device degradation and wavelength shifts, and the other is the low beam divergence that does not give uniform illumination in all directions. These two drawbacks can be largely overcome by introducing direct de-ionized water cooling and by
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using a polymer coating with embedded scatters on the laser facets in order to uniformly disperse the laser light. Edge emitting semiconductor lasers emitting at a wavelength of 635 nm were immersed in de-ionized water, resulting in significant increases in output power and wavelength stability. Different types of scattering elements were used with a transparent polymer matrix. The optimum volume, shape and size of scattering elements in the matrix material to provide uniformly distributed light was calculated using Mie theory. The optimal polymer thickness was determined by finding the dependence of the uniformity of the radiation pattern on the length of the scattering element. The theory and experimental results show that TiO2 is the best material for this application. This study has shown that introducing scattering elements and de-ionized water cooling improve the performance of semiconductor diode laser in light delivery systems for PDT.
H2 – Contributed Paper DEVELOPING MULTIFUNCTIONAL NANO-ARCHITECTURES: 247 LUMINESCENT AND SUPERPARAMAGNETIC Benoit Simard and Dongling Ma, National Research Council of Canada, Steacie Institute for Molecular Sciences, 100 Sussex Drive, Ottawa, ON K1A 0R6, Canada; [email protected]; [email protected]
This work aims at combining three useful functionalities: superparamagnetism, luminescence and readily functionalizeable suface into one multifunctional nano-architecure. The strategy we used is designing a nanostructure containing a superparamagnetic core and a luminescent silica shell, which encapsulates thousands of dye molecules. Traditional Stöber and reverse microemulsion methods have been tried seperately and the results are not satisfactory. Agglomerations, many core-free silica nanoparticles and irregular over-microscale hybrid networks were observed. To achieve the desired nanostructure with better control, we have developed a new two-step approach. In the first step, the first silica shell with specific thickness is coated onto iron oxide nanoparticles through a modified Stöber method. Next, the dye is doped into a second silica shell through a reverse microemulsion method. The synthesis yields core-shell nanoaprticles with improved morphology over the above two methods used independently. The key factor leading to the success in synthesizing this delicate nano-architecture resides in the use of “disguised” silica nanoparticles in the reverse microemulsion for the dye doping process. The “disguised” silica nanoparticles actually contain magnetic cores, but the core-core dipolar interactions are shielded by the first silica shell. Therefore, the same or slightly modified reverse microemulsion conditions should be applicable to various magnetic nanoparticles for dye doping as long as they are already covered by a silica shell thick enough to isolate their magnetic interactions. We consider this new approach as an important step towards developing a universal and simple synthesis for silica based, multifunctional core-shell nanostructures. We have worked on several dyes using this approach such as tris(2,2’-bipyridine) ruthenium (II) chloride and fluorescein-5-isothiocyanate. The novel hybrid nanomaterials demonstrate marked advantages over the uncoated materials, including enhanced collodial stability, increased luminescence density and reduced magnetic blocking temperatures. Herein, we report their synthesis, magnetic and photophysical properties as well as their applications.
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H2 – Invited Paper CANCER NANOTECHNOLOGY: DESIGNING MULTIFUNCTIONAL 248 NANOSTRUCTURES FOR TARGETING TUMOR CELLS AND VASCULATURES Shuming Nie, Gloria J. Kim, Departments of Biomedical Engineering, Chemistry, Materials Science and Engineering, Hematology and Oncology, 101 Woodruff Circle, Suite 2001, Atlanta, GA 30322, Fax: 404-727-3567, [email protected]
Significant progress has been made in developing new agents against cancer and in formulating them for in-vivo delivery and targeting. Research in genomics and proteomics continues to uncover biomolecular signatures (biomarkers) that are unique to cancer development and progression. Yet, the major challenge to target and selectively kill cancer cells remains. Linked with targeting moieties such as tumor-specific ligands or monoclonal antibodies, nanoparticles can be used to target tumor cells and tumor vasculatures. We have attained dramatic in vivo tumor shrinkage and tumor vasculature disruption using ternary biomolecular nanostructures. For hydrophobic anti-cancer agents not amenable to chemical conjugation, we have used the principles of molecular self-assembly to engineer targeted nanoparticles. These nanoparticles are also applicable to delivery and targeting of diagnostic and imaging agents. In particular, bioconjugated quantum dots make it possible to track movement of individual labeled molecules inside a cell or a small number of cancer cells growing in a living animal. Multifunctional nanostructures that can serve as phototherapeutic agents to selectively diagnose and treat cancer are currently under development. The ability to design multifunctional nanostructures has opened new opportunities in early cancer diagnostics, molecular profiling, and integration of cancer therapy and imaging.
H2 – Contributed Oral La0.75Sr0.25MnO3-BASED MAGNETIC NANOPARTICLES AS POTENTIAL 249 HEAT MEDIATORS IN ONCOLOGY Sebastien Vasseur, Etienne Duguet, Institute of Condensed Matter Chemistry of Bordeaux (ICMCB), University of Bordeaux, 87 ave Dr Schweitzer, 33608 Pessac Cedex, France; Eva Hadova, Emil Pollert, Institute of Physics (FZU), Academy of Sciences of the Czech Republic, Cukrovarnicka 10, 16253 Praha 6, Czech Republic; [email protected]
Hyperthermia consists in heating a cancerous tissue either to destroy directly tumour cells by necrosis or to improve drug or radiation efficiency. One promising route uses alternative magnetic fields allowing to magnetic materials to absorb electromagnetic energy and convert it into heat. In particular, magnetic fluid hyperthermia takes advantage of magnetic nanoparticles dispersion which could be administered by intravenous injection. Among the numerous requirements, two are of great importance : (i) the temperature must be controlled in vivo in order to avoid safe tissue overheating and (ii) the nanoparticles must be surface-derivatized not only for ensuring their stealthiness in the blood compartment towards macrophages (e.g. pegylation) but
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also for being labelled with appropriate targeting ligands. Our strategy is to take advantage of the temperature-dependence of the magnetic properties of manganese perovskites La1-xSrxMnO3, whose Curie temperature may be tuned from –130 to 100°C by varying substitution value x. So, as soon as medium temperature reaches Curie temperature, particles lose their magnetic properties and therefore their heating ability. Such particles would be both heaters and fusers. La0.75Sr0.25MnO3 nanoparticles were synthesized by citrate route, followed by annealing and milling. Optimal annealing conditions were determined as a compromise between high crystallinity, high magnetic properties and low size. In these conditions, 60-nm particles were synthesized. Under a 108 kHz / 88 mT alternative magnetic field, they exhibited specific heating power of 40 W/gMn and reached a maximal heating temperature of about 46°C, making such particles suitable for safe hyperthermia applications. Silica encapsulation of La0,75Sr0,25MnO3 particles led to core-shell particles with reduced cytotoxicity and opened the way to their surface functionalization.
H2 – Invited Paper PHOTOSENSITISER-MACROMOLECULE CONJUGATES FOR 250 TARGETED PHOTODYNAMIC THERAPY Ross W. Boyle, Nela Malatesti, Robert Hudson, Karen Smith and John Greenman, Department of Chemistry & Clinical Biosciences Institute, University of Hull, Kingston-upon-Hull, East Yorkshire, HU6 7RX, UK; [email protected]
Photodynamic Therapy (PDT) is a treatment for the eradication of unwanted or inappropriate tissue, including solid tumours and neaovasculature. PDT relies on the combination of a photosensitising drug, high intensity light in the visible or near infrared regions and molecular oxygen. The therapeutic effect is dependent on all three factors being optimised. Laser based PDT light sources and fibre optics now allow precise delivery of high intensity light to the treatment area, and little can be done to influence levels of tissue oxygenation, thus the major opportunity for further optimisation of PDT is by more accurate targeting of photosensitisers. Conjugation of PDT sensitisers to macromolecules, both biological and synthetic, has the ability to alter biodistribution and pharmacokinetic parameters, thus improving targeting and enhancing the therapeutic effect. Synthetic strategies for conjugation of porphyrin based photosensitisers to macromolecules, including antibodies, will be presented and properties of the resulting conjugates, including target cell binding, internalisation, intracellular distribution, and mechanism of photocytotoxicity, will be discussed.
H2 – Contributed Paper INDOCYANINE GREEN-LOADED NANOPARTICULATE DELIVERY 251 SYSTEM FOR SUPERFICIAL TUMOR DIAGNOSIS AND TREATMENT Jun Shao, Vishal Saxena, Mostafa Sadoqi, St. John’s University, 8000 Utopia Parkway, Jamaica, NY 11439, USA; [email protected]
The objective of this project was to develop an intravenously administrable
196 Oral Abstracts poly(dl-lactic-co-glycolic acid) (PLGA) nanoparticulate delivery system for Indocyanine Green (ICG), to enhance the potential for ICG use in tumor imaging and therapy. For this purpose PLGA nanoparticles entrapping ICG were engineered by spontaneous emulsification solvent diffusion method. ICG entrapment in nanoparticles was determined and physicochemical characterization of nanoparticles was performed. The stability of ICG in nanoparticles formulation under various conditions was determined. The intracellular uptake of ICG in nanoparticles by B16-F10 and C-33A cancer cell lines was studied in comparison with the free ICG solution. Anti-proliferation studies against cancer cells were performed to prove the photodynamic activity of ICG in nanoparticles. Biodistribution of ICG when delivered through nanoparticles and solution were evaluated in mice after tail vein injection. PLGA nanoparticles with a mean diameter of 350 nm and 0.2% ICG loading were obtained. The nanoparticles were nearly spherical in shape with negative zeta potential. The nanoparticle formulation significantly enhanced the overall stability to ICG in water with degradation half-lives of 3-5 days at various conditions as compared to 10-20 hr of free ICG solutions. The intracellular uptake of ICG through nanoparticles was directly proportional to time and extracellular nanoparticle concentration. The nanoparticles formulation enhanced about 100-fold intracellular uptake of ICG as compared to the free ICG solution. Nanoparticle formulation showed significant photodynamic effect at nano-molar ICG concentrations and very low light dose against the cancer cells in vitro. Biodistribution study on mice showed that the nanoparticle formulation enhanced the blood circulation time and retention time of ICG in various organs 2-5 times as compared to the free solution. A stable PLGA nanoparticlute delivery system was developed for ICG, which demonstrated the capability to enhance the potential for ICG use in tumor diagnosis and anticancer therapy.
D6 – Keynote Paper MAGNETIC BIOSENSORS IN BIODIAGNOSTICS 252 J. Manuel Perez, Nanoscience Technology Center, Department of Chemistry and Biomolecular Science Center, University of Central Florida, 12424 Research Parkway, Suite 400, Orlando, Fl 32826, USA; [email protected]
Designing activatable nanoagents to sense molecular markers, molecular interactions and pathogens associated with disease could result in the development of more sensitive molecular diagnostic agents. Toward this goal, we have developed an assay to sense molecular targets using magnetic nanosensors and nuclear magnetic resonance (NMR). These magnetic nanosensors consists of biocompatible iron oxide based magnetic nanoparticles capable of detecting a molecular target by changes in the NMR signal of the solution as the nanoparticles self-assemble in the presence of the target. We have shown that these magnetic nanosensors can detect various molecular interactions (DNA-DNA, protein-protein, protein-small molecule, and enzymatic reactions) in solution with high sensitivity and selectivity using NMR. The assay does not require purification of the samples and the observed target-induced change in NMR signal is detectable in turbid media or whole-cell lysate. We will present data showing the use of this technique to detect molecular targets involved in cancer, and viruses and toxins in solution. Furthermore, recent advances toward improving the detection limit, speed of detection and portability of the magnetic assay will be discussed.
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D6 – Invited Paper MULTIFUNCTIONAL QUANTUM DOTS FOR MOLECULAR IMAGING 253 AND THERAPEUTICS Xiaohu Gao, University of Washington, Department of Bioengineering, Seattle, WA 98195, USA; [email protected]
The development of high-sensitivity and high-specificity probes beyond the intrinsic limitations of organic dyes and fluorescent proteins is of considerable interest to many areas of research, ranging from in vitro ultrasensitive detection to in vivo medical imaging. Recent advances have shown that nanometer-sized semiconductor quantum dots (QDs) can be covalently linked with biorecognition molecules such as peptides, antibodies, nucleic acids, and small-molecule inhibitors for use as fluorescent probes. In comparison with organic fluorophores, QDs exhibit unique optical and electronic properties such as size- and composition-tunable fluorescence emission, large absorption coefficient, and significantly improved brightness and photostability. Due to their broad excitation profiles and narrow/symmetric emission spectra, high-quality QDs are also well suited for optical multiplexing, in which multiple colors and intensities are combined to encode thousands of genes, proteins, and small-molecule libraries. In this context, I present recent developments in bioconjugated QD probes and their applications in ultrasensitive molecular, cellular imaging and drug delivery. Despite their relatively large sizes (2-6 nm in diameter), bioconjugated QD probes behave like fluorescent proteins, and do not suffer from serious kinetics or steric-hindrance problems. In this “mesoscopic” size range, QDs and other types of nanoparticles also have more surface areas and functionalities that can be used for linking to multiple diagnostic (e.g., radioisotopic or magnetic) and therapeutic (e.g., anticancer) agents. These recent developments toward nanomedicine are expected to open new opportunities in molecular imaging, multiplexed profiling, disease diagnosis and treatment.
D6 – Contributed Paper PLASMON-ENHANCED FLUORESCENCE OF DYES ATTACHED TO 254 POLYELECTROLYTE COATED METAL NANOPARTICLES. Robert Nooney, Ondra Stranik, Colette McDonagh, Brian D. MacCraith. National Centre for Sensor Research, Dublin City University, Glasnevin, Dublin 9, Ireland; [email protected]; [email protected]
We report on the enhancement of fluorescence that can result from the proximity of fluorescent dyes to metal nanoparticles (NPs). This plasmonic enhancement which is a result of the localised surface plasmon resonance (LSPR) at the metal surface, can be exploited in order to improve the efficiency of optical biochips and thereby lower the limit of detection. The fluorescence enhancement, which depends on the dye-NP separation was measured and the optimum thickness of the polyelectrolyte shell determined. A model system was chosen, consisting of NPs surrounded by a polyelectrolyte spacer shell, to which was attached a layer of dye molecules where the LSPR wavelength was tailored to coincide with the absorption band of the dye. Colloidal silver and silver / gold alloy
198 Oral Abstracts nanoparticles with monodispersed spherical or triangular shapes and predetermined LSPR absorption bands were prepared using both wet chemistry and photoconversion techniques. The nanoparticles were coated with a polyelectrolyte shell using a layer by layer technique and the thickness of the coating was measured using transmission electron microscopy and UV absorption spectroscopy. Inorganic and organic dyes of different lifetimes and quantum efficiencies were attached either ionically or covalently to the polyelectrolyte shell. The enhancement was calculated by measuring the fluorescence of the NPs using a microplate reader and comparing to that of the pure dye. The LSPR contribution to the enhancement was validated by removing the metallic core using potassium cyanide and measuring the fluorescence of the remaining polyelectrolyte vesicle. The nanoparticle / polyelectrolyte layers were then deposited on a plastic substrate using a layer-by-layer approach with layer number matching the optimum value determined from the colloidal system above. Again a range of dyes were attached and the enhancement was determined. Ultimately, this plasmon enhancement system will be used as the basis of an enhanced bioassay platform.
D6 – Contributed Paper NANO-BIOSENSOR FOR DETECTION AND TREATMENT OF DISEASE 255 Tao Liu, Hong Zhao, No.2 Bei Yi Jie, Zhong Guan Cun, Beijing,; and Long Jiang, Key laboratory of Colloid and Interface, Center of Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences; China; [email protected], [email protected]
Investigation indicates that the appearance of the malignant tumor is highly correlative with the DNA mutation. It has become an important topic of the cancer study to detect the gene mutation and look for the relation between mutation and pathological change at the molecular level. Rapid detection for trace gene mutation can provide basic data for diagnose disease. Therefore, looking for a rapid and simple method used for detecting trace mutation is important and pressing. QCM is a simple, rapid and real-time measurement of DNA binding and hybridization at the sub-nanogram level. The nanogold particle has many special properties, for example high density, simple operation, and easy size-controlled. Combining these two techniques, i. e., nanoparticle modification of QCM surface and the application of gold narnoparticle amplifier, we improved the detection limit of DNA. This method makes it possible to detect single base mutation less than 10-16 mol / L.
D6 – Contributed Paper BIOFUNCTIONALIZATION OF WATER-DISPERSIBLE GOLD 256 NANOPARTICLES Hilde Jans1,2, Kristien Bonroy1, Filip Frederix1, Karolien Jans1,2, Bieke Van de Broek1,2, Gustaaf Borghs1, Guido Maes2; 1IMEC, MCP-ART, Belgium; 2K.U.Leuven, Department of Chemistry, Belgium, [email protected]
In recent years, gold nanoparticles have become the subject of intensive research for the
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detection and labeling of biomolecules. Due to their Localized Surface Plasmon Resonance properties, their UV-Vis absorption spectrum is extremely sensitive to changes in the nanoparticle size, the shape and the external dielectric environment in close proximity of the nanoparticles. This property has led to the development of novel classes of nanoscaled affinity biosensors. The aim of this work is to synthesize stable water-dispersible gold nanoparticles with different sizes that can be used for the development of biorecognition assays. The nanoparticles are functionalized with different mixed Self-Assembled Monolayers of thiols (SAMs). This enables to realize nanoparticles with enhanced stability. In a next step, biomolecules are immobilzed on top of these SAMs using covalent coupling procedures. The applied synthesis route consists of a very easy “one-step in-situ synthesis method”, which allows to realize well-defined gold nanoparticles functionalized with two different kinds of thiols: HS-(CH2)11-PEO6-COOH and HS-(CH2)11-PEO3-OH. In this approach the thiol molecules are mixed with the gold salt and subsequently reduced with NaBH4. By changing the ratio of the reagents, the size of the nanoparticles can be controlled. Next, anti-PSA antibodies are covalently coupled to the carboxylic groups of the SAMs onto the nanoparticles. The poly-ethylene oxide moieties prevent the non-specific binding and increase the water solubility. This synthesis route of gold nanoparticles allows to increase the nanoparticle stability, the anti-PSA binding efficiency and the activity of the immobilized anti-PSA antibodies. These nanoparticles can therefore be used to create very sensitive nanoscaled affinity biosensors.
D6 – Invited Paper A NEW PARTICLE-BASED IMMUNOASSAY THAT DEPENDS ON THE 257 QUANTITATIVE DETECTION OF MAGNETIC PARTICLES Stéphane Legastelois, Guillaume Renoud : Indicia Biotechnology, 33 avenue de la Californie, 69600 Oullins, France; [email protected] ; and Luc Lenglet, Magnisense, Tour de Rosny 2, 112 avenue du général De Gaulle, 93118 Rosny ss bois cedex, France
Magnetic particles are a powerful and versatile tool in biology. Bound to a suitable antibody, they are used to label specific molecules or microorganisms. A new assay system has been developed for the rapid and sensitive detection of proteins, virus or bacteria in biological, environmental or food samples. This system incorporates the use of coated superparamagnetic particles in a lateral flow or flow through format. The devices are analyzed by a laptop reader that detects and counts the number of superparamagnetic particles that react with the solid phase. The innovative detection technology is based on the non-linear behavior of superparamagnetic bodies in the presence of a magnetic field. This physical response is strictly proportional to the amount of magnetic material. Both lateral flow membranes or flow through syringes are introduced into a coil for the magnetic analysis. A exceptional dynamic range of six orders of magnitude is observed. In this study, we have demonstrated that lateral flow tests can be turned into a quantitative assay, by replacing gold colloid or latex beads by nanosized magnetic particles. The low detection threshold enables an enhanced sensitivity of 15 to 70 fold compared to conventional lateral flow tests. Data recording also eliminates potential subjectivity associated with visually-interpreted tests. Another format based on a flow through device was also
200 Oral Abstracts investigated. A cylindric solid phase built into a syringe allows the processing of large volumes of sample, thus reducing the time of the culture phase of food or waterborne bacteria. Assays carried out on legionella pneumophila exhibit a detection threshold of 1500 bacteria. Basically, data demonstrate that this technology is simple, accurate, and exhibits a low limit of detection. The robustness of the method allows measurements in almost any environmental conditions.
D6 – Invited Paper CANCER BIOMARKR DETECTION BASED ON 258 PHOTOLUMINESCENCE OF SEMICONDUCTOR QUANTUM DOTS AND SERS OF METAL NANOSTRUCTURES Jin Z. Zhang, Adam M. Schwartzberg, Tammy Oshiro, Leo Seballos, Abraham Wolcott, Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA 95064 USA; Chad E. Talley, Thomas R. Huser, Department of Chemistry and Materials Science, Lawrence Livermore National Laboratory, Livermore, CA 94550 USA; Rebecca Sutphen, Department of Interdisciplinary Oncology, College of Medicine and H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, Fl, 33612 USA; and Yiping Zhao, Department of Physics and Astronomy, University of Georgia at Athens, GA 30602, USA; [email protected]
Detection of cancer biomarkers at low concentration, especially for early stage cancers, is a major challenge in cancer research, diagnosis, and treatment. Detection platforms based on nanomaterials, including semiconductor quantum dots and metal nanostructures, offer promising hope in developing sensitive and selective methods for cancer biomarker detection. For example, rational design and study of new surface enhanced Raman scattering (SERS) substrates is key to advancing chemical and biological sensing based on SERS. The next generation of biological probes will ideally be single, small, SERS active nanostructures able to penetrate the inner workings of cells. To this end, we have developed various metal nanostructures based on aggregates, nanorods, and nanoshells with the goal to optimize their SERS activities. We have very recently demonstrated SERS from single, hollow nanostructures. Exceptional sample homogeneity leads to a nearly tenfold increase in signal consistency over standard silver substrates. At 30 nm in diameter, this is the smallest confirmed single SERS active particle ever reported, representing a major step in advancing sensing technology based on SERS. In the meantime, we have applied some of the substrates developed in the detection of cancer biomarkers and have achieved high sensitivity and molecular selectivity. The results have shown that SERS is extremely promising for chemical and biological sensing and imaging applications. To control the structure of the nanomaterials and thereby their optical absorption as well as SERS properties is critical for these emerging technological applications.
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D6 – Contributed Paper PARTICLE POPULATION CLASSIFICATION BY MICRO-FLOW 259 IMAGING Dave Thomas, David King, Peter Oma, Brightwell Technologies Inc., 195 Stafford Road West, Ottawa, Ontario, Canada, K2H 9C1; [email protected]
The paper will describe a new particle analysis technology which employs automated digital image analysis to classify particulate/cell suspensions. The technology involves drawing sample through a sheathed or unsheathed micro-fluidic cell. Sections of the flowing fluid are illuminated with a pulsed high-intensity source, magnified and imaged frame-by-frame on the camera’s pixel array. The system software identifies, in real time, pixels in each frame which are wholly or partially contained within a particle image. This information is used to isolate particles and measure particle parameters such as count, diameter, area, perimeter, circularity, and transparency. The volume of fluid sampled in each frame is accurately known, therefore particle concentrations are determined absolutely. The database of images and parameter values is interrogated by the user to produce histograms, scattergrams and to isolate sub-populations of interest. Micro-Flow Imaging (MFI) technology will detect and classify particles ranging from 0.8 to 100 microns in size over a concentration range of 0 to 100K’s per ml. The technique is insensitive to the particle’s optical properties and therefore offers distinct advantages over laser diffraction and light obscuration in terms of anlyzing heterogenous populations. The data set is intermediate between flow cytometry and manual microscopic analysis providing the automation, statistical validity and reproducability of the former while preserving the visual insight gained by the latter. Continuous sample introduction and time-resolved image analysis permitting high resolution reaction monitoring and trend charting. The paper will present experimental results applying MFI technology for general purpose particle fabrication process control with particular emphasis upon characterization and optimization of specific cell-related bio-processes including fermentation, viability, purification, dissolution, preservation, and protein aggregation. Application of the technology for detecting unwanted particulate within process fluids will also be presented, as well as use of the technology for realtime chemical reaction monitoring.
D6 – Invited Paper DEVELOPMENT OF COLLOIDAL SEMI-CONDUCTOR 260 NANOCRYSTALS AS BIO-IMAGING PROBES Kui Yu, Steacie Institute for Molecular Sciences, National Research Council Canada, 100 Sussex Drive; Ottawa, Ontario Canada K1A 0R6; [email protected]
Colloidal semiconductor quantum dots (QDs), which are spherical in shape with diameter on the order of 1 – 10 nm, have attracted a lot of attention recently, due to their size-dependent properties, for instance, CdSe nano-crystals emit from blue to red depending on their size, and due to their promise of practical applications to substitute
202 Oral Abstracts fluorescent dyes for bio-imaging. This presentation will address our comprehensive study on the synthetic factors as well as the dispersing media affecting the PL properties of quantum dots such as CdSe, in addition to our recent study on the bio-distribution of nano-crystals inside live mice. Furthermore, post-treatments aiming at high performance will be dealt with. The post-treatments for better surface passivation involve growing an inorganic shell hetero-epitacitically; during the post-treatment, radiative recombination of light-generated charge carriers increases. The insights gained from our comprehensive study facilitate the development of imaging probes based on a deeper understanding of the fundamental chemistry and physics of high-quality nanocrystals from large-scale production with a high degree of synthetic reproducibility.
I3 – Keynote Paper NANOMEDICINES FOR EFFICIENT DRUG AND GENE DELIVERY 261 Ijeoma F. Uchegbu, Xiaozhong Qu, Woei Ping Cheng, Andreas G. Schätzlein1, Nanomedicines Research Centre, Department of Pharmaceutical Sciences, University of Strathclyde, Glasgow, UK. 1Nanomedicines Research Centre, Cancer Research UK Centre for Applied Pharmacology and Medical Oncology, University of Glasgow, Glasgow G61 1BD; [email protected]
Our objectives are the design and fabrication of functional nanosystems based on amphiphilic polymers and poly(propylenimine) dendrimers, which enhance the bioavailability of hydrophobic drugs and anti-cancer genes. Linear soluble polymers grafted with hydrophobic and hydrophilic groups result in amphiphiles, which via hydrophobic associations yield various nanostructures such as dense nanoparticles, bilayer vesicles and polymeric micelles; all of which may be used for drug delivery. Water soluble amphiphilic carbohydrates self assemble into polymeric micellar clusters which improve aqueous levels of propofol by 2000 fold and prednisolone by 8 fold. Optimum drug/ polymer ratios in aqueous media are 48: 1 for propofol and 67: 1 for prednisolone; values that are between 16 and 300 times higher than those shown by the Pluronic block copolymers. Polymeric micellar clusters, when compared to commercial formulations produce a 10 fold increase in topical ocular prednisolone levels in the rabbit model and a 10 fold increase in intravenous propofol pharmacodynamic activity in the mouse model. Additionally, other amphiphilic polymers facilitate the oral absorption of cyclosporine, giving similar bioavailability levels to the commercial formulation Neoral®. Low molecular weight polypropylenimine dendrimers (MW < 1700 Da), by virtue of their high nitrogen density are excellent DNA carriers and gene transfer agents. Interestingly, systemic gene transfer in tumor bearing animals results in tumor gene expression which is 3 fold that produced by either Exgen-500 (linear polyethylenimine, Mw = 25,000 Da) or naked DNA. Additionally these dendrimers are tumoristatic agents and have thus been used to make an efficient tumor necrosis alpha gene medicine, which shows tumor ablation in the A431 tumor mouse model and excellent tumoricidal activity in the LS174T, B16F10 and C33a mouse tumor models. These data demonstrate that polymeric and dendrimer nanosystems may be exploited to yield efficient nanomedicines.
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I3 – Invited Paper NOVEL POLYMER TECHNOLOGY FOR THE DELIVERY OF 262 BIOLOGICAL MACROMOLECULES William G. Turnell, Medivas, LLC., 6275 Nancy Ridge Drive, CA 92121 USA; [email protected]
Synthetic analogs of naturally occurring macromolecules (“biologics”) constitute a large and important class of therapeutic compounds that have come to underpin a multi-billion dollar industry, despite the majority of these products having to be administered by injection. This presentation examines the fundamental barriers associated with delivery of biologics, citing oral insulin and vaccine formulations as examples. Data will be presented to show that amino acid based poly(ester amide) and poly(ester urethane) polymers help protect peptides and whole proteins from bio-degradation, whilst preserving native structure and bio-activity. This protective and controlled delivery of the biologic is achieved in concert with complete bio-degradation of residual polymer. For efficient protein-polymer coupling, both covalent, via amide or ester bond formation, and non-covalent conjugation schemes are described. The latter is designed for His6-tagged recombinant proteins coupled to each repeating unit of a nickel-decorated version of the polymer. This formulation strategy forms the basis of a rapid response program for the provision of vaccines against newly emerging pathological viral stains, such as both seasonal and pandemic influenza.
I3 – Invited Paper MICELLAR NANOPARTICLES 263 Robert W. Lee and Dinesh B. Shenoy, Novavax, Inc. 508 Lapp Road, Malvern, PA 19355, USA; [email protected]
This paper describes Novavax’s proprietary Micellar Nanoparticle drug delivery technology. Novavax, Inc. is a biopharmaceutical product development company focused on the research, development and commercialization of products utilizing its proprietary drug delivery and biological technologies for large and growing markets. Micellar Nanoparticle (MNP) technology (US Patent #5,629,021) is the basis for the only FDA approved product, ESTRASORB®, consisting of a nano-technology-based, cosmetically appealing lotion-like topical dosage form. MNP’s represent a composite vehicle that presents the drug in a variety of fractions: aqueous or hydro-alcoholic solution, micellar solubilized form, oil fraction and as surface-stabilized micro-/nano-crystals. This formulation approach creates a microreservoir-dissolution-controlled system that is capable of incorporating both water-soluble and water-insoluble active pharmaceutical ingredients and delivering them either topically (skin being the target tissue) or transdermally (systemic delivery). The physicochemical attributes of MNP’s will be described in relationship to what makes this drug delivery technology unique.
204 Oral Abstracts
I3 – Invited Paper TISSUE TARGETED NANOPARTICLES FOR SYSTEMIC DELIVERY OF 264 NUCLEIC ACID AGENTS ENABLES siRNA THERAPEUTICS Martin Woodle, Intradigm Corporation, 12115 Parklawn Drive, Suite K, Rockville, MD 20852, USA; [email protected], [email protected]
RNAi, and in particular the short dsRNA oligonucleotide active intermediate called siRNA, has revolutionized gene function research by providing an effective tool to selectively inhibit gene expression. This capability has altered dramatically drug target discovery and validation, but largely in cell culture. Intradigm now has developed tissue targeted nanoparticle systems that are proving to be an effective means to utilize siRNA oligonucleotides in vivo and via systemic administration, both as a powerful method for drug target research and most importantly as a means to use siRNA as a therapeutic modality. Application of a modular polymer chemistry approach has succeeded in development of unique nanoparticles that can achieve the different functions required to solve the long standing challenge of intracellular delivery of nucleic acid agents for therapeutic applications. The system has been developed for use of siRNA for drug target discovery and validation, operating directly in clinically relevant animal models, which generates the most useful gene function information. Results will be presented showing discovery in extremely rapid fashion a portfolio of novel cancer therapeutic targets and validation of a few for monoclonal antibodies and other classes of therapeutics. The presentation will also present development of a nanoparticle-siRNA system combining tissue selective delivery with gene selective inhibition by the siRNA payload, opening the door to dual-targeted therapeutics. Intradigm is developing a first product in this class, ICS-283, for inhibition of neovascularization and angiogenesis in cancer, ocular diseases, and other critical diseases. In addition, Intradigm is developing this system with collaborators in other therapeutic areas.
I3 – Invited Paper POLY-L-GLUTAMIC ACID AS A BUILDING BLOCK FOR 265 NANOMEDICINE: FROM THEAPEUTICS TO MULTIMODALITY DIAGNOSTICS Chun Li, Department of Experimental Diagnostic Imaging, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030, USA; [email protected]
Poly(L-glutamic acid) (PG) is a biodegradable polyamino acid possessing excellent physicochemical properties as a drug carrier for both diagnostic and therapeutic agents. PG-paclitaxel conjugate (PG-TXL) developed in our laboratatory accumulated selectively in solid tumors due to enhanced permeability and retention effect of macromolecules, and the conjugate demonstrated remarkable antitumor activity in preclinical studies. Owing to prolonged release of paclitaxel from PG-TXL in the tumors, PG-TXL used in combination with radiotherapy results in significant improvement in tumor control compared with
205 Oral Abstracts
individual treatment and in comparison with other preclinical in vivo data for combined chemo- and radiotherapy. PG-TXL has now advanced to clinical phase III studies. Conjugation of gadolinium p-aminobenzyl-diethylenetriaminepentaacetic acid (Gd-DTPA) to PG resulted in a macromolecular magnetic resonance imaging (MRI) contrast agent that could be used not only for blood-pool imaging but also for assessing the extent of necrosis because it selectively localized to necrotic areas of tumors 2-5 days after intravenous injection. Our data suggest that the enhanced permeability and retention effect of PG-based polymeric chemotherapeutic agents is attributable to their selective accumulation and retention in the necrotic areas of the tumors. To further enhance delivery efficacy, we synthesized and characterized dendritic PG that allows attachment of multipe targeting moieties at the termini of dendritic polymer chains. Using a near-infrared fluorescence dye indocyanine green (ICG) as a model diagnostic agent and folic acid as a model targeting moiety, we showed that the resulting nanometric conjugate, PAMAM16-PG-(ICG)-folate, exhibited selective binding to KB cells that overexpress folate receptors but not to SK-Br3 cells that do not express folate receptors. Thus, active targeting using PG-based dendrimer is a promising approach towards targeted delivery and molecular imaging.
I3 – Invited Paper FORMATION OF REVERSE MICELLAR TYPE VERSUS SURFACE 266 MODIFIED SOLID LIPID NANOPARTICLES Meike Harms, Martin A. Schubert, Ingo Friedrich, Christel C. Müller-Goymann, TU Braunschweig, Institute for pharmaceutical Technology, Mendelssohnstr. 1, 38106 Braunschweig, Germany, [email protected]
Solid lipid nanoparticles (SLN) - an alternative to other colloidal drug delivery systems like, emulsions, liposomes and polymer nanoparticles – possess inherent low drug loading capacities. In this study the amphiphilic lipid lecithin was mixed with triglycerides in order to increase drug solubility within the lipid matrix (LM). Transmission electron microscopy (TEM) was applied to characterize different lipid matrices. For binary mixtures of a triglyceride and lecithin typical lamellar structures were observed. For triglycerides with comparable low melting points additional areas with spherical structures could be detected. This might be attributed to the existence of a solidified reverse micellar solution (SRMS). Drug loading capacity of hydrocortisone (HC) and estradiol (EST) within these LM was increased significantly. Preparation of SLN where the SRMS is preserved within the particles probably results in higher drug loading capacities. In order to obtain these SLN of a reverse micellar type high-pressure homogenization at a temperature close to the melting temperature was applied. Therefore at least a part of the particles might stay in solid state and its structure including the SRMS remains unchanged. Drug loading capacities of these systems for EST was the same as for the LM without lecithin, but for HC it was significantly increased. Surface modified SLN are prepared by applying high-pressure homogenization at a temperature above the melting point. This enables rearrangement of lecithin to the surface of the oil droplets and formation of a mono- and/or multilayer around a triglyceride
206 Oral Abstracts enriched core, resulting in surface modified SLN after recrystallisation. This model could be proved by means of NMR and DLS measurements. In both cases liposomes resulting from lecithin leakage into the aqueous phase could not be observed by means of TEM.
M2 – Keynote Paper UTILIZATION OF DENSE GAS TECHNOLOGIES FOR THE 267 FABRICATION AND FORMULATION OF NANO-BIOMATERIALS Neil R. Foster, School of Chemical Engineering and Industrial Chemistry, The University of New South Wales, Sydney, NSW 2052, Australia; [email protected]
The term “nano-biomaterials” is used to describe a diverse range of materials including tissue engineering scaffolds, implant materials and drug formulations, all of which have at least one dimension of nano- scale. Conventional methods for the fabrication of nano-biomaterials usually involve high temperature or high shear process conditions and/or have significant organic solvent requirements. Many of these disadvantages can be overcome by using gas expanded liquids (GXLs) as processing media. Typically, a gas expanded liquid is a conventional organic solvent that has been expanded with a dense gas, such as CO2. The solvent, physical and chemical properties of GXLs are extremely tunable and, consequently, GXLs provide an extremely powerful tool for the processing and fabrication of complex nano-biomaterials. Fundamental characteristics of GXLs will be described as will the methods for tuning the properties of these systems. Several examples of nano-biomaterials processed using GXL technology will be presented.
M2 – Invited Paper MICROENCAPSULATION THROUGH HYBRID 268 AQUEOUS/SUPERCRITICAL CARBON DIOXIDE PROCESSING OF POLYMER COLLOIDS Matthew Yates, W. Yin, X. Chen; University of Rochester, Department of Chemical Engineering, Rochester, NY 14627; [email protected]
Several years ago, our group reported the development of a microencapsulation process based on swelling of aqueous polymer colloids by compressed carbon dioxide. Swelling by carbon dioxide causes the colloidal particles to enter the liquid state, facilitating mass transport in the polymer phase. Small molecule additives can then diffuse into the polymer particles. When carbon dioxide is slowly removed by venting, the particles return to the glass state, entrapping the additives inside. Provided colloidal stability is maintained throughout the process, the size and shape of colloidal particles are not significantly altered by carbon dioxide processing. The technique represents an organic solvent-free approach to microencapsulation, and is effective with a variety of polymers including biodegradable poly(lactic acid). The talk will give an overview of the process, including examples of encapsulating small molecule hydrophobic drugs into biodegradable colloids. The challenges and progress in extending the process to hydrophilic compounds and improving encapsulation efficiency will be discussed.
207 Oral Abstracts
M2 – Contributed Paper EXPLORING SUPERCRITICAL FLUID TECHNOLOGY FOR THE 269 DEVELOPMENT OF CONTROLLED DRUG DELIVERY Ana Rita C. Duarte,*1 Ana Luisa Simplicio, 1 Herminio C. de Sousa,2 Teresa Casimiro,3 Ana Aguiar Ricardo,3 M. Margarida Cardoso,3 Arlette Veja Gonzalez4, Pascale Subra4, Catarina M. M. Duarte1; 1 Nutraceuticals and Delivery Laboratory, ITQB/IBET, Aptd. 12 – 2781-901 Oeiras, PT; 2 Dep. de Eng. Química, FCT, Universidade de Coimbra,Pólo II – Pinhal de Marrocos, 3030-290 Coimbra, PT; 3 REQUIMTE/CQFB – Departamento de Química, FCT-UNL, 2829 – 516 Caparica, PT; 4 Laboratoire d’Ingénierie des Materiaux et des Hautes Pressions (LIMHP), CNRS, Institut Galilée, Université Paris XIII, 99 Avenue Jean Baptiste Clément, 93430 Villetaneuse, France; [email protected]
Controlled drug delivery products, using biocompatible or biodegradable polymers, have received considerable attention in the last years. These substances provide in general a more controlled release rate of assumption of the drug by the body improving its therapeutic action. In fact, there is a growing interest of the pharmaceutical industry in the development of these systems. Impregnation using supercritical fluid technology has already proven its feasibility. In this technique the drug component is dissolved in a compressed gas (carbon dioxide) that is used as a mobile phase and that, also, swells and stretches the polymer matrix, facilitating the diffusion of the drug, and increasing the rate of impregnation. A high purity product, free of residual solvents is obtained, since no organic solvents are involved in the impregnation process. In this work, the development of successful controlled release systems is presented: supercritical impregnation of contact lenses with ophthalmic active drugs for glaucoma treatment and the preparation of controlled drug release systems for the delivery of a different anti-glaucomatous drug and an anti-inflammatory drug, namely coprecipitation of Eudragit RL 100 and RS 100 with acetazolamide and naproxen-loaded ethylcellulose/methylcellulose microspheres. Co-precipitation of Eudragit RL 100 and RS 100 with acetazolamide was performed in a semi pilot-scale apparatus, built for particle formation by rapid crystallisation using supercritical carbon dioxide. Furthermore the preparation of molecularly imprinted polymers (MIPs) using supercritical fluid technology was evaluated. Poly(diethylene glycol dimethacrylate), was synthesised in scCO2 in the presence of different concentrations of two different template drug molecules, salicylic acid and acetylsalicylic acid. Results suggest that molecular imprinted polymers were successfully prepared by supercritical polymerisation and then impregnated with the template in order to prepare controlled release systems.
M2 – Invited Paper LIPOSOME FORMATION WITH SUPERCRITICAL CO2 AS AN 270 ALTERNATIVE SOLVENT.
208 Oral Abstracts
Katsuto Otake, Nat. Inst. Adv. Ind. Sci. Tech. (AIST), Tsukuba, Ibaraki 305-8565, Japan; and Hideki Sakai, Masahiko Abe, Faculty of Science and Technology, Tokyo University of Science, Yamazaki 2641, Noda, Chiba 278-8565, Japan; [email protected]
Supercritical carbon dioxide (scCO2) dissolves into phospholipids (PLs) to lower the melting point by loosening the molecular interactions. With the surfactant like natures of the PLs, essentially water –insoluble PLs become soluble in water in contact with scCO2 through the formation of CO2-in-water emulsion. The CO2-in-water emulsion becomes liposomal dispersion by releasing the pressure due to the phase inversion. Based on this phenomena, we have developed a process for generating liposomes that uses scCO2 as a solvent, and named the method as “improved supercritical reverse phase evaporation (ISCRPE)” method, from the similarity of the method with the conventional reverse phase evaporation method. The liposomes prepared by the ISCRPE method exhibited high trapping efficiency for both water-soluble and oil-soluble compounds. Freeze fractured TEM observations, osmotic shrinkage measurements, and DSC measurements revealed that the liposomes prepared by the ISCRPE method are unilamellar vesicles with loosely packed PLs. Comparison of nitrogen with scCO2 revealed that the presence of CO2 is necessary for the formation of liposomes. Further, scCO2 in contact with water dissolves to form carbonic acid, causing a decrease in pH. Using these characteristics of a CO2/H2O biphasic system, chitosan coated cationic liposomes of L-α-dipalmitoylphosphatidylcholine were successfully prepared by the ISCRPE method. Liposome-chitosan complexes carrying a positive charge were prepared in a single step procedure without using acid or organic solvent, including EtOH. The liposomal dispersion was stable at room temperature in a sealed tube for over 30 days. Details of the ISCRPE method and its applications will be presented in the conference.
M2 – Invited Paper SOLID DISPERSIONS BY COMPRESSED FLUID ANTISOLVENT 271 PRECIPITATION Gerhard Muhrer, Novartis Pharma AG, Chemical & Analytical Development, 4002 Basel, Switzerland; Francesco Fusaro and Marco Mazzotti, ETH Swiss Federal Institute of Technology, Institute of Process Engineering, 8092 Zurich, Switzerland, [email protected]
The classical anticonvulsant drug phenytoin has been used as a model compound in the assessment of the possibility of enhancing the dissolution rate of poorly water-soluble drugs using compressed fluid antisolvent techniques. In a first step, microcrystals of neat phenytoin have been precipitated using both the gas antisolvent (GAS) and precipitation with compressed antisolvent (PCA) processes. Process performances were assessed by working out the effect of the major operating parameters on product quality, thereby elucidating similarities and differences between the two techniques. In a second step, the PCA process has been used to generate co-formulations of phenytoin and the hydrophilic carrier poly(vinyl-pyrrolidone)-K30 (PVP-K30). The effect of temperature, pressure, flow rate ratio, and drug to polymer ratio in the starting solution was investigated, yielding a
209 Oral Abstracts variety of product morphologies. At operating conditions below the mixture critical locus and at drug to polymer ratios of 1:2 and below, spherical nanoparticles were obtained, and combined analysis by Raman spectroscopy, XRD and DSC revealed that phenytoin is indeed molecularly dispersed in the polymer matrix. Results from in-vitro dissolution rate measurements showed a substantially better performance of the co-formulations if compared to unprocessed, neat phenytoin crystals. A comparison of the product quality of phenytoin–PVP solid dispersions with amorphous co-formulations obtained by spray drying again convincingly underlines the potential of dense gas antisolvent techniques for the production of pharmaceutical formulations with enhanced oral bioavailability. Interestingly at conditions with a drug to polymer ratios above 2:1, coated microcrystals were obtained, which may open up new areas of application for dense gas precipitation in drug product development.
M2 – Invited Paper THE USE OF COMPRESSED CARBON DIOXIDE TO BROADEN THE 272 APPLICABILITY OF HOT STAGE EXTRUSION FOR DRUG DELIVERY Geert Verreck1, Annelies Decorte1, Koen Heymans1, Albertina Arien1, Jef Peeters1, Dehua Li2, David L. Tomasko2, Guy Van den Mooter1, Marcus E. Brewster1, 1 Johnson & Johnson Pharmaceutical Research & Development, Turnhoutseweg 30, B-2340 Beerse, Belgium; [email protected] 2 Department of Chemical Engineering, Ohio State University, 125 Koffolt Lab, 140 W. 19th Ave., Columbus, OH 43210, USA; [email protected]
Using a Leistritz Micro 18 intermeshing co-rotating twin-screw melt extruder, it was shown that an optimal extruder design and screw configuration resulted in the injection of pressurized carbon dioxide without leakage. Pressurized CO2 acted as a temporary plasticizer during the hot stage extrusion of a number of pharmaceutical acceptable polymers, including PVP-VA 64, eudragit® E100 and EC 20 cps. The same effect was observed for solid dispersions with different drug loadings, consisting of itraconazole with PVP-VA 64 and itraconazole with EC 20 cps. This plasticization was obtained under sub- and supercritical conditions allowing for a reduction in the processing temperature during the hot stage extrusion process up to 65°C for EC 20 cps. Depending on pressure and temperature, amorphous dispersions were obtained at low drug loadings, while higher concentrations resulted in solid dispersions consisting of a mixture of glassy and crystalline drug substance. This indicated that the morphology of the solid dispersion could be influenced when CO2 was injected during the hot stage extrusion process. Expansion of the injected carbon dioxide at the outlet of the extruder, resulted in a foamed extrudate. This change in macroscopic morphology after CO2 treatment, resulted in an increased specific surface area and porosity. As a consequence, milling efficiency was improved, resulting in a reduction of the particle size of the CO2 treated extrudates. The in vitro drug release of itraconazole from the 10 and 40% w/w solid dispersion could be controlled as a function of pressure and temperature by injection of carbon dioxide during the hot stage extrusion process.
210 Oral Abstracts
M2 – Invited Paper FABRICATION OF LIPID NANOPARTICLES FOR LOW MOLECULAR 273 WEIGHT AND BIOTECH DRUG DELIVERY BY GAS COMPRESSED TECHNIQUES 1Paolo Caliceti, 2Nicola Elvassore, 2Alberto Bertucco, 1Alessandra Semenzato, 1Stefano Salmaso, 1Sara Bersani – 1Pharmaceutical Sci. Dept. and 2Chemical Eng. Dept. – Universty of Padua – Italy – [email protected]
Micro- and nano-particulate solid lipid systems are promising pharmaceutical and cosmetic formulations. They are, in fact, flexible in composition, manageable, biocompatible and may be administered by various routes. Furthermore, solid lipid colloidal formulations may be properly used to increase the drug stability and modulate the release yielding successful drug delivery systems. Typically, the production of lipid nanoparticles involves the use of high temperature necessary for melting the lipids, high shear and pressure stresses (high shear homogenization, ultrasounds, high pressure homogenization), surfactants and/or organic solvents (solvent emulsification/evaporation) and lyophilization to obtain dry powders. This may limit the use of these systems in the case of fragile drugs. Alternative techniques based on supercritical near critical and compressed gas have been investigated. These methods operate under mild conditions without use of harsh chemicals, yield dry products and are environmentally compatible and industrially scalable. Typical example of this technique is Particle from Gas Saturated Solution (PGSS). In the recent years we focused our research interest in exploiting comprassed gas tachniques, namely PGSS, for preparation of colloidal lipid drug delivery systems. In order to make up an operative platform, experimental/in silico investigations aimed at defining thermodynamic parameters governing the processes were undertaken. Materials of pharmaceutical and cosmetic interest were considered and their physicochemical properties under the operative conditions were extrapolated by the elaboration of DSC, solubility studies and rheological determinations. These preliminary investigations addressed the selection of processable products and the set up of specific equipments. Either PGSS or GAMA (Gas Micro Atomization), a newly set up process, were appled to the nanoparticle production. Void particles as well as drug loaded nnaoparticles were produced and characterized to evaluate their physicochemical, morphological and biopharmaceutical properties. The results generated by this investigation were applied to obtain products which can be succesfully exploited for both pharmaceutical and cosmetic applications. Furthermore, the experimntatl data allow for drawing general considerations about the applicability of these techniques for industrial developmets.
211 Poster Abstracts
Abstracts of Poster Program
P1 PHARMACOKINETICS AND ANTILEISHMANIAL ACTIVITY OF 274 MANNOSE GRAFTED AMPHOTERICIN B LIPID NANOSPHERES Veerareddy P Reddy, Vobalaboina Venkateswarlu, Venisetty R Kumar University College of Pharmaceutical Sciences, Kakatiya University, Warangal, INDIA and Nahid Ali, Indian Institute of Chemical Biology, Kolkata, INDIA; [email protected]
Colloidal carriers of drugs such as liposomes, nanoparticles and solid lipid nanoparticles, have been studied as delivery systems with the aim of achieving better therapeutic efficacy and limiting side effects of the drugs. Amphotericin B lipid nanospheres (LN-A) and mannose grafted lipid nanospheres (LN-A-MAN) were prepared. Amphotericin B (dissolved in methanol), mannose, egg lecithin and cholesterol were dissolved in soybean oil, heated to 70°C on a water bath, and stirred until the system is clear. Glycerol, sucrose and sodium oleate were dissolved in sufficient amount of distilled water and the aqueous phase was added to the oil phase at the same temperature. A coarse emulsion was prepared by homogenizing at 6000 rpm for 3 min and was subjected to ultrasonication. The formulations were sterilized by autoclaving at 121oC. The pharmacokinetics and antileshmanial activity of LN-A and LN-A-MAN were compared with amphotericin B deoxycholate (Fungizone, marketed preparation). Same dose of each formulation (5 mg/kg) of amphotericin B was injected in the male wistar rats via tail vein. Plasma samples were collected at different intervals. Amphotericin B showed biphasic disposition. The pharmacokinetics of LN-A and LN-A-MAN were significantly different with fungizone. The peak plasma concentration of LN-A and LN-A-MAN was approximately 2.5 and 3.5 folds higher than fungizone. Antileishmanial activity of LN-A and LN-A-MAN was assessed in BALB/c mice infected with Leishmania donovani AG83 for 60 days. A single dose of LN-A (5 mg/kg), LN-A-MAN (5 mg/kg) and fungizone (2.5 mg/kg) was injected intravenously. Mice were sacrificed after 15 days of treatment with fungizone, LN-A, LN-A-MAN and Leishman Donovan Unit (LDU) is counted. Fungizone, LN-A and LN-A-MAN reduced 48 and 53; 75 and 66; and 95% and 90% of the parasite burden in liver and spleen respectively after 15 days of infection.
P1 FORMULATION AND QUALITY CONTROL OF GASTRORETENTIVE 275 TABLETS OF RANITIDINE HYDROCHLORIDE Janardan D, Veerareddy P Reddy, Venisetty R Kumar, Vobalaboina Venkateswarlu, UCPS, Kakatiya University, Warangal, INDIA. [email protected]
Gastroretentive dosage forms are the controlled release dosage forms that release the drug in a controlled manner thereby controlling the onset, duration and intensity of
212 Poster Abstracts pharmacological response by pharmaceutical means, promisingly elimate the frequent administration of dosage. Floating tablets are one of the important categories of drug delivery systems with gastric retentive behaviour. Ranitidine is a H2 blocker and absorbed from the upper part of GIT. Rapid metabolism after oral administration (bioavailability 50% approximately) and its need in stomach to exert local action, ranitidine hydrochloride required to formulate as gastroretentive controlled dosage form. Six formulations of floating tablets of ranitidine HCl were prepared by direct compression using different concentrations HPMC K4M (hydroxy propyl methyl cellulose K4M) with effervescent (sodium bicarbonate and citric acid), carbopol and both effervescent and carbopol. The formulations were coded as HE1, HE2, HC1, HC2, HEC1 and HEC2. The formulations were evaluated for weight variation, friability, hardness, content uniformity, thickness, floating lag time and floating time. All the formulations were subjected to in vitro drug release study and compared with that of marketed tablet. All the physical parameters evaluated for quality control were within the acceptable limits of Indian Pharmacopoeia. The content uniformity is in the range of 95 to 98%. The floating lag time of the prepared formulations is good except HC1 and the floating time for all the fomulations was >24 hours except HC1 which does not floated at all. The average percent drug release from the formulation HE2 was highest among others. In conclusion, effervescent is essential for the formulation to have good floating property and carbopol retards the drug release in floating formulations.
P1 SMART MEMBRANES FOR FLAVOR DELIVERY 276 Kevin A. Heitfeld, Dale W. Schaefer; University of Cincinnati, Chemical and Materials Engineering, ML 0012, Cincinnati, OH 45221-0012, USA; [email protected], [email protected]
Flavors often contain volatile compounds, so in the absence of encapsulation, they are vulnerable to premature release. Encapsulation also offers protection against environmental damage such as oxidation, light-induced reactions, etc. For processed foods, the ideal flavor would survive the temperature excursion during cooking, remain in the product for days to months and then be released instantaneously in the mouth. In the mouth, flavors are released due to mechanical disruption of the gelatin encapsulant and due to enhanced permeability of the gelatin in the moist environment. This work has focused on the use of temperature-responsive gels (TRGs) for flavor retention at cooking temperatures. TRGs are polymeric hydrogels with a large temperature-dependent change in volume. Specifically, we have studied a system with a lower critical solution temperature (LCST). This type of system exhibits a two phase region at elevated temperatures. A gel featuring a LCST exhibits swelling at low temperatures and collapse at high temperatures. In the collapsed state, the polymer acts as a transport barrier, keeping the volatile flavors inside. We have successfully modified a cellulose polymer to exhibit this volume change and have encapsulated an oil phase inside the gel. Flavor transport through the particles is a major consideration. We have studied flavor transport utilizing a vertical diffusion cell. We have successfully modified a hydroxypropyl cellulose polymer to achieve diffusion coefficients on the same order as sodium alginate, which is currently used in flavor encapsulation. However, comparing the diffusivity differential of these two polymers between the high temperature and low temperature states, HPC performs better than alginate by a factor of five.
213 Poster Abstracts
P1 INCREASED OSTEOBLAST FUNCTIONS ON TI NANOPATTERNED 277 WITH PEPTIDES Ganesan Balasundaram, Thomas J. Webster, Division of Engineering, Brown University, Providence, RI 02912, USA; [email protected]
In some clinical cases, the use of titanium in orthopedic implants has resulted in poor osseointegration; specifically, the successful use of titanium as bone implants has been limited due to nonspecific implant-tissue adhesion and subsequent scar tissue formation. Peptide sequences such as lysine-arginine-serine-arginine (KRSR) bind transmembrane proteoglycans (e.g., heparin sulfate) of osteoblast cells and are therefore actively being investigated for orthopedic applications. Furthermore, nanophase materials, or materials with consistuent particle or grain sizes less than 100 nm, are promising for various implant applications since our tissues are composed of nanometer components (i.e., proteins and/or inorganics). The objective of this study was to combine these two promising approaches (peptide immobilization and the use of nanomaterials) to develop better orthopedic materials. Peptide functionalization was conducted by means of a three step reaction procedure: silanization with 3-aminopropyltriethoxysilane (APTES), cross-linking with N-succinimidyl-3-maleimido propionate (SMP), and finally peptide immobilization. The three step reaction procedure was characterized by X-ray photoelectron spectroscopy (XPS). Scanning electron microscopy (SEM) was used to probe the surface properties of the materials studied. Results of this study provided the first evidence of increased osteoblast adhesion on unmodified nanophase compared to conventional metals. For all titanium materials, results showed that the immobilization of the cell adhesive KRSR sequence increased osteoblast adhesion compared to those non-functionalized and those functionalized with the non cell adhesive control peptide (KSRR) after 4 hours. Further, because osteoblast adhesion is a necessary prerequisite for subsequent functions (such as deposition of calcium-containing mineral), the present study suggests that nanophase metals functionalized with bioactive peptides (such as KRSR) should be further studied for orthopedic implant applications.
P1 CORRELATION OF POLYMER ENERGETICS AND DRUG RELEASE 278 RATES Daniel J. Burnett, Joe Domingue, and Frank Thielmann, Surface Measurement Systems Ltd., 2222 South 12th Street, Suite D, Allentown, PA 18103 USA; and Sasa Baumgartner, Faculty of Pharmacy, University of Ljubljana, Askerceva 7, Ljubljana, Slovenia; [email protected]
Cellulose ether polymers are common matrix compounds in sustained release tablets. The goal of this study is to correlate surface energetic properties of the individual formulation components measured using Inverse Gas Chromatography (IGC) with drug release profiles of the final product. IGC is a well-established technique for quickly and accurately
214 Poster Abstracts measuring the surface energetics for a wide range of solids. Surface energies have been determined with various polar and non-polar vapour phase probe molecules. Hydroxypropyl (HPC), hydroxyethyl (HEC), and hydroxypropylmethyl (HPMC) cellulose were used as polymers and Pentoxifylline was taken as a model drug. Tablets were prepared by direct compression of a 3:1 polymer-drug mixture. Release rates were determined by USP 26, App. I at 37 C. Results from IGC measurements suggest that HPMC has the highest dispersive surface energy followed by HPC and HEC. Acid-base numbers calculated from specific interactions show the strongest basicity for HEC, followed by HPMC and HPC. The acid numbers show the opposite trend. These trends in the specific energy correlate well with the observed release rates. The higher the basicity and lower the acidity the faster the release . This suggests that hydrophilic basic sites of the polymer are responsible for the interaction with the aqueous medium. This is supported by the specific interaction energies for the drug which are significantly lower. Therefore, the drug-polymer interactions are less relevant for the release mechanism.
P1 INVESTIGATING THE KINETICS OF MOISTURE-INDUCED 279 CRYSTALLIZATION OF AMORPHOUS LACTOSE Daniel J. Burnett, Joe Domingue, and Frank Thielmann, Surface Measurement Systems Ltd., 2222 South 12th Street, Suite D, Allentown, PA 18103 USA; [email protected]
Many low molecular weight amorphous materials will revert to their thermodynamically stable, crystalline form. This timescale of this transition is dependent on both temperature and humidity (or solvent partial pressure). Therefore, understanding the kinetics of this transition over a wide range of temperatures and humidity values is important for the processing and storage of materials containing amorphous phases. Dynamic Vapor Sorption (DVS) is a well-established method for the determination of vapor sorption isotherms. It is based on a highly sensitive gravimetric system, which measures vapor adsorption and desorption. In the current study water sorption experiments were performed over a range of humidity and temperature conditions to investigate the crystallization kinetics of a spray-dried, amorphous lactose sample. At 25 °C the onset time to crystallization for spray-dried lactose was measured over the 48 to 60% relative humidity range. Above 53% RH, crystalline lactose formation occurred in one step, while a two-step process was observed at 51% RH and below. Similarly, experimetns at 51% RH over a range of temperatures showed a two-step process at low temperatures and a one-step mechanism at higher temperatures. Kinetic modeling indicates crystallization occurs in two steps: (1) auto-catalyzed crystallization and (2) 3-D diffusion limited water desorption.
P1 SCALABLE FABRICATION OF MONODISPERSE, SHAPE-SPECIFIC 280 ORGANIC NANOPARTICLES FOR USE AS DELIVERY VECTORS Ginger Denison Rothrock, Benjamin Maynor, Jason Rolland, Liquidia Technologies, P.O. Box 110085, Research Triangle Park, NC 27709, USA; and
215 Poster Abstracts
Joseph DeSimone, University of North Carolina, CB# 3290 Venable Laboratories, Chapel Hill, NC 27599-3290, USA; [email protected]
To date, there has been no general particle fabrication method available that afforded rigorous control over particle size, shape, composition, cargo and chemical structure. We have developed scalable, “top-down” nanofabrication methodologies for the fabrication of polymeric and organic nanostructures for nanobiotechnology and materials engineering, utilizing the method we refer to as Particle Replication In Non-wetting Templates, or PRINTTM. Using PRINTTM, we can fabricate monodisperse particles with simultaneous control over structure (i.e. shape, size, composition) and function (i.e. cargo, surface structure). PRINTTM is the first general, singular method capable of forming organic particles that: i) are monodisperse in size and uniform shape; ii) can be molded into any shape; iii) can be comprised of essentially any matrix material; iv) can be formed under extremely mild conditions (and are therefore compatible with delicate cargos such as proteins, DNA, and drugs); v) are amenable to post functionalization chemistry for the bioconjugation of targeting ligands; and vi) are initially in an addressable array (which opens up combinatorial approaches since the particles can be “bar-coded” using methods similar to DNA array technologies). As such, PRINTTM is a significant scientific and technological breakthrough which will allow the continuous fabrication of heretofore inaccessible populations of nanomaterials, composed of organic polymers, conducting polymers, biodegradable polymers, hydrogels, inorganic particles, natural products, and more.
P1 CHEMICAL FUNCTIONALIZATION OF POLY(NITROPHENYL 281 ACRYLATE-CO-METHACRYLAMIDE) MICROGELS WITH pH-SENSITIVE GROUPS AND A TARGETING LIGAND Virginia Saez-Martinez, Leyre Perez-Alvarez, Estibaliz Hernaez, M.Teresa Herrero, Issa Katime, Department of Physical Chemistry, Faculty of Science and Technology, University of the Basque Country, 48940 Leioa, Vizcaya, Spain; [email protected]
The spherical morphology and nanometric size of colloidal microgels permits them to travel in the blood stream and accumulate at tumor sites, which is interesting for anticancer therapies standpoint.They can be chemically modified to exploit the small diference in pH between tumors and normal tissues, and they can be coupled with targeting ligands that interact with receptors expressed preferentially among tumor cells. Microgels in the nanometer size range show a very fast response to environmental changes, moreover, there have been relatively few studies of acid-swellable microgels which can be very interesting for these therapies. In this study copolymeric microgels of nitrophenyl acrylate (NPA) and methacrylamide (MeAM) were functionalized by chemical modification of their reactive pendant group (NPA) in order to obtain acid-swellable and targeting micorgels. The crosslinked microgels of poly(NPA-co-MeAM) were synthesized by precipitation polymerization in acetonitrile. EGDMA was used as the crosslinker and AIBN as the
216 Poster Abstracts initiator. NPA is an active ester monomer that easily reacts with amines in soft reaction conditions. Chemical modification took place by aminolisis reaction of synthesized poly(NPA-co-MeAM) microgels with 2-aminomethyl pyridine and N-tert-butoxicarbonil-1,2-ethanediamine. The extent of these aminolisis reactions was followed by UV-Vis and 1H-NMR spectroscopy. Folic acid was used as the targeting ligand which was attached to the polymer backbone through N-tert-butoxicarbonil-1,2-ethanediamine groups that act as spacer molecules. Microgels swelling behavior as a function of pH was studied by Quasielastic Light Scattering (QELS), and folic acid attachment to polymeric network was quantified by UV-Vis spectroscopy.
P1 NANOPARTICLE PREPARATION OF α-LIPOIC ACID SUSCEPTIBLE 282 TO POLYMERIZATION Chul Ho Park, Jonghwi Lee*; Department of Chemical Engineering and Materials Science, Chung-Ang University, 221, Heukseok-dong, Dongjak-gu, Seoul 156-756, Korea (South); [email protected]
Among various antioxidants, the redox system of alpha-lipoic acid (ALA) and dihydrolipoic acid (DHLA) is an excellent antioxidant because both the reduced and oxidized forms retain their functions of protecting defects related to blood flow and neurotrophins. Recent medical studies confirmed their efficacy of not only anti-obesity but also appetite suppression, as an overdose of ALA is taken (over 600 mg/kg). The dose might be reduced into an acceptable range by improving the bioavailability of ALA. The reduction of crystal sizes to nanometers in a drug delivery system can directly improve its bioavailability. There are several restrictions in utilizing the general processes to prepare ALA nanoparticles such as high shear homogenizing and ball milling, because cyclic five-membered disulfide is susceptible to free radical ring-opening polymerization by friction heat. In wet comminution, ALA of less than 0.01 M was reduced to DHLA whose thiol (-SH) can attack the cyclic disulfide (SN2 reaction) and act as the initiator for ionic polymerization. Therefore, this study focuses on how to prepare the nanoparticles of ALA without any side chemical reactions like the polymerization. The temperature of surroundings during ball milling was preserved at 4 °C in order to control that of ALA particles below 20 °C for 4 hours. As a result, we could produce ALA nanoparticles having the mean particle size of 300 nm with using a stabilizer, PVP (polyvinylpyrrolidone). To prove the possible bioavailability improvement, nanoparticles and microparticles of ALA were injected to standard rats. After 6 hours, the average amount of cumulative food intake of rats on ALA nanoparticles was half as much as that of rats on ALA microparticles.
P1 SYNTHESIS, FUNCTIONALIZATION AND TRANSPORT PROPERTIES 283 OF MESOSTRUCTURED PARTICLES Boon Sing Ng, Petr Vasiliev, Robert Hodgkins, Lennart Bergström. Department of Inorganic Chemistry, Arrhenius Laboratory, Stockholm
217 Poster Abstracts
University, Stockholm SE-10691, Sweden; [email protected]
Functionalized mesoporous spherical particles with a high surface area, mechanical rigidity and chemical inertness have a great potential for a number of applications, e.g. bioseparation and controlled drug release. There are several routes to produce these particles where the rapid single-step, aerosol-assisted method is able to give excellent control over the mesostructure, pore size and also offer facile and versatile approaches for functionalization. The possibility to tune the particle size and the pore size and mesostructure through the choice of templating amphiphilic molecule and the synthesis conditions will be described. Characterization of the materials involves the use of diffraction, microscopy, gas adsorption measurements and magnetic susceptibility. An attempt will be made to relate the three-dimensional pore structure, determined from TEM, to the transport properties of small molecules using confocal laser microscopy. An example will also be given how superparamagnetic iron oxide nanocrystals with a suitable surface modification can be incorporated into the mesostructured particles. The possibility to tune the magnetic susceptibility by the amount and type of the magnetic nanoparticles opens up many interesting applications, e.g. bioseparation. The large surface area and extremely well defined pore size of the mesoporous material together with the possibility to transport the particles by a magnetic field give us a very interesting possibility for separation of specific molecules or targeted delivery.
P1 MOLECULARLY IMPRINTED HYDROGEL MICROSPHERES FOR 284 PHOSPHATE REMOVAL Anika A. Odukale, Christopher D. Batich, University of Florida, Department of Materials Science and Engineering, Edward A. Ross, University of Florida, Department of Nephrology, Gainesville, Florida 32608, USA; [email protected]
The ability to distinguish among molecules in solutions is central to many areas of research and technology. One way to achieve such a system is via the use of molecularly imprinted polymers (MIPs) that possess selective binding properties due to recognition sites within the polymer matrix. Recently, there has been a growing appreciation for the need to avoid calcium based oral phosphate binders in patients with end stage renal disease as to prevent long term complications associated with elevated serum phosphate levels. Current therapeutic agents used to alleviate this condition overwhelmingly involve the use of nonabsorbable synthetic polymers to bind excess phosphate. However, the cost of these agents makes it an alternative not affordable to the greater general public. Thus, MIPs potential for enhanced stability, and selectivity that mimic natural binding phenomena, make them an attractive cost effective option. We describe a novel technique for developing molecularly imprinted gelatin microspheres as oral phosphate binding agents. Using a modified general emulsion polymerization schema, and genipin as a crosslinking agent, phosphate imprinted microspheres on the order of 20 – 100 µm can be synthesized. After extraction of the template molecule, an imprint cavity of specific shape and memory remains in the polymer. This cavity contains functional groups that enable targeted,
218 Poster Abstracts high-affinity binding for phosphate to the polymer. Phosphate uptake using the gelatin MIPs is evaluated in an in-vitro milieu that simulates human gastric conditions. The characterization of imprinted polymers was performed using UV-VIS, light microscope,and scanning electron microscopy techniques. Potentially therapeutic MIPs are thus synthesized with effective phosphate imprinting, thereby demonstrating feasibility of this novel low cost technology.
P1 SYNTHESIS AND CHARACTERIZATION OF SILICA 285 NANOPARTICLES WITH HIGH FLUORESCENCE Michihiro Nakamura, Kazunori Ishimura, The University of Tokushima Graduate School, Institute of Health Biosciences, Dep. of Anatomy and Cell Biology, Kuramoto-cho, Tokushima 770-8503, Japan; Hirokazu Miyoshi, The University of Tokushima, Radioisotope Research Center, Kuramoto-cho, Tokushima 770-8503, Japan; and Hiroshi Satake, The University of Tokushima, Center of Cooperative Research, Minami Jousanjima, Tokushima 770-8506, Japan;[email protected]
Stable dispersions of monodisperse silica nanoparticles with high fluorescence have been obtained using an efficient conjugation method between fluorescent dye and 3-aminopropyltriethoxysilane(APS). Fluorescent dye and APS were conjugated covalently using chemical reaction. The silica nanoparticles were prepared from tetraethoxysilane in mixtures of ammonia, water, ethanol, and APS-fluorescent dye conjugate. The intensities of fluorescence of silica nanoparticles were very high and stable, and were controllable by the change of the concentration of APS-fluorescent dye conjugate in the mixture. In addition, silica nanoparticle with two kinds of fluorescence, e.g. fluorescein and rhodamine G, have been also prepared successfully. The fluorescent silica nanoparticles were characterized by fluorescence spectroscopy, fluorescence microscopy, and electron microscopy. They showed high intensity of fluorescence, stable monodispersion, controlled size, and mesoporus surface of nanoparticles. And some advantages of our nanoparticle with high fluorescence will be discussed in comparison with quantum dots.
P1 ENZYMATIC SYNTHESIS AND CHARACTERIZATION OF NANOGELS 286 BY SELF-ASSEMBLY OF HYDROPHOBIZED POLY(L-LYSINE)-AMYLOSE CONJUGATES Nobuyuki Morimoto, Mio Yamazaki, Kazunari Akiyoshi, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-surugadai, Chiyoda-ku, Tokyo 101-0062, JAPAN; [email protected]
Nanosize hydrogels (nanogels), which are composed of submicrometer-scale gel particles, have attracted growing interest in many biotechnological and biomedical applications. We reported self-assembly methods for preparing physically cross-linked nanogels by the
219 Poster Abstracts
controlled association of hydrophobically modified polymers in water. For example, cholesterol-bearing polymers such as cholesteryl group-bearing pullulan (CHP) or cholesteryl group-bearing poly (L-lysine) form nanogels in dilute aqueous solution by intermolecular self-assembly. Amylose, a linear polysaccharide consisting of α-1, 4 linked glucose units, is an interesting macromolecule that forms complex with various hydrophobic guest molecules in the helical cavity in water. Amylose derivatives can be obtained from enzymatic polymerization by phosphorylase with maltopentaose derivatives as primers. We reported that methoxy poly(ethylene oxide)-block-amylose (MPEO-amylose) is a novel amphiphilic polymer having the amylose chain as a molecular recognition site. In this study, hydrophobized poly (L-lysine) grafted maltopentaoses (CH-MaPLL) were synthesized as amylose-primers. The partial modification of maltopentaose-poly (L-lysine) (MaPLL) by hydrophobic cholesteryl groups leads to the formation of nanogels (50 nm by DLS) by their self-associations. CH-MaPLL copolymers were enzymatically polymerized in the presence of phosphorylase b and α-D-glucose-1-phosphate. The degree of polymerization (DP) was linearly increased in accordance to the polymerization time. Characterization and function of nanogels by self-assembly of CH-Amylose-PLL were reported.
P1 – Invited Poster CONTROL OF CRYSTALLIZATION FOR MEDICAL APPLICATIONS: I 287 CONTROL OF NUCLEATION AND GROWTH OF MAGNETITE PARTICLES FOR CANCER HEAT IMMUNOTHERAPY. II SPHERICAL PARTICLE-ASSEMBLY BY TWO-SOLUTION METHOD FOR DRUG DELIVERY SYSTEM. Yoshitake Masuda, Nagoya University, Chikusa, Nagoya, 464-8603, Japan. AIST, National Institute of Advanced Industrial Science and Technology, Moriyama, Nagoya, 463-8560, Japan; [email protected]
We developed novel solution processes to control crystallization of Fe3O4 (magnetite) and colloidal crystal. Control of nucleation and growth of magnetite particles has attracted attentions for cancer heat immunotherapy. Spherical particle-assembly was fabricated by newly developed two-solution method and expected to be applied in novel applications such as photonic crystals or drug delivery system. Nucleation, growth and crystallization of Fe3O4 (magnetite) were successfully controlled by pH modification. Magnetite micropattern was fabricated by the site-selective deposition technique for demonstration of magnetite crystallization control using self-assembled monolayers (SAMs) as templates. Magnetite particles (crystallite diameter: 20 nm) were selectively deposited on Pd catalysts which were adsorbed to the -NH2-terminated surface of a SAM, in an aqueous solution containing iron nitrate and dimethylamine-borane. Pd colloid catalyst particles were adhered to the amino group of a patterned SAM by electrostatic interaction. Crystalline magnetite particulate films were then deposited on amino group regions by the effect of metal Pd catalyst. Site-selective deposition of crystalline Fe3O4 was thus realized in an aqueous solution. The deposition mechanism of Fe3O4 is discussed in detail in comparison with the deposition of c-FeOOH in the solution. We further proposed a novel process to fabricate spherical particle assemblies and their micropatterns. Hydrophilic regions of a
220 Poster Abstracts
patterned SAM were covered with methanol solution containing SiO2 particles and immersed in decalin to control the shape of droplets and gradually dissolve the methanol into decalin. Interfacing of methanol/decalin and shrinkage of methanol droplets were utilized to obtain meniscus force to form spherical particle assemblies; additionally, its static solution system allowed precise control of the conditions. Particles were assembled to form spherical shapes on hydrophilic regions of an SAM and consequently, micropatterns of spherical particle assemblies were successfully fabricated through self-assembly. These processes would contribute to the advancement of particle technology.
P1 SOLID LIPID NANOPARTICLES TO OVERCOME MULTI-DRUG 288 RESISTENCE IN CANCER THERAPY Eun-Jung Kim, Myung-Kwan Chun, Hyo-Kyung Han, Hoo-Kyun Choi, College of Pharmacy, Chosun University, 375 Seoseok-dong, Dong-gu, Gwangju, South Korea; and Cheol-Hee Choi, Research Center for Resistant Cells, Chosun University, 375 Seoseok-dong, Dong-gu, Gwangju, South Korea; [email protected], [email protected]
The failure of chemotherapy in cancer patient is often due to the development of resistance. Some tumor cells acquire drug resistance with exposure to structurally and functionally unrelated drugs. This multi-drug resistance (MDR) is mainly the result of overexpression of membrane-bound proteins that efflux drugs from the cells. MDR plays an important role in reducing the efficacy of a drug, causing the failure of chemotherapy for the many of cancer patients. Since most nanoparticles enter the cytosol by endocytosis, the efflux of drug due to membrane-bound proteins may be avoided. Among nanoparticles, the solid lipid nanoparticles (SLN) were selected for applying to anti-cancer drug delivery system because the SLN not only has physical stability and biocompatibility but also can incorporate a lipophilic drug by emulsification and subsequent recrystallization of the dispersed phase. The SLN was prepared from curdlan and cacao butter using phase inversion of curdlan by changing the pH of the solution. Curdlan, a naturally occurring polysaccharide, was used both as a surfactant and as a shell material. Cacao butter was used as a core material. Doxorubicin was chosen as a model anticancer drug. The in vitro release of the drug from SLN was sustained for 4 days and the amount of released drug at pH 5 was higher than that at pH 7.4. Even at the SLN concentration of 1 mg/ml, little cytotoxicity of the SLN was observed for AML-2/D100, which is a resistant cancer cell. The amount of doxorubicin accumulated in the cell from the doxorubicin-loaded SLN increased continuously with time. Based on this result, SLN could be used to overcome MDR.
P1 EFFECT OF NANO-SIZING CALCIUM PHOSPHATE/CISPLATIN 289 COORDINATION COMPLEXES ON IN VITRO DRUG RELEASE Liisa T. Kuhn and Xingguo Cheng University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030-1615; [email protected]
221 Poster Abstracts
Intratumoral treatments of chemotherapy and a drug delivery carrier effectively localize the chemotherapy, thereby minimizing systemic toxicity, while allowing for an increase in drug administration. Previously, we prepared a calcium phosphate (CaP) particulate drug delivery system for the chemotherapy drug cisplatin (CDDP) and evaluated the anti-tumor effects in a mouse model for human cervical cancer. The large (18 gauge) needle required to inject the micron-sized particles intratumorally led us to “nano-size” the CaP particles through the addition of Darvan during the CaP precipitation. After a 1 hr reaction time, dispersed 119 nm particulates were collected. The micro- and nano-particulates were characterized TEM, FTIR, XRD, particle size analysis and zeta potential measurements. Complexes of CaP and CDDP were prepared through electrostatic binding of an aquated species of CDDP to the CaP particulates in a chloride-free phosphate buffer. The drug loading was determined by platinum atomic absorption spectroscopy. Drug release studies were completed at 4 hrs, 1d, 3d, 7d, 10d and 15 days. The nano-complexes released only slightly more drug over the 15 day release assay (53% of the loaded drug vs. 40%) as compared to the micron-sized complexes; however, there was desirable reduction in the drug burst release and an extension of the sustained release. The toxicity of the released drug from both types of CaP/CDDP was compared to that of the free drug CDDP in vitro with the CellTiter cell proliferation assay using a mouse carcinoma cell line. IC50 values of both complexes were found to be statistically equivalent to pure CDDP indicating no adverse reaction between the CDDP and the nano-CaP. Lastly, nano-sizing the complexes increased the injectibility from an 18 gauge to a 26 gauge needle which will be adequate for animal testing. The nano-CaP/CDDP complexes are therefore ready for additional efficacy testing in a mouse tumor model. Funded by a BCTR grant from the Susan G. Komen Breast Cancer Foundation.
P1 BIODEGRADABLE POLYMERIC AGGREGATES PREPARAED BY 290 AMPHIPHILIC POLY(HYDROXYETHYL ASPARTAMIDE-CO-PROPYL ASPARTAMIDE) GRAFTED WITH POLY(LACTIDE) FOR DRUG DELIVERY Jianhua Hu, Wei Chen, Haoran Chen, Wuli Yang, Changchun Wang, Department of Macromolecular Science, Fudan University, and the Key Laboratory of Molecular Engineering of Polymers, Ministry of Education, Shanghai 200433, China; [email protected]
In recent years, poly(aspartic acid) (PASP) has been studied in detail as a typical hydrophilic biodegradable polymer. Its derivatives, such as poly(hydroxyethyl aspartamide) (PHEA), poly(asparagine)-g-poly(caprolactone) et al., are totally biodegradable, their potential applications in biological and pharmaceutical area have explored recently. Yokoyama and his coworkers modified PASP by hydrophilic chain poly(ethylene glycol) (PEG) at first, and then incorporation of an hydrophobic anticancer drug to PEG-PASP to obtain amphiphilic block copolymers, which could form nanoparticles in aqueous medium. Kim et al. and Nakato et al. reported hydrophobic modification of PASP by grafting hydrophobic octadecyl chains and dodecyl chains onto
222 Poster Abstracts the backbone of PSI polymers with aminolysis, respectively. After the hydrolysis of the residual succinimide, amphiphilic copolymers were obtained, and these two amphiphilic random copolymers could self-aggregate in water. In this paper, we report the synthesis of new type of amphiphilic graft copolymers with a hydrophilic P(HEA-co-PA) backbone and a hydrophobic PLA graft. Their aggregative behaviors in aqueous medium were studied in detail by dynamic light scattering (DLS). The experimental results showed that the grafting degree of the copolymers have a little influence on the size of the aggregates, the particle size is around 100-200 nm. During the preparation of the aggregates, when water was dropped into the DMF solution of P(HEA-co-PA)-g-PLA copolymers, the PLA chains should be interact each other to form tight microdomains by either intermolecular or intramolecular association and then the stable aggregates were formed. TEM of the aggregates of P(HEA-co-PA)-g-PLA were conducted and clearly indicate the spherical shape of aggregate particles. These biodegradable amphiphilic graft copolymers could be used for drug delivery.
P1 SYNTHESIS, CHARACTERIZATION AND LOCALIZED SURFACE 291 PLASMON RESONANCE STUDY OF SILVER ORGANOSOL Sudip Nath1, Tarasankar Pal2 and J. Manuel Perez1; 1Nanoscience Technology Center, University of Central Florida, 12424 Research Parkway, Orlando, FL 32826,USA; 2Department of Chemistry, Indian Institute of Technology, Kharagpur 721 302, India; [email protected]
A simple and reproducible technique for the synthesis of silver nanoparticles in organic solvent (i.e. organosol) is reported from specific a silver precursor, solid silver acetate. Hexadecylamine in molten condition acts as a solvent for silver acetate and finally imparts stability to the evolved nanoparticles. The amine-capped organosol shows unique stability as neither agglomeration nor oxidation takes place over one year. The synthesized silver particles have been characterized by UV-visible, TEM, XRD and XPS studies. The hexadecylamine stabilized silver organosol was employed to examine the altered optical properties in different solvent systems and ligands by accounting the changes in localized surface plasmon resonance (LSPR) spectrum. It was observed that the position of the surface plasmon band of silver nanoparticles is greatly affected by the solvents and ligands under consideration. The quantitative alteration of the LSPR spectrum involving encapsulated nanoparticles in a dielectric ligand shell has been rationalized from Mie theory. It has also been shown that cationic and anionic surfactants of different chain lengths induce significant changes in the optical properties of silver nanoparticles. The λmax of the LSPR gradually shifts to red with the increase in chain length of both the cationic and anionic surfactants, indicating specific binding of the surfactant molecules around silver nanoparticles. Finally the affinity of the synthesized silver nanoparticles with amine molecules has been accounted from HSAB principle into consideration.
223 Poster Abstracts
P1 3+ WATER-SOLUBLE FLUORESCENT LAF3:LN NANOCRYSTALS FOR 292 BIO-APPLICATIONS Yong Zhang, Feng Wang, and Wee Beng Tan, Division of Bioengineering, Faculty of Engineering, National University of Singapore, 9 Engineering Drive 1, Singapore 117576; [email protected]
Use of fluorescent labeling agents has greatly assisted the study of complex biological interactions in the field of biology. Besides commonly used fluorescent labeling agents such as organic fluorophores and quantum dots (QDs), lanthanide-doped nanocrystals have shown great potential to be a promising new class of fluorescent labeling agents recently. However, lanthanide-doped nanocrystals are usually made at high temperatures or in organic solvents, yielding products with a low processability, and as such their bio-applications are very limited. In this study we presented a simple method for synthesizing water-soluble lanthanide-doped LaF3 nanocrystals in aqueous solution at low temperature. The nanocrystals had a nearly spherical shape and an average size of below 30nm. They consisted of well crystallized hexagonal phases. The LaF3 nanocrystals doped with different lanthanide ions (Eu3+, Ce3+, Tb3+, and Nd3+) were synthesized, showing strong luminescence in the visible (VIS) and near-infrared (NIR) spectral regions. In 3+ particular, the LaF3:Nd nanocrystals, with both the major excitation and emission peaks located within the spectra wavelength range of 600-1100 nm (the “optical window” for cells and tissues), have a good potential to be used as fluorescent imaging probes for cell and animal studies.
P1 FLUORESCENT SILICA BEADS FOR DETECTION OF CERVICAL 293 CANCER Igor Sokolov, S. Iyer, Yaroslav Kievsky, Dept. of Physics and Dept. of Chemistry, and C. Woodworth, Dept. of Biology, Clarkson University, Potsdam, NY 13699, USA; [email protected]
Recently we have synthesized super-bright present fluorescent silica (glass) particles, beads. The beads are a few microns glass nanoporous particles which contain florescent dyes imbedded in the pores. The pores are self-closed, so the dye does not leak out being sealed inside the pores. The fluorescent activity of the beads is so high that there is no need in special fluorescent microscopy. Even ordinary dark-field microscopy mode is enough to detect the beads. The beads are stable for many days even under direct sun light. Having silica outside, the beads are biocompatible and can be used for labeling in-vivo. Here we report one interesting application of these particles for detection of cervical cancer. The cells from three different individuals (3 normal and 3 tumor) were tested for affinity. We have developed two different methods for detecting the affinity in a Petri-dish. After simple precipitation of the beads onto the cells, the unbounded beads removed by washing. The next method involved using centrifugation for the removal of the unbounded beads. Both methods show unambiguous identification of malignant versus normal cells.
224 Poster Abstracts
P1 DNA-TEMPLATED SELF-ASSEMBLY OF TWO DIMENSIONAL AND 294 PERIODICAL GOLD NANOPARTICLE ARRAYS Jaswinder Sharma, Rahul Chhabra, Yan Liu, Yonggang Ke, and Hao Yan*, Department of Chemistry and Biochemistry and Biodesign Institute, Arizona State University, Tempe, AZ 85287, USA; [email protected]
Nanoparticles (NPs) are being actively developed as building blocks for electronic, photonic and spintronics devices. When they are organized into well-defined ensembles, their collective properties depend critically on the inter-particle spacings and their hierarchical organization. Up to now, methods to control these parameters are scarce. DNA-templated self-assembly provides a unique solution to meet the above challenge, due largely to the following features of DNA and DNA-based nanostructures: DNA has well-defined geometry and most predictable, diverse and programmable intra/inter-molecular interactions; DNA can be modified by different chemical groups, which act as linkers to covalently attach DNA oligomers to the NPs; the design of stiff DNA-based nanostructures and their self-assembly have become a routine method to construct one- and two-dimensional DNA lattices of well-defined patterns. More specifically, when NPs are assembled onto self-assembled DNA lattices, the periodicities and interparticle spacings defined by the DNA scaffolds can be readily adjustable, with nanometer spatial precisions. This level of precision provides exquisite control in the construction of rationally defined NP architectures. Here we report the first time assembly of 5 nm Au NP functionalized with a single DNA strand on three different patterned structural templates that are constructed from self-assembled DNA tiles. We show that the periodicity and interparticle spacings of the NP nanoarrays can be precisely controlled by varying the DNA tile dimensions. In our strategy, the NP-conjugated strand first participates in the formation of a single DNA tile structure, this Au NP-bearing DNA tile was subsequently used to assemble with another DNA tile to form three different lattice structures, each with a well defined periodical pattern. This novel system is amenable both to the DNA lattice formation and preventing non-specific aggregations between the Au NPs. This development paves the way to assemble more complex nanoparticle arrays on DNA nanoscaffolds for future device applications.
P1 SYNTHESIS AND CHARACTERIZATION OF POLYMERIC 295 MICROSPHERES OF NARROW SIZE DISTRIBUTION CONTAINING IODINE FOR X-RAY IMAGING APPLICATIONS Anna Galperin, Rina Ben Shabat, Shlomo Margel, Department of Chemistry, Bar-Ilan University, Ramat-Gan 52900, Israel; [email protected], [email protected]
Polystyrene template microspheres of narrow size distribution ranging between approximately 0.2 and 5.0 μm were prepared by dispersion polymerization of styrene in a
225 Poster Abstracts
mixture of ethanol and 2-methoxy ethanol, under various polymerization conditions. Uniform radiopaque polystyrene microspheres of ca. 2.3 ± 0.2 µm were prepared by a single-step swelling of 2.3 ± 0.2 µm polystyrene template microspheres, dispersed in an aqueous solution with methylene chloride emulsion droplets containing 2,3,5-triodobenzoylethyl ester. After completing the swelling process, the methylene chloride was evaporated in order to lock the 2,3,5-triodobenzoylethyl ester in the polystyrene microspheres. The influence of the weight ratio [2,3,5-triodobenzoylethyl ester]/[polystyrene] on the % entrapped 2,3,5-triodobenzoylethyl was elucidated. Characterization of the radiopaque polystyrene microspheres was accomplished by light microscope, FTIR, TGA, SEM, XPS and elemental analysis. This novel encapsulation method of 2,3,5-triodobenzoylethyl ester within polystyrene microspheres by a single-step swelling process may be used as a model for encapsulation of various hydrophobic radiopaque drugs within uniform polystyrene template particles of various diameters for different x-ray imaging applications. Likewise, 2,3,5-triiodobenzoic acid was converted to its acrylic derivate by esterification with 2-hydroxyethyl methacrylate. The monomer, 2-methacryloyloxyethyl(2,3,5-triiodobenzoate) was then homopolymerized, using dispersion polymerization method, in 2-methoxyethanol in presence of polyvinylpyrrolidone as stabilizer and benzoyl peroxide as initiator, to get poly-(2-methacryloyloxyethyl[2,3,5-triiodobenzoate]) microspheres of 0.70 µm ± 0.05. The microspheres were characterized by FTIR, TGA, DSC, SEM, BET and elemental analyses. The effect of some reaction parameters on various aspects of dispersion polymerization of MAOETIB was studied. The radiopacity of the microspheres of both types was demonstrated by an imaging technique based on x-ray absorption usually used in hospitals. This novel radiopaque microspheres could be used for various x-ray imaging needs, e.g. blood pool, body organs, embolization, dental composition, implants, protheses and nanocomposites.
P1 INCORPORATION OF MAGNETIC PARTICLES IN POLYMER 296 SYSTEMS OF XYLAN Amanda K. A. Silva, Érica L. da Silva, Juliana F. de Carvalho, Elquio E. Oliveira, Karlyle Nalena da S. Santos, Toshiyuki N. Junior, Luiz Alberto L. Soares, E. Sócrates T. Egito, Universidade Federal do Rio Grande do Norte, Departamento de Farmácia, Laboratório de Sistemas Dispersos (LASID), Rua Gal. Gustavo Cordeiro de Farias, s/n, 59010-180, Natal/RN, Brazil. [email protected]; and Artur S. Carriço, Universidade Federal do Rio Grande do Norte, Departamento de Física Teórica e Experimental, Campus Universitário, s/n, 59072-970, Natal/RN, Brazil.
Magnetic systems with magnetite particles have been proposed for oral use as magnetic resonance contrast agents and magnetic markers for monitoring gastrointestinal motility. As magnetite is soluble in acid, particles may undergo dissolution at gastric pH. Regarding pharmaceutical technology, protecting compounds from gastric environment is a key issue. In fact, many approaches have been proposed, namely a strategy that relies on the resistance of some polysaccharides to the digestive action of gastrointestinal enzymes. The
226 Poster Abstracts matrices of polysaccharides are assumed to remain intact in the physiological environment of stomach and small intestine. Once they reach the colon, bacterial polysaccharidases come into play, and degradation of the matrices takes place. Such group of polysaccharides is comprised of amylase, chitosan, pectin, dextran, inulin, chondoitrin, xylan, etc. The aim of this work was to develop xylan-coated magnetic microparticles in order to protect magnetite form gastric dissolution. Such polymer-based magnetic microparticles were produced by emulsification/crosslinking method. The sample characterization was performed by laser scattering particle size analysis, optical microscopy, thermogravimetric analysis and vibrating sample magnetometry. Characterization data showed that polymeric superparamagnetic particles were successfully produced. Polymer/magnetite ratio was 75:25. In vitro dissolution tests at gastric pH for 2h were evaluated for both magnetic particles (MP) and polymeric magnetic particles (PMP). Dissolution rate was nearly 30% and 3% for MP and PMP, respectively. In conclusion, the obtained results have demonstrated the feasibility of the presented method to coat, and protect magnetite particles from gastric dissolution. Such systems may be very promising for oral administration.
P1 MAGNETITE PARTICLE DISSOLUTION AT GASTRIC pH 297 Amanda K. A. Silva, Érica L. da Silva, Juliana F. de Carvalho, Elquio E. Oliveira, Karlyle Nalena da S. Santos, Toshiyuki N. Junior, Luiz Alberto L. Soares, E. Sócrates T. Egito, Universidade Federal do Rio Grande do Norte, Departamento de Farmácia, Laboratório de Sistemas Dispersos (LASID), Rua Gal. Gustavo Cordeiro de Farias, s/n, 59010-180, Natal/RN, Brazil. [email protected]; Artur S. Carriço, Universidade Federal do Rio Grande do Norte, Departamento de Física Teórica e Experimental, Campus Universitário, s/n, 59072-970, Natal/RN, Brazil.
Magnetite particles have been proposed for oral use as magnetic resonance contrast agents and magnetic markers for monitoring gastrointestinal motility. Despite the promising properties, magnetic particles may dissolve in acid media. The normal transit time in the stomach (pH 1–2) is 2 h. Due to the low pH, magnetite particle dissolution may take place during the period in which particles are in the stomach. The aim of this work was to carry out a dissolution test to mimetize the gastric environment in order to evaluate magnetite dissolution. First, magnetic particles were produced by coprecipitation of iron salts in alkaline medium; then, sample characterization was performed. Characterization data showed that the produced sample consisted of magnetite particles in the size range of 0.5-5 µm. In-vitro dissolution test was performed at 37 °C by using a rotating paddle at the rotation speed of 100 rpm in a 0.1 M HCl solution. At regular intervals of time, aliquot samples were withdrawn and the amount of free iron in the medium was determined by measuring the UV absorbance at 512 nm (phenanthroline method). According to the dissolution test, magnetite particles presented the highest dissolution rate in the early beginning of the dissolution test, 8.8% of dissolution during the first 15 min. Then, dissolution presented an almost linear pattern from 15 to 120 min, without reaching a plateau. It was found that almost 30% of the sample was dissolved by 120 min. In fact, this
227 Poster Abstracts is a striking result, considering that such high particle loss in gastric environment will compromise particle use. In order to overcome this drawback, an enteric coating on particle surface may be a promising approach. It is believed that coating the surface of the magnetite particles with such material can prevent their dissolution.
Session P1 COMPOSITE THERMORESPONSIVE MICROGEL CORE DECORATED 298 WITH MAGNETIC NANOPARTICLES J.E. Wong, A.K. Gaharwar, W. Richtering, Institute of Physical Chemistry, RWTH-Aachen, Germany; D. Bahadur, Dept. of Metallurgical Engg. and Mats. Sci., IIT-Bombay, Mumbai, India and D. Müller-Schulte, Magnamedics GMBH, Aachen, Germany; [email protected]
Magnetic nanoparticles have recently received tremendous attention due to their potential application, especially in the biomedical field. The driving force behind this work lies in the possibility of encapsulating drugs or other macroions within a matrix, to then use magnetic field to target, magnetic induction to release the drug in a controlled and diffused manner as determined by the permeability of the shell around the matrix. We report the synthesis and characterization of a new type of composite core-shell microcapsule. We used the layer-by-layer technique to (i) alternately adsorb charged polyelectrolytes onto a thermoresponsive core of a microgel, then (ii) decorate the shell consisting of polyelectrolyte multilayers with oppositely charged magnetic nanoparticles. The magnetic iron oxide are synthesized by co-precipitation technique and the microgel used is Poly(N-isopropylacrylamide) (PNiPAM), synthesized by batch polymerization. The polyelectrolytes used are poly(diallyldimethylammoniumchloride) (PDADMAC) and poly(styrenesulfonate) (PSS). Depending on the nature of the outermost polyelectrolyte layer, oppositely charged magnetic nanoparticles can be decorated on the polyelectrolyte-coated microgel. Dynamic light scattering (DLS) reveals a hydrodynamic radius of about 200 nm for the coated microgel and electrophoretic measurements confirm charge reversal at each deposition step (+30 mV and -30 mV on deposition of PDADMAC and PSS respectively), showing that each polyelectrolyte layer is successfully adsorbed on the microgel. DLS confirms the overall diameter of the magnetic core-shell composite to be around 600 nm (nanoparticles being about 90 nm in diameter). We shall show additional characterization results such as X-Ray Diffraction (XRD), Superconducting Quantum Interference Device (SQUID), Electron Microscopy, and thermogravitometry analysis (TGA). Such core-shell composite particles with unique and modified properties have promising applications in controlled, extended, and targeted delivery vehicles for drugs, proteins, vaccines, cosmetics and food supplements.
P1 MAGNETIC NANOPARTICLES - POLYELECTROLYTE 299 INTERACTION : A LAYERED APPROACH FOR BIOMEDICAL APPLICATION A.K. Gaharwar, J.E. Wong, W. Richtering, Institute of Physical Chemistry,
228 Poster Abstracts
RWTH-Aachen, Germany; D. Bahadur, Dept. of Metallurgical Engg. and Mats. Sci., IIT-Bombay, Mumbai, India and D. Müller-Schulte, Magnamedics GMBH, Aachen, Germany; [email protected]
Magnetic nanoparticles (MNPs) offer some unique advantages as sensing, image enhancement, and site-specific drug delivery. However, the size, charge, and surface of these MNPs strongly affect their applicability in the majority of the envisioned biomedical applications. It is important that the systems are biocompatible and capable of being functionalized for recognition of specific target sites in the body, hence the surface modification of the MNPs with polyelectrolytes (PEs). In this work we report two different surface modification methods. One approach is to prepare the MNPs from iron salts in aqueous solutions in the presence of PEs to impart an in-situ PE coating. Uncoated MNPs have positively charged surface, so the PEs used are either negatively charged (poly(acrylic acid) (PAA) and poly(styrenesulfonate) (PSS)) or neutral (dextran). Charge overcompensation is observed in the former case as confirmed by charge reversal obtained from electrophoretic measurements. However in the latter case, the zeta potential is neutral, showing that the dextran sheath effectively shields the charges on the MNPs. Transmission electron micrographs (TEM) show that the MNPs remained discrete. The X-ray diffraction (XRD) patterns indicate that binding of PEs do not result in any phase change. Magnetic measurement reveals the MNPs are superparamagnetic, and their saturation magnetisation is only slightly reduced after PE binding. The second approach is to coat the MNPs with PE multilayers using the layer-by-layer technique. The PEs used are polyethylenimine (PEI), poly(diallyldimethylammoniumchloride) (PDADMAC) and PSS. Electrophoretic measurements confirm charge reversal at every stage of layering. After every PE layer we observe not only an increase in size of the particles but also in the size distribution. Measurements from dynamic light scattering (DLS), superconducting quantum interference device (SQUID) and thermogravitometry analysis (TGA) will also be discussed. Preliminary results obtained by cell incubation tests using tumor cells and yeast cells show promising results. Fluorescence micrograph shows immediate attachment of MNPs to the yeast cells, and they successfully aligned themselves in the presence of an external magnetic field.
P1 FUNCTIONALIZED SUPERPARAMAGNETIC NANOPARTICLES AS 300 PATHOGEN CAPTURE PROBES Arnold J. Kell and Benoit Simard, Steacie Institute for Molecular Sciences, National Research Council, 100 Sussex Drive, Ottawa, Ontario, Canada K1A 0R6 and Gale Stewart, Ann Huletsky, Maurice Boissinot, and Michel Bergeron, Centre de Recherche en Infectiologie, 2705 Boul. Laurier (RC-709), Ste-Foy, Québec, Canada, G1V 4G2; [email protected]
Superparamagnetic nanoparticles are garnering a lot of attention in the fields of biodiagnostics and medicine, where the development of water-soluble nanoparticles with universal linkers allows for the covalent attachment of essentially any biological moiety to their surfaces. Recently we have prepared silicon dioxide covered iron oxide nanoparticles
229 Poster Abstracts
(SIONs) and modified the surface of these nanoparticles with amine groups capable of undergoing a variety of bio-conjugation reactions to endow the interface with many useful functional groups where our goal is to develop SIONs capable of capturing pathogens from biological samples. As such, we will briefly describe the preparative conditions employed to optimize the amine surface coverage and stability, but the thrust of the presentation will focus on the ability of these nanoparticles to be incorporated into a number of pathogen capture assays. We will show that these amine-modified SIONs (A-SIONs) can efficiently capture polystyrene (PS) beads (bacteria mimics) from aqueous solution upon covalent attachment to the PS surface where the linking chemistry is easily controlled. A-SIONs have also been modified with vancomycin, an antibiotic well known to interact with the glycopeptide chains present on the surface of gram positive bacteria. These vancomycin-modified SIONs (V-SION) are capable of magnetically confining vancomycin antibody-modified PS beads (another bacteria mimic) as well as both gram positive and gram negative bacteria (E. faecalis S. epidermidis and E. coli) from aqueous solution. Though the interactions between the bacteria (and bacteria mimic) and the SION in this case are entirely noncovalent (hydrogen bonding and electrostatic interactions), both modified PS beads and bacteria can be readily captured through the appropriate modification of the SION nanoparticles. Together these results indicate that SIONs hold promise as a means of isolating pathogenic species from biological samples.
P1 CORE-SHELL NANOPARTICLES WITH COVALENTLY LINKED TNF 301 FOR CELL SIGNALLING INVESTIGATIONS Marc Herold, Thomas Schiestel, Herwig Brunner, and Günter E. M. Tovar. Laboratory for Biomimetic Surfaces, Fraunhofer Institute for Interfacial Engineering and Biotechnology, Nobelstr. 12, Stuttgart, 70569, Germany, Peter Scheurich, Klaus Pfizenmaier, Institute for Cell Biology and Immunology, University Stuttgart, Allmandring 31, D-70569 Stuttgar; [email protected]
Cytokines play a key role in cell-cell communication. Normally they are investigated in a soluble form or (over)expressed on the surface of a cell line. Both ways are often erroneous as the first is an improper simplification whereas the latter usually leads to a complex response pattern due to multiple cell-cell interactions. Objectives of this work was the built-up of synthetic-bionic hybrid systems consisting of organically modified silica nanoparticles and the cytokine Tumor Necrosis Factor-α (TNF) to create a defined system for the investigation of membrane-TNF signaling. Silica particles with a size of about 100 nm were synthesized. We have modified the nanoparticles with different functional shells to provide maleinimide groups at their surface. Free cysteine groups were introduced into the N-terminus of TNF by recombinant methods. This approach leads to a covalent and site-directed coupling of the protein. The resulting hybrid particles were characterized by physicochemical and biological methods. The particles initiate cellular responses otherwise only seen for cellmembrane-bound TNF. Thus, the hybrid Nanoparticles are clearly showing new and cell analogous properties. These particles are thus referred to as NANOCYTES® and are a valuable tool for biological research with high potential as a new drug for cancer therapeutics.
230 Poster Abstracts
P1 NOVEL NANOPARTICLES WITH ACTIVATED ESTER SURFACE 302 PREPARED BY EMULSIONPOLYMERIZATION OF POLYMERIZABLE SURFACTANTS Marc Herold, Maria Håkanson, Herwig Brunner, and Günter E. M. Tovar, Institute for Interfacial Engineering (IGVT), University of Stuttgart & Fraunhofer Institute for Interfacial Engineering and Biotechnology (IGB), Nobelstr. 12, D-70569 Stuttgart E-Mail: [email protected]
The present contribution describes the synthesis of two surfmer molecules (polymerizable surfactants) and their use to synthesize in a one-stage reaction polymer nanoparticles for bioconjugation. The surfmers bear activated ester groups which ensure the reactivity of the final particles. The surfmers p-(11(acrylamido)undecanoyloxy)-phenyldimethyl sulfoniummethylsulfate (AUPDS) and p-(11(methacrylamido)undecanoyl oxy)-phenyldimethylsulfoniummethylsulfate (MUPDS) were characterized and their polymerisation behaviour in emulsion copolymerisation with methylmethacrylate (MMA) and styrene was examined. Both compounds supported the formation of positively charged nanoparticles with a diameter in the range 100 nm to 170 nm. The composition of the copolymer p(AUPDS-co-MMA) was determined by 1H-NMR spectrometry and elemental analysis. Scanning electron microscopy confirmed a narrow particle size distribution. The polymer particles were conjugated with streptavidine (SAv) and a streptavidine peroxidase conjugate (SAv-POD) to evaluate the reactivity of the activated ester that was covalently linked to the particle surface. Up to 28 mg Streptavidin was immobilized per 1 g particles, binding up to (2000 ± 360) µmol biotin ligands per 1 g particles.Various other molecules bearing N-nucleophilic groups, such as amines or hydrazides, e.g. fluoresceineamine, n-butylhydrazide, remazol-brilliant-blue®, were immobilized at the particle surface.
P1 PRODUCTION OF NOVEL MULTICOMPONENT PHARMACEUTICAL 303 NANO AND MICROPARTICLES WITH AN AEROSOL FLOW REACTOR METHOD Janne Raula, David P. Brown, Anna Lähde, Esko I. Kauppinen, Center for New Materials and Laboratory of Physics, Helsinki University of Technology, P.O. Box 1602, FIN-02044 VTT, Finland, and Hannele Eerikäinen, Orion Pharma, Pharmaceutical Product Development,P.O. Box 65, FIN-02101, Espoo, Finland; [email protected]
Good flowability of pharmaceutical powders is an important feature for powder handling. Moreover, the particles should remain stable and intact to preserve their chemical and physical properties over extended storage periods. To achieve these goals, excipient materials such as polymers and surface-active materials are used, not only to stabilize a drug particulate formulation, but also to reduce the interaction forces between particles. This work presents a novel aerosol flow reactor method to produce nano and micronsized
231 Poster Abstracts
particles with matrix-type structures as well layered structures via molecular self-assembly. In the method, the starting solution containing an active material and expicient(s) are atomized and fed into a heated laminar flow reactor with the aid of dry or solution-saturated carrier gas. By controlling the gas flow rate, residence times up to tens of seconds are achieved, enabling molecular ordering such as self-assembly and crystallization to take place. We show the production of ketoprofen and naproxen nanoparticles stabilized with pharmaceutical polymers such as Eudragits and core-shell type salbutamol and salt particles coated with the biocompatible peptide L-leucine at various synthesis conditions. The shape of the latter particles varied from wrinkled to rounded with hard and crystalline L-leucine nanocrystals on the surface. These L-leucine crystals dramatically reduced the contact area between fine particles thus improving flowability. Downstream of the reactor, particle size distributions were determined with aerosol size spectrometers. Particle morphology was characterized with SEM and TEM.
P1 SIZE AND CONCENTRATION MONITORING OF HFA SUSPENSIONS 304 Philippe G. Rogueda, AstraZeneca R&D Charnwood, Bakewell Road, Loughborough, LE11 2HD, U.K., [email protected]. Vitaly Buckin, Evgeny Kudryashov Ultrasonic Scientific, 1 Richview Office Park, Clonskeagh, Dublin 14, IRELAND.
Pressure metered dose inhalers (pMDI), extensively use din the treatment of asthma, are solutions or suspensions of micronised drugs in HFA (hydro fluoro alkanes) propellant. The monitoring of the suspension properties of these formulations has always been an issue. These could not be studied by traditional techniques (light based technologies), because of their concentration and the fact that the dispersing phase is a pressurised liquid. High Resolution Ultrasonic Spectroscopy (HRUS) has been developed to monitor the phase separation phenomenon in these suspensions. HRUS was adapted to work with HFA propellants, and tested on a selection of commercial pMDI suspensions: Ventolin, Flixotide and Salbuhexal. The variation of the average particle size (Dv50) and the volume concentration as a function of time were recorded over a duration of one hour. All suspensions flocculated before sedimenting. The kinetics of the aggregation was very fast, a couple of minutes, whereas full sedimentation was much slower (in excess of one hour). It is the first time that the kinetics of the variation of the size and concentration of HFA suspensions can be recorded quantitatively with accuracy. The application of the HRUS technique extends beyond bench testing of HFA suspensions, and could be used for concentration and stability monitoring of solution pMDIs, and for on-line measurements during production. The suspension properties were compared with the delivery efficiency of the pMDI.
P1 DISPERSION OF NOVEL INHALATION DRUG POWDERS WITH 305 NANOCRYSTALLINE L-LEUCINE COATING David P. Brown, Janne Raula, Esko I. Kauppinen, Center for New Materials
232 Poster Abstracts
and Laboratory of Physics, Helsinki University of Technology, P.O. Box 1602, FIN-02044 VTT, Finland, and Juha A. Kurkela, Aerosol Technology Group, VTT Processes, P.O. Box 1602, FIN-02044 VTT, Finland; [email protected]
High flowability and efficient dispersion are important requirements for inhalable pharmaceutical powders delivered by dry powder inhalers (DPI). Reduced contact area and hard particle surfaces are known to decrease forces between fine particles and significant research has been conducted to control and optimize physical surface properties. This work presents a novel aerosol flow reactor method to produce inhalable drug particles (size≤5 μm) with a core-shell structure in a one step process. The composite particles consist of salbutamol sulphate and the biocompatible peptide L-leucine. In the aerosol reactor, the molecular arrangements such as self-assembly and crystallization of L-leucine molecules take place resulting in the spherical drug particles becoming coated with hard and crystalline L-leucine leafy structure. These L-leucine crystals significantly reduced the contact area between fine particles, thus, improving flowability. This work also demonstrates the feasibility of a novel deagglomeration device that enables to study the deagglomeration behavior of the powders in a turbulent flow field with a constant and continuous powder feed. Jet Reynolds numbers deagglomerating the powders varied from 8000 to 48000. The size distributions of the deagglomerated fine powders were determined with aerosol size spectrometers. Particle morphology was characterized with SEM and TEM. The sample powders were fed into the deagglomeration device with and without lactose carrier particles. Salbutamol particles with L-leucine coating showed very good deagglomeration even without carrier particles, having a fine particle fraction (FPF) up to 0.47. Moreover, these coated powders were easily fed without carriers indicating good flowability. In general, the deagglomeration from the carrier and neighboring particles improved with increased flow rate. However, the influence of L-leucine coating on deagglomeration was more pronounced with less L-leucine. This is interpreted as the interplay between the beneficial effects of surface roughness and the detrimental effects of interlocking L-leucine leaves.
P1 QUANTITATIVE HARDNESS AND SURFACE CHARACTERISATION 306 BY ATOMIC FORCE MICROSCOPY FOR USE IN EARLY STAGE SCREENING OF PHARMACEUTICAL FORMULATIONS M. C. Perkins, J. Zhang, S. Y. Luk, C. E. Madden, C. J. Roberts*, N. Patel, and A. Parker, Molecular Profiles Ltd., 8 Orchard Place, Nottingham Business Park, Nottingham, NG8 6PX, UK; *Laboratory of Biophysics and Surface Analysis, School of Pharmacy, University of Nottingham, Nottingham, NG7 2RD, UK; [email protected]
Understanding the physical properties of any new active pharmaceutical ingredients (API) is crucial in understanding the how that API will behave during processing and formulation. As such tools that can predict these properties at early stages of development are crucial. Atomic Force Microscopy (AFM) has been used extensively as a tool for understanding the adhesive and cohesive properties of numerous API’s and offers a
233 Poster Abstracts
particular advantage as it has the ability to screen small quantities of material. The analysis of these surface mediated solid-solid interactions can be extended further to provide information on a range of important surface properties. Determination of deformation mechanics, the accurate quantification of hardness and determination of surface energy of pharmaceutical components from the interaction curves are demonstrated. Here we apply nano-deformation measurements to crystals of α-lactose monohydrate to derive a quantitative value of young’s modulus (E). This data can also be used in a qualitative manner to describe the type of deformation a material undergoes, providing an indication of elastic, viscoelastic and plastic deformations. These curves are also applied spatially to locate and characterise the presence of amorphous and crystalline materials on the surface of a modified sorbitol crystal. In the final section, we detail the measurement of the dispersive component of the surface free-energy for purely crystalline α-monohydrate and spray-dried amorphous lactose. The γD determined from a single crystal ((0,-1,-1) face) of α-lactose monohydrate was 23.3 ± 2.3 mJm-2 and for the amorphous form 57.4 ± 7.9 mJm-2. These values clearly delineate the crystalline and amorphous forms of lactose. Furthermore, these values are consistent with data previously obtained from Inverse Gas Chromatography (IGC) and contact angle measurements. However, unlike contact angle measurements that produce a surface measurement and IGC that biases the high energy sites the AFM data provides access to localised measurements, such as individual faces or in the identification and location of components within heterogeneous samples (e.g. surface amorphous material within a crystalline sample). The AFM data presented here indicates new opportunities for material characterisation in particular for the characterisation of small quantities of material (ie single particles). Taken with current advances in the application of AFM, this approach may be used to aid candidate selection through improvements in early stage screening of API physical properties.
P1 PARTICLE SIZE DISTRIBUTION CHARACTERIZATION OF 307 SUB-MICRON SUSPENSIONS USING ELECTRON MICROSCOPY Ben Lich, Hans Kruesemann, FEI Electron Optics B.V., 5 Achtsteweg Noord, Eindhoven, 5600 KA, Netherlands; and Arjen Tinke, Johnson & Johnson Research, Turnhoutseweg 30, B-2340 Beerse, Belgium; [email protected]
In the pharmaceutical industry there is a growing tendency to develop less soluble active ingredients (API). In order to assure acceptable in-vivo dissolution and bioavailability, a clear trend is observed of going towards a larger specific surface area of the active substance and thus to smaller drug particles. In order to prevent agglomeration of small particles below ca. 1 μm, they are generally stabilized in a suspension by means of the addition surfactants. In these cases where the dissolution of a suspension is the dominant factor in the release mechanism of the drug, to keep the product under control it is necessary to characterize the sub-micron or nano-size particles in terms of their particle size distribution (PSD). Since the shape of a particle can be considered as its size manifestation in three dimensions and since the surface/volume ratio exponentially increases with a smaller size of the particles, for sub-micron and nano-size particles the characterization of shape becomes increasingly important . Current size measurement
234 Poster Abstracts techniques (e.g., Laser Diffraction) generally rely on an indirect measurement of the particle size, and reporting of PSD data generally occurs based on the assumption that the particles are spherical. As pharmaceutical actives are typically obtained by crystallization and milling processes, particles are definitely non-spherical and hence there is a scientific interest in characterizing the accurate particle size and shape. The latter yields a better prediction and understanding of the solubility of the active ingredients and hence will lead to improved drug formulations. In this study for rather wide size distributions of a sub-micron suspension in water we present very promising results based on expanding the “seeing is believing” addagium, i.e. scanning electron microscopy images are used not only for qualitative purposes, but also for the quantitative particle size distribution analysis of a pharmaceutical sub-micron suspension. The effects of sample preparation, image acquisition conditions and image processing are evaluated. In particular, the consequences related to the choice of filters, imaging vacuum mode and detector technology will be discussed in more detail.
P1 METHODS FOR CHARACTERIZATION OF MICRO- AND NANO-SIZED 308 PARTICLES AND CAPSULES Holger Woehlecke, Dietmar Lerche, Dr. Lerche KG, Rudower Chaussee 29, D-12489 Berlin, Germany; and Rudolf Ehwald, Humboldt-University, Institute of Biology, Invalidenstr. 42, D-10115 Berlin, Germany; [email protected]
Microparticles and microcapsules have found a broad spectrum of applications, each of which requiring special physical properties as mechanical behavior and permeability characteristics. A complex of methods is presented to analyze these important properties. For complex characterization of size exclusion limits (cut-off) and size dependent permeability of permeable molecules the authors developed a fast and convenient method based on high-resolution gel permeation chromatography. The permeability of narrow size fractions of a polydisperse polymer solution is measured synchronously by this method. Different texture properties of microcapsules and –beads in a diameter range from 500 to 5000 µm can be easily characterized by compression analysis of single particles by means of the LUMiTexture. This includes e.g. burst point and apparent interfacial tension of microcapsules and -spheres. Furthermore we report on a new method for evaluation of the elasticity/deformability of capsules and small particles to quantify packing, compression and dilatation behavior. This method is based on sedimentation analysis by means of an analytical centrifuge LUMiSizer®. This device has an integrated optic-electronic sensor system and allows for analysis of spatial and temporal changes of light transmission during centrifugation of particle suspensions. This system also allows for measuring of particle size. The presented methods can be useful for screening of new particle and capsule membrane materials for special applications and for the characterization and certification of new products. The authors are interested in co-operation. Work was supported in part by project “Werkzeuge zur Applikation von 3-D-Zellkulturen” EFRE and Berlin (Profit 10129304).
235 Poster Abstracts
P1 CHARACTERISING BICONTINUOUS CUBIC NANOPARTICLES BY 309 CRYO FIELD EMISSION SCANNING ELECTRON MICROSCOPY Shakila Rizwan1, Charlie Dong2, Ben Boyd2, Sarah Hook1 and Thomas Rades1 1New Zealand’s National School of Pharmacy, University of Otago, Dunedin 2Department of Pharmaceutics Victorian College of Pharmacy, Monash University, Victoria Lyotropic liquid crystals are complex systems formed by the assembly of amphiphilic molecules in solvent rich systems (usually water) into various geometries. The most common geometries include the lamellar, the cubic and the hexagonal phase. The phase we are interested in is the bicontinuous cubic phase, which in the presence of excess water may be dispersed into nanoparticles known as cubosomes. Cubosomes are a novel lipid particulate delivery system currently being investigated for drug delivery purposes. They offer distinctly different properties over conventional lipid based particulate systems such as liposomes or emulsions. The particles have two water channels, a hydrocarbon chain region and an interfacial head group that allows for incorporation of hydrophobic, hydrophilic or amphiphilic compound. Previously cubosomes have been characterized by cryo transmission electron microscopy (cryo TEM) with small angle x-ray diffraction (SAXS) confirming the bicontinuous liquid crystalline type. The present study aims to characterize bicontinuous liquid crystalline nanoparticles by the novel method of cryo field emission scanning electron microscopy (cryo FESEM). Bulk cubic phase and cubosomes were prepared and analysed using cryo FESEM and SAXS. The results show the tortuous, bicontinuous nature of the cubic phase with a non intersecting network of water channels similar to the mathematical description of the bicontinuous cubic phase. Particles also show the same underlying tortuous structure entirely consistent with that of the bulk cubic phase. Cryo FESEM provides valuable insight into the morphological features, which to date have only been demonstrated mathematically. We are able to show with this technique the unique details of these systems and provide strength to support the nodal surface representation of bicontinuous cubic liquid crystalline systems.
P1 CHARACTERIZATION OF METAL DEPOSITION ON 310 NANOPARTICLES AT SINGLE PARTICLE LEVEL Grit Festag, Andrea Csáki, Thomas Schüler, Robert Möller, Wolfgang Fritzsche, Institute for Physical High Technology (IPHT), Photonic Chip Systems Dep. A.-Einstein-Str. 9, 07745 Jena, [email protected]
Metal nanoparticles, especially gold nanoparticles have been used as labels in electron and light microscopy for many years. Nowadays, they have generated considerable interest for numerous processes in molecular construction as well as in various kinds of bioanalytical methods. Thereby, specific reductive deposition of silver or gold on the nanoparticles has a tremendous importance for signal enhancement. Although a variety of metal enhancement methods have been established there are still few data about the rate, intensity, and
236 Poster Abstracts specificity of their enhancement effect. For characterizing we non-specifically immobilized gold nanoparticles on silicon oxide and enhanced them by different commercial and homemade metal salt solutions. Atomic force microscopy (AFM) was used to study this process at the single particle level in order to reveal the influence of parameters like particle size, composition of enhancement solution, length of incubation etc.. Because on-line measurements were not feasible, techniques were developed to relocate certain positions at the sample in order to image particle arrangements after every enhancement step. We could demonstrate that the examined metal enhancement methods considerably differed in enhancement velocity, homogeneity, and specificity. We extended these studies to an investigation of enzymatic metal deposition. Thereby an enzyme complex catalyzes the growth of a metal layer. We compared the enzymatic and the particle-induced deposition regarding growth kinetics and specificity of the reaction with special emphasis of a possible application in an electrical chip-based DNA detection.
P1 ADHESION FORCES BETWEEN HYBRID COLLOIDAL PARTICLES AND 311 CONCANAVALIN A L. B. R. Castro1, 2 and D.F.S. Petri1, 1Instituto de Química, Universidade de São Paulo, Brazil; 2Max Planck Institut for Polymer Research, Mainz, Germany; [email protected]
Hybrid particles of poly(methyl methacrylate) and carboxymethylcellulose, PMMA/CMC were attached to atomic force microscopy cantilevers and probed against Concanavalin A (ConA) films formed either on Si wafers or on CMC substrate. Regardless the substrate, the approach curves showed different inclinations, indicating that the probe first touches a soft surface and then a hard substrate. The distance corresponding to the soft layer was estimated as (20 ± 10) nm and was attributed to the CMC layers attached to the hybrid particles surfaces. Probing PMMA/CMC particles against ConA adsorbed onto Si wafers yielded retract curves with a sawlike pattern. The average range of adhesion forces (maximum pull-off distance) and mean adhesion force were estimated as (100 ± 40) nm and –(11 ± 7) nN, respectively, evidencing multiple adhesions between CMC sugar residues and ConA. However, upon probing against ConA adsorbed onto CMC substrates the mean pull-off distance and mean adhesion force were reduced to (37 ± 18) nm and –(3 ± 1) nN, respectively, indicating that the ConA molecules immobilized onto CMC films are less available to interact with the hybrid particle than the ConA molecules adsorbed onto Si wafers. Another set of experiments, where PMMA/CMC particle probed against ConA-covered Si wafers in the presence of mannose, showed that the addition of mannose led to a considerable decrease in the mean adhesion force from – (11 ± 7) nN to – (3 ± 1) nN. Two hypotheses have been considered to explain the effect caused by mannose addition. The first suggested the desorption of ConA from the substrate, so that the hybrid particle would probe bare Si wafer (weak adhesion). The second proposed the adsorption of mannose onto ConA layer and onto CMC at the surface of hybrid particle, so that mannose layer would probe against another mannose layer, leading to low adhesion forces. In situ ellipsometry and capillary electrophoresis have been applied to check the hypotheses.
237 Poster Abstracts
P1 INVESTIGATION OF THE PARTICLE SIZE DISTRIBUTION AND ZETA 312 POTENTIAL OF ALUMINUM HYDROXIDE ADJUVANT FOLLOWING FREEZE DRYING AND FREEZE THAWING Amber Clausi, Scott Merkley, and Theodore Randolph, Department of Chemical and Biological Engineering, UCB 424, University of Colorado, Boulder, CO 80309; [email protected]
Protein-based therapeutics often require a lyophilized formulation to ensure adequate long-term stability. However, it has been reported that aluminum-based adjuvant salts, an essential component of vaccine formulations, aggregate upon freezing and thus preventing their use. The purpose of this study is to examine the effect of cooling rate on the particle size distribution and zeta potential of aluminum hydroxide adjuvant particles. Two cooling rates were utilized: a slow cooling utilizing a lyophilizer with a 0.5 ºC/min ramp rate, and a faster cooling rate obtained by dripping the solution into liquid nitrogen. Samples were analyzed following a freeze/thaw cycle and following freeze drying utilizing both cooling methods. It was found that the slower cooling rate results in aggregation of the aluminum hydroxide particles with an order-of-magnitude increase in particle diameter. These effects are minimized through the faster cooling rates of the dripped solutions with a particle size distribution similar to that of the liquid formulation. The aggregation in the tray cooled samples is strongly dependant on the trehalose concentration in the formulation, with high trehalose concentrations preventing aggregation, but the ionic strength of the solution has little effect. No additional aggregation is seen following drying or reconstitution of the dried product, indicating that it is induced during freezing. The aggregation in the tray frozen samples is accompanied by a decrease in the zeta potential of the particles, whereas no significant decrease in zeta potential was seen for the drip freeze dried samples when compared to the liquid. It was also seen that the zeta potential of the aluminum hydroxide particles in succinate buffer decrease over a two-week aging period indicating a possible surface substitution of hydroxyl and succinate groups when stored as a liquid.
P1 INCORPORATION OF HYDROPHILIC COMPOUNDS IN SOLID LIPID 313 PARTICLES USING SUPERCRITICAL FLUID TECHNOLOGY A.R. Sampaio de Sousa1, Ana A. Matias1, Hermínio C. de Sousa2, Catarina M.M. Duarte1*; 1Nutraceuticals and Delivery Laboratory, ITQB/IBET, Aptd. 12 – 2780 Oeiras, Portugal; 2Departamento de Engenharia Química, FCT-Univ.Coimbra, Polo 2 – Pinhal de Marrocos, 3030-290 Coimbra, Portugal; [email protected], [email protected]
Supercritical fluids (SCF) are a clean and environmentally friendly technology which has been shown to be a viable option in the formulation of particulate delivery systems, such as microparticles and nanoparticles, liposomes, and inclusion complexes, which control the delivery and/or enhance the stability of labile compounds. The advantages of SCF technology include use of mild operating conditions, the use of benign nontoxic materials (such as CO2), and the minimization of organic solvent use and production of particles with controllable morphology, narrow size distribution, and low static charge. The technique used in this work, PGSS® -
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Particles from Gas Saturated Solutions seems to be one of the most promising methods of particle formation with supercritical fluids, allowing the generation of particles from a great variety of substances that need not to be soluble in supercritical carbon dioxide. In this method, CO2 is incorporated in the bulk of a melted matrix, whose melting point can be significantly decreased by the presence of CO2, creating a gas-saturated solution that can be expanded through a nozzle generating solid particles. The aim of this study was to incorporate hydrophilic compounds, such as ascorbic acid and powerful antioxidants obtained from olive by-products, in lipid particles. Lipids are extremely promising carriers that can improve the in vivo properties of bioactive ingredients and facilitate the incorporation of the compounds in hydrophobic media as oily products. All the lipid matrices used, as well as emulsifiers, are considered to be GRAS excipients. The particles obtained were analysed in terms of structure (SEM), particle size (PCS), water content and active compound content.
P1 ACETAZOLAMIDE CONTROLLED RELEASE FROM MICROPARTICLES 314 PREPARED BY SAS Ana Rita C. Duarte, Catarina M. M. Duarte1, Arlette Vega-González*, Pascale Subra2; 1Nutraceuticals and Delivery Laboratory, ITQB/IBET, Aptd. 12 – 2780 Oeiras, Portugal; 2Laboratoire d’Ingénierie des Matériaux et des Hautes Pressions, C.N.R.S., Institut Galilée, Université Paris XIII, 99 Avenue Jean Baptiste Clément, 93430 Villetaneuse, France ; [email protected]; [email protected]
Microparticles of Eudragit RS and Eudragit RL co-precipitated with acetazolamide were prepared using the supercritical anti-solvent technique (SAS). Composite particles of Eudragit RS 100 with acetazolamide and Eudragit RL 100 with acetazolamide, as well as mixtures of the polymers (50:50; 70:30 and 30:70) were coprecipitated with acetazolamide. In all experiments the ratio polymer:drug was 2:1. The drug release of acetazolamide from the particles prepared was evaluated. Results suggest that a controlled release system for this drug was successfully prepared and the rate of drug release could be controlled to some extent by the addition of different polymer ratios. The release profiles were modelled with Fick’s law of diffusion and Power law. The systems studied present a release mechanism controlled by drug diffusion, which complies with Fick’s law of diffusion.
P1 PHARMACOLOGICAL AND HISTOLOGICAL EVALUTION OF 315 HYDROXYPROPYL-β-CYCLODEXTRIN-BUPIVACAINE INCLUSION COMPLEX Daniele R. de Araujo1, Cíntia M. S. Cereda1, Giovana R. Tofoli2, Leonardo F. Fraceto 1,3, José Ranali2, Angélica F. A. Braga4 and Eneida de Paula1; 1 State University of Campinas, Dept. of Biochem., Cidade Universitária Zeferino Vaz s/n, Campinas, SP, Brazil; 2 State University of Campinas, Dept.of Physiological Sciences, Av. Limeira 901, Piracicaba, SP, Brazil; 3 University of Sorocaba, Dept. of Pharmacy, Rod. Raposo Tavares, Km 92,5; Sorocaba, SP, Brazil; 4 State University of Campinas, Dept. of Anesthesiol., Cidade Universitária Zeferino Vaz s/n,
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Campinas, SP, Brazil; [email protected]; [email protected]
Bupivacaine (BVC) is probably the most extensively used local anesthetic (LA) in surgical procedures worldwide. Development of LA in carriers such as beta-cyclodextrin (β-CD) has been used as an alternative for the treatment of pain, prolonging the anesthetic effect and reducing its toxicity. In the search for safer LA formulation we have prepared a complex of BVC in hydroxypropyl-β- CD (HPβ- CD). This study compared the anesthetic effect of this inclusion complex as against BVC in solution. The complex was obtained by mixing equimolar amounts of HPβ- CD and BVC to a final 0.5% BVC concentration. Motor and sensory blockades induced by plain and complexed BVC were assessed after intrathecal administration in rats, using the paw-pressure test. A histological analysis of the kidneys was performed in animals treated with 0.5% BVCHPβ- CD , BVC or HPβ- CD, in order to evaluate the nephrotoxicity of HPβ- CD. In vivo experiments showed that the latency time for motor blockade was significantly reduced after treatment with BVCHPβ- CD (p<0.001 when compared with BVC), and no changes in: time for recovery, time for maximum effect and total effect. Moreover, BVCHPβ- CD enhanced the intensity (1.5 fold, p<0.001) and prolonged the duration of analgesia in relation to BVC. Histological images did not reveal any morphologic changes after treatment of the animals with HPβ- CD, BVC or BVCHPβ- CD . The BVCHPβ- CD inclusion complex improved the differential nerve blockade without evoking nephrotoxicity in rats.
P1 IN VITRO TESTING OF VASCULAR SMOOTH MUSCLE CELLS 316 EXPOSED TO UNMODIFIED MULTIWALLED CARBON NANOTUBES Tammy Oreskovic, Paul Rice and Natalia Varaksa, National Institute of Standards and Technology, Materials Reliability Division, Boulder, Colorado 80305, USA; [email protected]
Carbon nanotubes (CNTs) have superior structural, mechanical and electrical properties that are of great interest for medical and biological applications. Promising areas include drug delivery, therapeutics, tissue engineering, biosensors and probes. For most applications, modification of the CNT surface is required to improve stability and ease of dispersion in aqueous solutions. This surface modification often alters the physical properties of the CNTs and may obscure the actual interaction of pristine CNTs with living systems. Additionally, this new surface (i.e., chemically modified) may change once introduced into the biological environment through metabolic interactions and/or enzymatic breakdown of the surface. As a precursor to evaluating surface modification of CNTs we first exposed unmodified multiwalled carbon nanotubes (MWCNTs) to vascular smooth muscle cells (VSMCs). Varying densities of MWCNTs were applied by a spraying technique to glass substrates. VSMC cell growth was monitored over the course of days with a proliferation assay to assess cell metabolic activity. Cell cultures were examined by optical and scanning electron microscopy. From these studies we have found that cells did not attach to MWCNTs as well as controls (glass substrates). However, VSMCs still remained viable based on the proliferation tests and microscopic imaging. Further studies will include use of different cell types and various markers of cell proliferation to better quantify changes in cells over extended periods of time.
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P1 NANOCARE - CHANCES AND RISKS OF NANOMATERIALS 317 Regine Hedderich, Forschungszentrum Karlsruhe, NanoMat, PO Box 3640, Karlsruhe D76021, Germany; [email protected]
One of the main challenges facing nanotechnology is to investigate the health effects of nanomaterials. NanoMat, based at the Forschungszentrum Karlsruhe (www.nanomat.de), has about 25 partners from research institutions and industry working on the theme: “The synthesis and investigation of nanostructured metals and ceramics and the materials and functions arising from their nanoscale nature.” NanoMat is ideally placed as: • A meeting point for research and industry • An independent institution oriented to the public interest • All the relevant subject areas are represented at the Forschungszentrum Karlsruhe (Synthesis and Characterisation of Nanomaterials, Toxicology, Technology Assessment) complemented by activities of the other partners. • Optimisation of the lifecycles of nanomaterials (sustainable development, toxicology, and economic use) • NanoVision (2003, 2004, 2005, 2006) Interdisciplinary workshop annually NanoCare is a project funded by the BMBF and will aid the generation of new scientific knowledge on possible health effects of nanoparticles. Sixteen partners from research and industry are involved. NanoCare is coordinated by the Karlsruhe Research Center and NanoMat. In order to establish a unique information base “Environmental and health effects of nanoparticles” a most comprehensive collection of relevant data will be made, which will be assessed and presented online in an easy access format. NanoCare has a three stage approach: (1) Knowledge Generation - Collection of primary data of health effects of nanoparticles in the workplace. Information from particle analyses, biological test systems and deagglomeration behaviour; (2) Knowledge Management - The collection and categorisation of characteristic parameters for the creation of a scientific knowledge base. In a second stage the information obtained will be catalogued according to target groups (journalists, scientists, public) and made available in the form of a data bank; and (3) Knowledge Transfer - The data bank will be made public in a user friendly form. Seminars, conferences and workshops will ensure an active dialogue between research, industry and the general public (www.nanopartikel.info).
P2 SURFACE FUNCTIONALIZATION OF MAGNETIC NANOPARTICLES 320 VIA SURFACE-INITIATED ATOM-TRANSFER RADICAL POLYMERIZATION (ATRP) Shy Chyi Wuang, Feixiong Hu, Koon Gee Neoh*, En-Tang Kang, Department of Chemical and Biomolecular Engineering, National University of Singapore 10 Kent Ridge, Singapore 119260; [email protected]
The potential of using superparamagnetic magnetite nanoparticles for biomedical
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applications can be increased by grafting with biocompatible polymers. The application of the ATRP technique to achieve grafting of two different polymeric brushes, poly(poly(ethylene glycol) monomethacrylate) (P(PEGMA)) and poly(N-isopropylacrylamide) (P(NIPAAM)), was demonstrated with different initiators. The modified nanoparticles were characterized using Fourier transform infra-red spectroscopy, X-ray photoelectron spectroscopy, vibrating sample magnetometer and thermogravimetric analysis. The results indicate that ATRP is an effective way of controlling the grafting of biocompatible polymers. The P(PEGMA)-functionalized nanoparticles were assessed for their ability to reduce phagocytosis by macrophages in vitro. It was found that the uptake of these functionalized nanoparticles by macrophages was greatly reduced from 158 pg/cell for the pristine magnetite nanoparticles to <2 pg/cell after grafting with P(PEGMA). Since increasing plasma half-life is a challenge encountered in in vivo applications of nanoparticles, the P(PEGMA)-functionalized nanoparticles offer a distinct advantage in this aspect. No cytotoxicity effects were observed with the macrophages after incubation with the P(PEGMA)-functionalized nanoparticles. On the other hand, the P(NIPAAM)-functionalized nanoparticles can serve as an anchor for the subsequent attachment of other biomolecules such as heparin. These results demonstrate how magnetite nanoparticles can be surface modified via ATRP to tailor for potential biomedical applications.
P2 COLLOIDAL DELIVERY SYSTEMS FOR FUNCTIONAL FOODS 321 Krassimir P. Velikov, UFHRI, Unilever R&D Vlaardingen, The Netherlands; [email protected]
The growing demand for functional foods is largely driven by the increasing knowledge of the ingredients and their impact on physiological functions and health. The design of functional foods, however, brings enormous challenge for the food industry. The delivery of nutraceuticals required to design functional foods is a technological challenge for food industries, as similarly encountered in the pharmaceutical industry. The incorporation of nutraceuticals (e.g. nutrients or bioactive molecules) can compromise the product functionality. The issues are often related to solubility, taste, and stability of the functional ingredient or unwanted changes in the product stability, appearance, and taste due to interactions with other ingredients. The incorporation of minerals supplement in food products, for example, can cause changes in taste, lipid oxidation, color changes, and physico-chemical instability. In this talk, some examples will be given on how some of the above mentioned issues can be addressed by using colloidal delivery systems. The fabrication, characterization, properties, and stability of colloidal delivery systems for iron will be discussed in detail.
P2 TEMPLATE-ASSISTED SELF-ASSEMBLY OF POLYMERIZABLE LIPID 322 NANOTUBES Ali M. Alaouie, Alex I. Smirnov, Department of Chemistry, North Carolina
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State University, 2620 Yarbrough Drive, Campus Box 8204, Raleigh, NC 27695-8204, USA; [email protected]
Mixtures of certain synthetic phospholipids, such as, for example, diacetylenic and short-chain saturated lipids, are known to form ribbons and nanotubes in solution. Formation of these unusual bilayer phases is known to be dependent upon lipid concentration, structure and other experimental conditions. The lipid in these structures could be polymerized by exposing the sample to an ultraviolet light. Previously, we have shown that under certain conditions many phospholipids would self-assemble into a nanotube when placed inside a nanopore. For macroscopically homogeneous and uniformly packed nanopores, such as anodic aluminum oxide (AAO) these lipid nanotubes form arrays that could be used in combinatorial assays. Here we demonstrate that the mixtures of diacetylenic and short-chain saturated lipids could be deposited inside nanoporous AAO and then polymerized. Moreover, the AAO substrate can then be removed either partially or completely releasing structures formed. We characterize formation of AAO-templated polymerized lipid assemblies with UV-vis and SEM. The nanopore diameter of nanoporous AAO substrate could be varied and this could be utilized to attenuate phospholipid self-assembly. We also discuss potential use of template-assembled polymerized lipid nanostructures. DOE Contract DE-FG02-02ER15354 to A.I.S.
P2 THE UPTAKE MECHANISM OF AN APOE-DERIVED PEPTIDE 323 DEPENDS ON ITS AGGREGATIONAL STATE Eik Leupold, Sandro Keller, Heike Nikolenko, Michael Bienert, and Margitta Dathe, Leibniz Institute of Molecular Pharmacology (FMP), Robert-Rössle-Str. 10, 13125 Berlin, Germany; [email protected]
Specific and controllable drug targeting has long been recognized as a crucial issue but also as a major obstacle in the application of numerous therapeutics. Supramolecular structures, particularly micelles and liposomes equipped with uptake mediating address compounds, have attracted much attention as pharmaceutical formulations for the administration of otherwise only poorly soluble, rapidly degradable, or even toxic effector molecules. Here we introduce a novel peptide vector derived from the low-density lipoprotein receptor (LDLr)-binding domain of apolipoprotein E (apoE). The highly cationic tandem peptide (A2) acquires upon coupling of two hydrophobic palmitoyl chains (P2) detergent-like properties, such as a strong tendency for self-association and the ability to integrate into lipid bilayers. Light scattering and isothermal titration calorimetry studies shed light on lipopeptide (P2A2)-lipid interactions and yielded a phase diagram with three well-confined areas ascribed to micellar, transition, and vesicular ranges, permitting mastery of the colloidal state. Owing to its straightforward synthesis, its unique physicochemical properties, and its adaptability to a broad spectrum of targets, P2A2 is a highly promising candidate for the development of micellar and liposomal drug delivery systems. Confocal laser scanning microscopy demonstrated the efficient internalization of free A2, micellar P2A2 and liposome bound P2A2 into mouse brain capillary endothelial cells. For further
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elucidation and comparison of the corresponding uptake mechanisms we performed uptake experiments under various conditions, e.g. low temperature, energy depletion, and in presence of specific endocytosis inhibitors. These revealed a clear energy dependent uptake mechanism for A2 in all three aggregational states, but also striking differences in the individual endocytotic pathways, providing a basis for target specific drug administration.
P2 QUANTIFYING LOW LEVELS OF AMORPHOUS CONTENT USING 324 VAPOR SORPTION TECHNIQUES Daniel J. Burnett, Joe Domingue, and Frank Thielmann, Surface Measurement Systems Ltd., 2222 South 12th Street, Suite D, Allentown, PA 18103 USA; [email protected]
Amorphous materials in pharmaceutical formulations yield complex and challenging problems concerning the performance, processing, and storage of these products. The presence of amorphous materials can be wanted or unwanted, depending on the desired or undesired unique properties of the amorphous phase. For these reasons, determining the amount of amorphous material is critical in the formulation of pharmaceutical powders. Dynamic gramivetric vapor sorption (DVS) is a well-established method for the determination of vapor sorption properties. It is based on a highly sensitive gravimetric system, which measures the adsorption and desorption of extremely small amounts of probe molecule. There are several gravimetric methods available in the literature to quantify amorphous contents. One method measures the vapor uptake of a (partially) amorphous sample before and after a solvent-induced crystallization event. Another method compares the relative uptakes of the amorphous and crystalline phases without crystallizing the amorphous material. A third method necessitates the formation of a hydrate or solvate to quantify amorphous contents. In the current studies vapor uptakes were collected on trehalose samples with various amounts of amorphous material. The various available methods will be compared and discussed. All methods were able to detect amorphous contents below 5%.
P2 INVESTIGATING THE EFFECTS OF PROTEIN CONCENTRATION ON 325 THE GLASS TRANSITION BEHAVIOR OF LYOPHILIZED PROTEIN-SUGAR FORMULATIONS Daniel J. Burnett, Joe Domingue, and Frank Thielmann, Surface Measurement Systems Ltd., 2222 South 12th Street, Suite D, Allentown, PA 18103 USA; Andrew Ingham and Kevin Ward, Biopharma Technology Ltd., Winnall Valley Road, Winchester SO23 0OL UK; [email protected]
The successful utilization of proteins as pharmaceutical formulations presents significant challenges in maintaining formulation stability. As a result, proteins are often co-lyophilized in sugar matrices for added stability. Ultimately, the interactions between
244 Poster Abstracts the incorporated sugars and the protein determine the formulation stability. The formulation’s glass transition (Tg) is commonly used to evaluate the physical stability of amorphous materials. The Tg can be greatly affected by the moisture content; therefore investigation of the water sorption behavior for these protein-sugar formulations is paramount in determining the ultimate product stability. Dynamic Vapor Sorption (DVS) is a well-established gravimetric method for the determination of vapor sorption properties. In the current study water sorption and glass transition behavior of freeze-dried sucrose, mannitol, and maltose samples with varying BSA concentrations were investigated at 25 °C. BSA had an anti-plasticizing affect on the lyophilized maltose and sucrose formulations. The pure maltose sample showed a glass transition at 50% relative humidity (RH). As the BSA content was increased from 11% to 33%, the Tg increased from 57% RH to 79.5% RH. Similarly, pure sucrose exhibited a Tg at 37%RH, while a 33% BSA-sucrose mixture had a glass transition above 55% RH. Further, the BSA showed a stabilization effect on sucrose structure collapse. Pure lyophilized sucrose crystallized at 55% RH, while the 33% BSA-sucrose formulation crystallized above 90% RH. Increasing BSA content in BSA-mannitol samples allowed the formation of higher mannitol amorphous contents.
P2 SURFACE MODIFIED SOLID LIPID NANOPARTICLES 326 Meike Harms, Martin A. Schubert, Christel C. Müller-Goymann, TU Braunschweig, Institute for pharmaceutical Technology, Mendelssohnstr. 1, 38106 Braunschweig, Germany; [email protected]; [email protected]
In this study solid lipid nanoparticles (SLN) are prepared from mixtures of triglycerides and the amphiphilic lipid lecithin. For the production of SLN high pressure homogenization at temperatures above the melting point is employed. During homogenization an emulsion is formed. This can result in an rearrangement of lecithin into the aqueous phase in order to form micelles or liposomes. Furthermore lecithin can stabilize the surface of the lipophilic droplets or stay within the lipophilic phase. SAXS and TEM showed that no leakage of lecithin into the aqueous phase took place during preparation. Furthermore NMR and DLS were performed to obtain information about the chemical environment and mobility of the lecithin molecules. The incorporation of lecithin into the lipid matrix (up to 30%) resulted in a decrease of the particle size of the SLN. Further increase of the lecithin content (up to 50%) did not change the particle size. NMR measurements showed a rather low mobility of the lecithin molecules up to a concentration of 30% within the LM. Contents up to 50% led to a significant increase of the mobility. From these results a model of surface modified SLN can be deduced. The particles consist of a triglyceride enriched core, which is covered by lecithin – depending on the content either as a monomolecular or multimolecular layer. The nonionic emulsifier is incorporated into that layer.
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P2 HYBRID MICROGEL PARTICLES WITH ZINC SULPHIDE INCLUSIONS 327 Jessica Hain, Andrij Pich, Hans-Juergen P. Adler, Institute of Macromolecular Chemistry and Textile Chemistry, Dresden University of Technology, Mommsenstraße 4, 01069 Dresden, Germany; [email protected] de
We report on the preparation of hybrid microgels filled with ZnS inclusions. Temperature-sensitive poly-(N-vinylcaprolactam)-co-poly-(acetoacetoxyethyl methacrylate) (PVCL/PAAEM) microgel particles have been used as reaction containers for preparation and deposition of ZnS nanoparticles by reaction of zinc acetate and thioacetamide under ultrasonic agitation. Zinc sulphide was directly deposited into the microgel leading to formation of composite particles which exhibit colloidal stability and temperature-sensitive properties. Dynamic Light Scattering measurements show an interesting influence of incorporated ZnS on the particle size and position of the phase transition temperature. Electrophoretic mobility measurements and microscopic investigations with SEM, TEM and AFM were performed to determine the structure of the generated hybrid microgel particles and their stabilization. Composite particles contain up to 20 wt.-% of zinc sulphide and lose their colloidal stability with increasing inorganic filler content. Obtained hybrid microgels exhibit the ability to form organized arrays on different substrates during drying. This self organization process can be easily controlled by adjusting the temperature or the pH-value. This interesting feature can be used for preparation of patterned surfaces on nano-scale and is useful for numerous applications in catalysis, electronics and material engineering.
P2 EXPLOITING EMERGING TECHNIQUES IN IMPRINT LITHOGRAPHY 328 TO MAKE HIGHLY UNIFORM, SHAPE-SPECIFIC DRUGS, IMAGING AGENTS, AND NANO-CARRIERS Larken E. Euliss, Julie A. Dupont, Christopher M. Welch, Benjamin W. Maynor, Stephanie E. Gratton, Jason P. Rolland, Ginger M. Denison, Ashish A. Pandya, Rudolph L. Juliano, Klaus M. Hahn, and Joseph M. DeSimone University of North Carolina at Chapel Hill, Department of Chemistry, CB #3290, Venable and Kenan Laboratories, Chapel Hill, NC 27599; [email protected]
One of the current trends in the delivery of therapeutic, detection and imaging agents for the diagnosis and treatment of cancer patients is the utilize nano-carriers such as liposomes, micelles, dendrimers, biomolecules, polymer particles, and colloidal precipitates. While these materials afford some control over load ability and structure, each suffers from serious drawbacks with regard to stability, flexibility, polydispersity, or functionality. Considerable effort has been devoted to the design and fabrication of these materials, but it is clear that the scientific community has only “scratched the surface” with respect to nanostructure complexity, composition, and function. For these reasons, we are interested in developing a process that will allow the rigorous control over particle size,
246 Poster Abstracts shape, composition, cargo, and chemical structure. Taking this all into account, we have developed a new method of particle fabrication by borrowing the materials useful for imprint lithography—an emerging technology adapted from the microelectronics industry—in that we have a method that is extremely versatile and flexible for the direct fabrication and harvesting of monodisperse, shape-specific nano-biomaterials. By utilizing the method we have designed referred to as Particle Replication In Non-wetting Templates, or PRINT, we have fabricated monodisperse particles with simultaneous control over structure (i.e. varying aspect ratios, varying size regimes, composition) and function (i.e. cargo, surface structure). We have accomplished this by utilizing low-surface energy, chemically-resistant fluoropolymers as molding materials and patterned substrates to produce these designer materials. Unlike other particle fabrication techniques, PRINT is delicate and general enough to be compatible with a variety of important next-generation cancer therapeutic, detection and imaging agents, including various cargos (e.g. DNA, proteins, chemotherapy drugs, biosensor dyes, radio-markers, contrast agents), targeting ligands (e.g. antibodies, cell targeting peptides) and functional matrix materials.
P2 DEVELOPMENT AND CHARACTERIZATION OF INTERACTIVE 329 MIXTURES WITH A FINE PARTICULATE, MUCOADHESIVE CARRIER FOR NASAL DRUG DELIVERY Nelly Fransén, Erik Björk and Christer Nyström. Department of Pharmacy, Uppsala University, Sweden; [email protected]
A new way of producing mucoadhesive delivery systems for nasal administration is suggested by the creation of interactive mixtures with a small-size carrier particle. Interactive mixtures are created by dry mixing a fine particulate substance with coarse carrier particles. This results in a very homogenous mixture through deposition of the smaller particles on the surface of the carriers. Most interactive mixtures have previously been created with a carrier particle size exceeding 100-200 µm; this study shows that it is possible to use a mucoadhesive carrier as small as 30 µm, which is well suited for nasal administration. Sodium starch glycolate (SSG), which is commonly used as a superdisintegrant in tablets, was used as carrier material because of its swelling capacity and good flowability. SSG was fractioned into 4 size ranges to evaluate the effect of carrier particle size on the mixture quality. Furthermore, mixtures with 4 different weight percentages of the micronized model substance (sodium salicylate, SS) were prepared and interactive mixtures could be obtained with as much as 4 % SS, corresponding to 20 % surface coverage. However, this limit will most likely be substance specific. The emphasis of the study was laid on mixture homogeneity evaluation and the experimental results were compared with Pharmacopoeia regulations and theoretical calculations for an interactive system with a small-size carrier. It was concluded that it is not possible to distinguish between an interactive and a randomised mixture by sample analysis: the small particle size does theoretically and practically give very homogeneous samples regardless of the mixture quality. Visual inspection by scanning electron micrographs is therefore required. The presented method does, in its simplicity, offer advantages compared with
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microparticles, since no dissolution step is necessary in the production and the surface deposition of the drug could lead to a faster onset of effect.
P2 MONODISPERSE MICROGEL SYNTHESIS INSIDE MICROFLUIDIC 330 DEVICES Bruno G. De Geest, Jo Demeester, Stefaan C. De Smedt, Laboratory of General Biochemistry and Physical Pharmacy, Department of Pharmaceutics, Ghent University,Harelbekestraat 72, 9000 Ghent, Belgium; and John Paul Urbanski, Todd Thorsen, Department of Mechanical Engineering, Massachusetts Institute of Technology, Mass. Ave. 72, Cambridge, MA 02139, USA. [email protected]
Hydrophilic microgels have gathered large interest in biomedical field due to their biocompatibility and the possibility to make them stimuli responsive by incorporation of functional groups such as pH, temperature, electric field, glucose, etc responsive moieties. However, there is no general method available for the synthesis of monodisperse microgels, although this should offer considerable advantages compared to polydisperse ones with respect to monitoring, predicting and modelling of their behaviour. In this poster we demonstrate the fabrication of biodegradable monodisperse microgels inside a soft-lithography based microfluidic device. The polymer used in this study is dextran-hydroxyethyl methacrylate (dex-HEMA), this polymer has a Mw~19kDa and is biodegradable by hydrolysis of the carbonate esters connecting the methacrylate groups with the polymer backbone. An in-line droplet generator is used for the formation of water-in-oil emulsion droplets wherein the water phase is a 30% solution of dex-HEMA. Afterwards the dex-HEMA is polymerized by UV-irradiation yielding microgels with a diameter of 10 µm.
P2 MODIFIED NANO-BENTONITE ANTI-VIRAL ACTIVITY AGAINST 331 THE HERPES SIMPLES VIRUS TYPE 1, RHINOVIRUS TYPE 37, AND ROTOVIRUS Jerald W. Darlington, Amcol International, Inc., 1500 West Shure Drive, Arlington Heights, IL 60004, USA: [email protected]
Bentonite, the commercial name for montmorillonite, is the active mineral derived from weathered volcanic ash. It has the unusual properties of a platelet structure with extremely large surface area and an inherent negative charge in the platelet. One especially interesting feature of the clay is the surface of the platelet can be chemically modified to enhance the clay’s ability to absorb and bind toxins, poisons etc. Previous presentations have reported the results of modifications of the clay against the Influenza Virus (H3N2 and H5N1) and Human Immunodeficiency Virus (HIV-1IIIB), The previous summary was that modified clay R-0088 (0.01% w/v) exhibited reductions in infectivity of >90% and >99% for H3N2 Influenza and HIV-1IIIB viruses respectively. Modified clays R-100,
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R-101 and R-102 all exhibited between 80% (0.0001% w/v) and >99% (0.01% w/v) reductions in infectivity for Influenza H3N2 Virus. The current studies investigated modified nano-bentonites against the Herpes Simplex Virus (Type 1), the Rhinovirus (Type 37), and the Rotovirus. Each modification was incubated with the viruses for different times and at different concentrations. Modified clays R-0402 exhibited reductions in infectivity between 80% and 99.9% for Herpes Simples Type 1 and Rhinovirus Type 37. Modified clays R-400 and R-402 exhibited between 90% and 99.9% reductions in infectivity for Rotovirus. The results suggest that different modifications to the bentonite clay alter the efficacy of the clay’s antiviral properties. It is very interesting to note that in multiple cases, the same modified clay had antiviral properties against two or more very different viruses. The potential uses of modified bentonite clay in the treatment and prevention of Influenza, HIV, Herpes Simplex, Rotovirus and Rhinovirus infections are significant. The current uses of bentonite clay for internal health benefit use as a formulation aid in oral suspensions are indicative of the minerals extensive use by humans. As an antiviral, the clay could be utilised for topical applications (i.e. vaginal gels for protection against HIV infection and nasal sprays for treatment of Influenza infections) and food supplements for humans (i.e. treatment against diarrhoea-causing enteric pathogens such as rotaviruses) and animals (i.e. treatment against avian Influenza bovine shipping fever).
P2 NOVEL CROSSLINKED CHITOSAN AND DEXTRAN GELS AS DRUG 332 DELIVERY SYSTEMS José F. Almeida1,2, Armando Alcobia3, António Lopes2, Maria H. Gil1; 1Dept. Eng. Química - FCTUC, P-3030-290 Coimbra, Portugal; 2Inst. Tecnol. Química e Biológica - UNL, P-2781-901 Oeiras, Portugal; 3Hosp. Garcia Orta, P-2801-951 Almada, Portugal; [email protected]
Controlled release from biocompatible materials has received recently much attention for its biomedical applications. Due to their biocompatibility and biodegradability, gluocopyranosides such as chitosan and dextran appears as promising polymer materials if one is able to regulate their rheological properties and the encapsulation/release efficiency. In this work we prepared graft polymer gel particles from chitosan (“medium” Mw) and dextran (Mw=5-40×106). The starting polymers were dissolved in the approprite aqueous media (simple water for dextran, and 5% acetic acid for chitosan) in the suitable semi-dilute concentration range (1-4% w/v) and as graft polymer we used N-isopropylacrylamide (NIPAAm). The reaction was initiated either using γ radiation or ammoniumcerium (IV)–nitrate radical initiator and as far as the gel rheology γ radiation gives more consistent gel particles. FT-IR data for the grafted Dex-NIPAAm systems revealed the presence of two peaks around ~1650 and ~1550 cm-1 which can be ascertained with the typical amide I and II bands of NIPAAm, revealing the success of the grafting reaction; this was also confirmed by 1H-NMR. As model drug we used a potent antiemetic substance used in the prevention of chemotherapy-induced nausea and vomiting, ondansetron, (±) 1, 2, 3, 9-tetrahydro-9-methyl-3-[(2-methyl-1H-imidazol-1-yl)methyl]-4H-carbazol-4-one
249 Poster Abstracts
(hydrochloride form) which was incorporated in the gel particles both before and after the co-polymerization. Swelling capacity and cycles of incorporation/release of the drug were evaluated with essays where PBS solution’s pH and temperature were varied in the physiological range of interest (withdrawing of aliquots and HPLC analysis). Results showed that, irrespective of the polymers, there is a positive correlation between co-polymer concentration and swelling, as well as with drug incorporation. Release essays show an initial burst followed by slow release but no significant pH dependence (in the range of interest) and a moderate dependence with temperature.
P2 SILICA NANOPARTICLE FORMATION IN CONFINED 333 ENVIRONMENTS VIA BIOMIMETIC CATALYSIS Christina A. Bauer, David Robinson, Darryl Y. Sasaki, Blake A. Simmons Sandia National Laboratories, Livermore, CA 94551-0969; [email protected]
In nature, silica formation occurs under ambient conditions with unprecedented control. This is performed with templating biomolecules via morphosynthesis, wherein the starting materials are spatially confined. In the case of diatoms, a set of cationic polypeptides (“silaffins”) and polyamines were found to promote and mediate silica patterning. The development of emulsions and micelles of a polyamine-containing organic phase are thought to direct the intricate structures. We have explored the use of reverse micelles for patterning, including scenarios whereby the amine is incorporated into a constrained environment or where the amine is present as a functionality on the headgroup. In reverse micelles, the resulting particles display smooth structures formed via aggregation of smaller particles, reminiscent of diatoms. Reaction conditions within micelles were found to differ from the bulk. Silica polymerization was monitored by a variety of methods, namely fluorophore uptake, dynamic light scattering (DLS), and scanning electron microscopy (SEM). After calcination, these samples reveal a high degree of porosity, supporting incorporation of amines within the final structure. Vesicles were also explored as confined reaction vessels and templates, whereby amines were included in the lipid head group or as a polyamine incorporated into the bilayer surface.
P2 NANOPARTICLE-POLYMER COMPLEXATION: ELECTROSTATIC 334 SELF-ASSEMBLY AS A ROUTE TO STABLE DISPERSIONS OF HYBRID NANOCOLLOIDS A. Sehgal, Y. Lalatonne, M. Morvan, Complex Fluids Laboratory, CNRS - Cranbury Research Center Rhodia Inc., 350 George Patterson Blvd., Bristol, PA 19007 USA and J.-F. Berret, Matière et Systèmes Complexes, UMR CNRS n° 7057, Université Denis Diderot Paris-VII, 140 rue de Lourmel - 75015 Paris – France ; [email protected]
The inherent instability of inorganic nanoparticle sols may be resolved by complexation
250 Poster Abstracts with ion containing polymers at the particle interface. We exploit a precipitation-redispersion (P-R) mechanism for complexation of short chain polyelectrolytes with cerium oxide nanoparticles to extend their stability over a wide pH range. Small angle X-ray scattering in conjunction with static and dynamic light scattering reveals that P-R may yield hybrid nanocolloidal complexes with a singlet inorganic core - charged corona structure. The electrostatic self-assembly of ionic stickers anchors the chains onto the surface resulting in a polymeric brush which provides steric and electrostatic stabilization. This simple strategy to achieve stable dispersions surmounts the critical limitation in the use of nanoparticles, allowing a facile means to translate the intrinsic properties of mineral oxide nanoparticles to a range of novel applications.
P2 ONE-STEP SYNTHESIS OF SPINEL IRON OXIDE-BASED 335 CORE-CORONA NANOPARTICLES FOR IN VIVO APPLICATIONS Julien Reboul, Franck Bertorelle, Corine Gérardin, Laboratoire de Matériaux Catalytiques et Catalyse en Chimie Organique UMR 5618 CNRS/Ecole de Chimie Montpellier/UM1Institut Gerhardt FR187, 8 rue de l'Ecole Normale, 34296 Montpellier cedex 5, France ; Corinne Chanéac, Laboratoire de Chimie de la Matière Condensée, Université Pierre et Marie Curie, 75252 Paris cedex, Etienne Duguet, Institut de Chimie de la Matière Condensée de Bordeaux UPR CNRS 9048, University of Bordeaux, 87 ave Dr Schweitzer, 33608 Pessac Cedex, France ; [email protected]
Core-corona nanoparticles made of iron oxide cores and hydrophilic macromolecules are of particular in vivo interest as MRI contrast agents or as hyperthermia mediators. The main objective of the present study was to develop a new chemical route for the synthesis of magnetic nanoparticles sterically stabilized in physiological medium and capable of circulating for a long time in the blood compartment. An original one-step aqueous synthesis based on the mineralization of hybrid supramolecular complexes composed of iron salts and double hydrophilic block copolymers was investigated. The polymers based-complex aggregates are obtained by complexation of Fe(2+) and Fe(3+) ions by block copolymers, which contain a complexing block (such as sodium polyacrylate) and a stabilizing block such as (polyethylene oxide, or polyacrylamide). The present complex aggregates are precursor colloids which play the role of nanoreactors in the preparation of the stabilized magnetite particles. Hydrolysis of the metal cations in the aggregates leads to the formation of spinel iron oxide–based colloids, which are stabilized by the block copolymers. Highly stable core-corona colloids could then be prepared. The stability towards the addition of monovalent salts was found to be excellent. The polymer neutral blocks, which form the diffuse corona of the particles, act as steric stabilizing agents and are expected to act as plasma protein repellents. Moreover, the size of the hairy magnetic nanoparticles was tuned by adjusting the copolymer-to-metal ratio, and the polymer block lengths. Magnetic measurements allowed checking their superparamagnetic behavior. Moreover, preliminary experiments showed promising results either as MRI contrast agents or as heat promoters.
251 Poster Abstracts
P2 FABRICATION AND CHARACTERIZATION OF SiO2 AND MgO 336 COATED Fe2O3 NANOPARTICLES FOR HYPERTHERMIA APPLICATIONS Ji-Hun Yu, Powder Materials Research Center, Korea Institute of Machinery and Materials, 66 Sangnam, Changwon, 641-011, South Korea; Yong-Ho Choa, Jai-Sung Lee, School of Materials and Chemical Engineering, Hanyang University, Ansan, 426-791, South Korea; and Heinrich Hofmann, Department of Materials Engineering, Swiss Federal Institute of Technology (EPFL), Lausanne, CH-1015, Switzerland; [email protected]
SiO2 and MgO coated γ-Fe2O3 nanoparticles have been synthesized by chemical vapor condensation and ultrasonic pyrolysis processes and their feasible possibilities on hyperthermic application were investigated in this study. The synthesized γ-Fe2O3 nanoparticles have a spherical shape and around 20 nm in size. And SiO2 and MgO oxide layers well coated on the surface of γ-Fe2O3 nanoparticles with around 5 nm in thickness. The chemical compositions of the nanoparticles were 10wt% γ-Fe2O3 in SiO2 coated and MgO coated γ-Fe2O3 nanoparticles. The generated heats from the composite particles under alternative magnetic field were measured and SAR value, specific powder loss of the composite particles, were calculated from the generated heats. The power loss of SiO2 and MgO coated γ-Fe2O3 nanoparticles which means the magnetic heating effect under alternative magnetic filed were much higher than the single phase γ-Fe2O3 nanoparticle due the very fine size under 20 nm and well dispersion in body compatible SiO2 matrix. Additionally, MgO coated γ-Fe2O3 nanoparticle has much higher powder loss value due to the higher thermal conductivity of MgO than SiO2 coating layer. The superparamagnetism and hyperthermic property of SiO2 and MgO coated γ-Fe2O3 nanoparticles were discussed in terms of microstructural development in this study.
P2 PREPARATION OF GOLD NANOCOMPOSITES WITH DIELECTRIC 337 MAGNETIC CORES FOR TARGETTED PHOTOTHERMAL THERAPHY Sang E. Park, Min Y. Park, Sang W. Lee, Dept. of Chemical and Bio Engineering, Kyungwon University, Bokjeoung-dong San 65, Soojeoung-gu, Gyeonggi-do 461-701, Korea; and Jae W. Lee, Jea M. Yang, Seung J. Haam, Dept. of Chemical Engineering, Yonsei University, Seoul 120-749, Korea; and Yong M. Huh, Dept. of Radiology, Yonsei University, Seoul 120-749, Korea; [email protected]
Metal nanoshells are a new type of nanoparticles consisting of a dielectric core covered by a thin metallic shell. In particular, gold nanoshell possesses unique biocompatible and optical proeprties for targetted drug delivery and plasmonically-modualetd applications. Magnetic nanoparticles have widely been used for biomedical imaging and cancer therapy of hyperthermia under magnetic field. Thus, hybrid magnetic cores covered by thin metallic shell can be a potential candidate for photothermal cancer therapy under magnetic resonance guidance. In this study,
252 Poster Abstracts gold nanocomposites with magnetic cores were fabricated by the three consequtive steps: i) synthesis of nano-scaled superparamagnetic iron oxide, ii) formation of spherical dielectic cores embeded by magnetites, and iii) the growth of thin gold shell over the spherical cores. Gold nanocomposites exhibited a strong NIR absorption at 800-1200 nm by adjusting the core/shell ratio. The higher concentration of organometallic precursor and reducing agents induced the stronger intensity of NIR absorption. However, the excessive addition of both chemicals consequently led to the broadening and lowering of maximum absorption peak, probably due to the flocculation of the hybrid nanoshells. The zeta potential and optical properties were characterized by ELS (Electrophoretic Light Scattering System) and UV-Vis spectroscopy. The surface morphology and size distribution were also characterized by TEM/SEM (Transmission/ Scanning Electron Microscopy), DLS (Dynamic Laser Scattering System), and FIB (Focused Ion Beam).
P2 DNA-TEMPLATED SELF-ASSEMBLY OF TWO DIMENSIONAL AND 338 PERIODICAL GOLD NANOPARTICLE ARRAYS Jaswinder Sharma, Rahul Chhabra, Yan Liu, Yonggang Ke, and Hao Yan*, Department of Chemistry and Biochemistry and Biodesign Institute, Arizona State University, Tempe, AZ 85287, USA; [email protected]
Nanoparticles (NPs) are being actively developed as building blocks for electronic, photonic and spintronics devices. When they are organized into well-defined ensembles, their collective properties depend critically on the inter-particle spacings and their hierarchical organization. Up to now, methods to control these parameters are scarce. DNA-templated self-assembly provides a unique solution to meet the above challenge, due largely to the following features of DNA and DNA-based nanostructures: DNA has well-defined geometry and most predictable, diverse and programmable intra/inter-molecular interactions; DNA can be modified by different chemical groups, which act as linkers to covalently attach DNA oligomers to the NPs; the design of stiff DNA-based nanostructures and their self-assembly have become a routine method to construct one- and two-dimensional DNA lattices of well-defined patterns. More specifically, when NPs are assembled onto self-assembled DNA lattices, the periodicities and interparticle spacings defined by the DNA scaffolds can be readily adjustable, with nanometer spatial precisions. This level of precision provides exquisite control in the construction of rationally defined NP architectures. Here we report the first time assembly of 5 nm Au NP functionalized with a single DNA strand on three different patterned structural templates that are constructed from self-assembled DNA tiles. We show that the periodicity and interparticle spacings of the NP nanoarrays can be precisely controlled by varying the DNA tile dimensions. In our strategy, the NP-conjugated strand first participates in the formation of a single DNA tile structure, this Au NP-bearing DNA tile was subsequently used to assemble with another DNA tile to form three different lattice structures, each with a well defined periodical pattern. This novel system is amenable both to the DNA lattice formation and preventing non-specific aggregations between the Au NPs. This development paves the way to assemble more complex nanoparticle arrays on DNA nanoscaffolds for future device applications.
253 Poster Abstracts
P2 DETECTION OF HYBRIDIZATION REACTIONS ON 339 MICROPARTICLES BY TEMPLATE DEPENDENT EXTENSION Sofia Martins, Miguel Prazeres, Luis Fonseca, Gabriel Monteiro, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisbon, Portugal; [email protected]
Manufacturers of plasmid DNA (pDNA) intended for gene therapy or DNA vaccination are required to comply with a set of rules established by regulatory authorities (e.g. FDA, EUDRA). For instances, in order to get their products approval, the content of genomic DNA (gDNA) from the producing cells must be below 10 ng/µg pDNA. Nucleic acid hybridization is the current established technique used to quantify gDNA in biopharmaceuticals. Microarray technology is a promising approach as it can provide the sequence specific information in a faster and simpler way. However, one of the major disadvantages of the miniaturized based hybridization reactions is the requirement for target labeling. This is time consuming and it can change the levels of targets originally present in the sample. In this work, we used a variation of primer extension reactions performed in CPG-microparticles in order to detect and quantify gDNA present in pure plasmid DNA preparations. A 20 nt oligonucleotide probe, complementary to the Escherichia coli 16S rRNA gene, is immobilized via the 5’ terminus by thiol chemistry. Microparticles are hybridized with complementary gDNA or plasmid DNA molecules for negative control. The hybrids are then detected by template mediated extension of the immobilized probe, at the sites where hybridization has occurred. The incorporation of Digoxigenin-labeled nucleotides or fluorescinated nucleotides enables the detection of the extended hybrids. Using this approach it was possible to directly quantify gDNA at nanogram level. Hybridization with non complementary plasmid DNA molecules produced no detectable signals. Therefore using this methodology it was possible to detect nucleic acid’s hybridization reactions with high specificity without the need for a pre-treatment of the target sample. The use of microparticles as the solid support for immobilization offered several advantages, namely increased immobilization efficiency and good hybridization kinetics in a miniaturized format.
P2 FORMATION OF HYBRID NANOPARTICLE-ALGINATE 340 MICROCAPSULES FOR BIO-DETECTION APPLICATIONS Roberta Brayner, Stephanie Truong, Nordin Felidj and Fernand Fiévet, Interfaces, Traitements, Organisation et Dynamique des Systèmes (ITODYS), UMR-CNRS 7086, Université Paris 7-Denis Diderot, case 7090, 2 place Jussieu, 75251 Paris Cedex 05, France; Thibaud Coradin and Jacques Livage, Laboratoire de Chimie de la Matière Condensée, UMR-CNRS 7574, Université Paris 6 - 4 place Jussieu, 75252 Paris Cedex 05, France ; [email protected]
Natural polysaccharides such as chitosan, alginate and carragheenan have some important properties such as mucoadhesivity, biocompatibility and nontoxicity, which render them
254 Poster Abstracts interesting biomaterials. From a physicochemical point of view, these polysaccharides have the special quality of gelling upon contact with cations (alginate and carragheenan) and anions (chitosan) under very mild conditions. In this work, poly-saccharidic alginate biopolymers have been used as templates for the controlled growth of several nanoparticles. Au3+, Ni2+ and Co2+ could be used to form alginate gels as spherical capsules. After reduction in mild conditions, metallic nanoparticles with narrow size distribution were obtained inside alginate microcapsules Hybrid Au-alginate was then immersed in methylene blue (MB) aqueous solution (used as a probe molecule) at a concentration of 10-5M. It was observed a wavelength plasmon band shift probably due to an electronic coupling between Au nanoparticles and MB molecule inside alginate network. It was also observed an important SERRS effect for MB molecule adsorbed on these Au nanoparticles compared to the same molecule adsorbed on a lithographycally designed 2D Au arrays film. Metallic Ni, Co and NiCo solid solutions nanoparticles were also obtained inside alginate matrix. In this case, SQUID measurements indicated that all samples are ferromagnetic. In addition, hybrid Ni-alginate presents a superparagnetic behavior with a blocking temperature TB = 290 K. In summary, these polyfunctional hybrid materials may be applied in bio-detection, drug delivery (because alginates have a great protein loading capacity), magnetic targeting or magnetic resonance immaging.
Session P2 MAGNETIC nanoparticles LOADED ERYTHROCYTES AS MAGNETIC 341 RESONANCE IMAGING CONTRAST AGENT Angelika Müller1, M. Brähler1, N. Buske2, R. Georgieva3, J. Pinkernelle1, U. Teichgräber1, A. Voigt1, Hans Bäumler1, 1Charité - Universitätsmedizin Berlin, D – 10098 Berlin, Germany, 2www.magneticfluids.de, 3Max-Planck Institute of Colloids and Interfaces, D-14476 Golm/Potsdam, Germany; [email protected]
Superparamagnetic iron oxide nanoparticles (SPION, size 3-20nm) are often used in magnetic resonance imaging (MRI), but their circulating half-life time of only two hours and their fast phagocytosis by the lymphocyte-macrophage system makes them not ideal for this indication. We encapsulated the SPIONs into Red Blood Cells (RBC). RBC can be loaded with a variety of substances. They can be prepared to show similar in vivo survival times as native RBC (120d). By loading the SPION into RBC, it is possible to circumvent the lymphocyte-macrophage system and prevent fast accumulation of SPION in spleen and liver. We used water based dispersions as loading agents, containing very well dispersed citrate coated magnetite SPIONs (so called magnetic fluids, pH = 7.2, the counter ion was potassium, content of Fe3O4 is 4vol.%, particle Ø 5-15 nm). For loading, the RBC´s membrane was opened by hypoosmotic dilution and subsequently resealed. Magnetic RBC (mRBC) show a strong attraction towards an external magnetic field gradient of a permanent magnet. Transmission electron microscope pictures show the distribution of SPIONs inside the cells and in the membrane. The relaxation time (Bruker minispec contrast agent analyzer 1,5T) of mRBC in comparison to native RBC was reduced to 96,9% for T1 and 94,3% for T2. To show the effect of mRBC on images obtained by MRI,
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different dilutions of mRBC in whole blood were examined. Contrast enhancement occured at dilutions of up to 1:20 (T2). A 10% dilution of mRBC in blood showed significant contrast enhancement in phantoms of 1mm diameter (Bruker Pharm Scan 7T; T2). 10% mRBC is the highest concentration of mRBC to be administered in vivo. With single cell MRI (Signa excite 1,5T; T2*) individual mRBC could be distinguished.
P2 MAGNETIC NANOPARTICLES FOR POSSIBLE SIMULTANEOUS MR 342 IMAGING AND DRUG THERAPY OF BRAIN TUMORS Beata Chertok, Bradford A. Moffat, Allan E. David and Victor C. Yang, Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, Ann Arbor, USA; [email protected]
This study intends to examine the plausibility of utilizing magnetic nanoparticles for imaging and drug targeting of brain tumors. Magnetic nanoparticles consisting of an iron oxide core and a starch shell were obtained from Chemicell® (Germany). The small hydrodynamic diameter (~100 nm) suggested the possibility of their extravasation in the tumor area through the EPR (enhanced permeability and retention) effect. High saturation magnetization of 94 emu/gFe, determined by SQUID magnetometry, indicated that these nanoparticles are highly responsive to the magnetic field. This magnetic responsiveness lays the ground for possible retention of the nanoparticles within the brain tumor tissue by application of an external magnetic field around the head, a strategy termed “magnetic -1 -1 targeting”. Due to a high T2 relaxivity of 43 s mM , the presence of magnetic nanoparticles at a particular spatial location in the brain manifests itself by reduction in the local signal intensity on T2-weighted MR images in vivo, enabling the monitoring of the distribution kinetics of these nanoparticles throughout the brain non-invasively. MRI-based monitoring was used to test the effect of magnetic targeting on the extent and selectivity of accumulation of the magnetic nanoparticles in the tumor of rats harboring orthotopic 9L-gliomas. Animals were intravenously injected with nanoparticles (12mgFe/kg b.w.) under application of a magnetic field of 0T (control) or 0.7T (test group) around their head for 30 min. MR images were acquired before the administration of nanoparticles and immediately after magnetic targeting at 1-hr intervals over a period of 4 hrs. Quantitative analysis of MR images revealed that magnetic targeting has resulted in a significantly higher glioma accumulation of the nanoparticles in the test animals over the control (p=0.005). Moreover, magnetic targeting also increased the target selectivity index of nanoparticle accumulation in glioma over the unaffected normal brain (17±5 vs 1±0.5 in targeted vs non-targeted animals, respectively).
P2 HYDROGELS AND NANOPARTICLES IN MICROARRAYS FOR FOOD 343 CONTROL Katarzyna Derwinska, Claudia Preininger, Ursula Sauer, ARC Seibersdorf Research GmbH, 2444- Seibersdorf, Austria; [email protected]
Microarrays have created new opportunities in medical, environmental and food
256 Poster Abstracts diagnostics. To date the technologies are mainly restricted to the research lab due to their high cost, lack of approved standards and certain technical hurdles, such as insufficent reproducibility and sensitivity, especially in cases of low probe and target concentrations. For achieving stronger signals and lower detection limits 3D hydrogels as well as nanoparticles were employed as immobilization matrix for proteins. 3D hydrogels allow extremely high immobilization capacities and low non-specific binding and furthermore, provide an optimum water-like environment for proteins to maintain their structure and thus, activity. On the contrary, immobilized particle arrays combine the advantages of suspension (e.g. array element is prepared in bulk; variety of surface chemistries) and planar arrays (e.g. high throughput), but are 6fold superior to suspension and 200fold superior to planar arrays. Herein, we compare 3D hydrogel surfaces and particle arrays to conventional 2D chip surfaces with respect to signal-to-noise ratio, detection limit and suitability in food diagnostics. Seven commercial poly(urethanes) (PU) of different swellability were tested both unmodified and modified with various additives (e.g. methacrylate, chitosan) or various crosslinkers, such as hexamethylene diamine (HDA), monochlortriazinyl-ß-cyclodextrin Na-salt (MCT) and cyanuric chloride (TsT). All hydrogel surfaces were characterized by analytical methods, such as light microscopy, AFM, profilometry, ellipsometry and contact angle measurements. ≥50 nm particles based on melamin, polystyrene and silica were modified, coated to IgG and printed in hydrogel solution providing multiple assay replicates. Both approaches were evaluated with regard to signal-to-noise ratio, fluorescence background and data reliability (percentage of data available for data anlysis, % CV, etc.) using a protein chip for detection of albumin in milk. Detection limits below 0.01 mg/ml albumin were easily achieved demonstrating the outstanding quality of the tested materials.
P2 PROTEIN MICROARRAYS AND ,SPATIALLY RESOLVED 344 PHOTOLUMINESCENCE IN BIO-CONJUGATED QUANTUM DOT-PROBES FOR EARLY CANCER DETECTION T. A. Zhukov, M . Dybiec, A. Zajac, W. Qian, C. Phelan, T. A. Sellers S. Ostapenko,.H. Lee Moffitt Cancer Center & Research Institute; University of South Florida, NNRC, 4202 E. Fowler Ave, 33620 Tampa, Florida, [email protected]
We propose a novel approach to increase the accuracy of early cancer detection through the application of nanotechnology, where luminescent semiconductor quantum dots (QDs) are conjugated with biomolecules. QD-bioconjugates offer unique opportunities of simultaneous (multiplexed) detection and analysis of panel extremely low marker levels and fluctuations. Quantitative measurement of biomarker on array can be achieved with QD-immunometric methodology utilizing spatially resolved (mapping) photoluminescence spectroscopy, and with confocal microscopy following with image analysis. We have constructed QDs probes by conjugation with antibodies specific to the selected lung, ovarian and prostate cancer serum and cell-based protein markers. Preliminary data show that proposed methodology allows monitoring of changes in biomarker concentration in nanoscale (pM-nM) range. We developed a prototype of a
257 Poster Abstracts
potential serologic assay with greater sensitivity than current methods and illustrate our method with several serologic biomarkers for cancer – CA125, IL-10, PSA, VEGF. The PL model in our study was developed to find and identify spectral signatures of QD-bioconjugates. We observed that conjugated QDs are reliably detected at substantially reduced concentration, which is critical for defining sensitivity limits of the luminescence metrology in multiplex assay. In our microarrays we print antigene at different concentrations on a functionalized substrate using MicroGrid microarrayer. Later microarray is processed with conjugated QDs where detector antibody recognizes printed antigene. We have modified the MicroGrid spotter to print Protein / Quantum Dots arrays on silicon substrates. The processed microarray undergoes imaging with confocal microscope and quantitation utilizing computerized dynamic analysis system (CDAS) for classifying micro arrays to measure the features on array spots: such as area and intensity. The quantitation of target antigen on the protein microarray spots show that the QDs luminescence intensity tracks the antigene (biomarker) concentration in analyte sample. Comparative analyses for sensitivity and specificity of biomarker detection with proposed methodology and techniques in relation to cancer diagnoses are underway.
P2 PRODUCTION OF BIOCOMPATIBLE FILMS BY CONTROLLED 345 GAS-PHASE SYNTHESIS AND DEPOSITION OF NANOPARTICLES
Karsten Wegner1,2, Massimo Gatelli1, Simone Vinati1, Paolo Piseri3, Gero Bongiorno3, Paolo Milani3 1) Tethis S.r.l., Piazzetta Bossi 4, I-20121 Milan, Italy; 2) Particle Technology Laboratory, Swiss Federal Institute of Technology, ETH Zentrum ML F22, Sonneggstrasse 3, CH-8092 Zurich, Switzerland 3) CIMAINA and Dipartimento di Fisica, Università di Milano, Via Celoria 16, I-20133 Milan, Italy; [email protected]
An integrated process for controlled one-step production of nanostructured biocompatible films was realized by combining gas-phase nanoparticle synthesis, aerodynamic particle manipulation, size selection and deposition. The manufacturing process merges high-temperature flame or plasma synthesis of nanoparticles and supersonic nozzle expansion, thus benefiting from combined technological advantages: First, flame reactors offer high level control over nanoparticle size, morphology, composition and crystallinity. They are commonly used for industrial production of metal-oxide and carbon nanoparticles. The employment of microplasma sources extends the spectrum of product particles to non-oxide ceramics and metals. Second, expansion of the aerosol through a system of nozzles and aerodynamic lenses allows gas-phase selection of a nanoparticle size fraction and focusing of particles to a beam for defined-area deposition. Particle as well as beam size can be controlled by the system pressure and the geometric arrangement of the nozzles. Third, the process separates the deposition zone from the high temperature ( >1000°C) nanoparticle synthesis reactor, enabling coating even of temperature-sensitive materials such as biodegradable polymers. Furthermore, integration of the technologies yields high intensity nanoparticle beams. Coating of a 1000 cm2 surface with a 100 nm thin
258 Poster Abstracts carbon film was demonstrated in less than 1 hour, even with a laboratory-scale apparatus. Routes for scale-up and continuous production are being developed. The integrated production technology for nanostructured thin films is presented by the example of biocompatible titania films that are used for culturing primary human melanocytes. The effect of process parameters in the different processing stages on nanoparticle and product film properties is investigated. Furthermore, it is demonstrated how the use of masks during deposition can even result in manufacturing of microarrays.
P2 SYNTHESIS OF MAGNETIC NANOPARTICLES BY THERMAL 346 DECOMPOSITION AND BIOCOMPATIBILITY Sang Im Park, ChongOh Kim, Chungnam Nat`l Univ., Dept. of Mater. Sci. and Eng., 220 Gung-dong, Yuseong-gu, Daejeon, Korea; and JongHwan Lim, Chungnam Nat`l Univ., Div. of Veterinary Pharmacology and Toxicology; and Nguyen Duy Ha, JongHee Kim, Chungnam Nat`l Univ., Research Center for Advanced Magnetic Materials; [email protected]
Novel properties of nanoparticles arise from the large fraction of atoms, which reside on the surface of the particles and from the finite number of atoms in core. Nanocrystals can be synthesized in a number of different ways, including grinding, electrodeposition, sputtering, metal evaporation, solution phase metal salt reduction, and neutral organometallic precursor decomposition. Two of the most common procedures for high quality nanoparticles are the thermal decomposition of a precursor molecule in the presence of coordinating solvent and a capping ligand. Smart application of nanomagnetic particles in biotechnology includes diagnosis and therapy as carrier to tumors. The application of magnetic particles for biological parts requires the biological experiments such as in vivo or in vitro certainly. In this work, the magnetic fluids with iron oxide that had the spherical shape with uniform size were prepared, and the biocompatibilities of them were investigated by using S. D. rat. Also, the surface of magnetic iron oxide was radioisotope labeled, and the discharging routes of them were then tracked in a rat.
P2 GENE SILENCING OF MUTANT K-RAS MEDIATED BY POLYCATION 347 LIPOSOME Tomohiro Asai, Satoshi Kido, Yuka Tsunoda, Kosuke Shimizu, Naoto Oku, Univ. of Shizuoka, Dept. of Medical Biochem., Yada, Suruga-ku, Shizuoka 422-8526 Japan; and Takehisa Dewa, Mamoru Nango, Nagoya Inst. of Tech., Mater. Sci. and Eng., Gokiso-cho, Showa-ku, Nagoya, 466-8555, Japan; [email protected]
Activating K-ras mutation is known as an ideal target for pancreatic cancer therapy, since a point mutation in codon 12 of the K-ras oncogene occurs in about ninety percent of the patients. RNA interference (RNAi) strategy for silencing mutant K-ras is promising approach to treat pancreatic cancer. In this study, we developed cetyl-polyethylenimine
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(cetyl-PEI)-modified liposome (polycation liposome; PCL) for efficient delivering of short interfering RNA (siRNA). PCL, composed of cetyl-PEI, dioleoylphosphatidyl-ethanolamine (DOPE), and cholesterol, was prepared, sized, and complexed with siRNA. Suit-2 human pancreatic carcinoma cell line with mutant K-ras and HT-29 human colon adenocarcinoma with wild-type K-ras were used for evaluating the effect of gene silencing. The results showed that PCL-mediated lipofection of mutant K-ras siRNA reduced the protein expression and subsequent phosphorylation of extracellular signal-regulated kinase (ERK) in Suit-2 cells. Furthermore, the knockdown of mutant K-ras resulted in the suppression of Suit-2 cell proliferation. This suppressive effect was significantly higher than the use of wild-type K-ras siRNA. In contrast, the silencing effects of mutant K-ras siRNA in HT-29 cells were weaker than those of wild-type K-ras siRNA. These results indicated that mutant K-ras siRNA/PCL system could specifically work on the target mRNA. Next, we focused on mutant K-ras siRNA-induced apoptosis of Suit-2 cells. Both DNA fragmentation assay and confocal microscopic study showed that the lipofection of mutant K-ras siRNA resulted in induction of apoptosis. In confocal microscopic observation, the number of TUNEL-positive cells was increased in mutant K-ras siRNA compared with wild-type K-ras siRNA. These data suggested that mutant K-ras siRNA could lead to apoptosis of Suit-2 cells by blocking mitogen-activated protein kinase (MAPK) pathway. The finding indicated that mutant K-ras siRNA/PCL system might be effective against pancreatic cancer.
P2 AMPEROMETRIC IMMUNOSENSOR BASED ON HIGHLY DISPERSED 348 GRAPHITE MICROPARTICLES FOR INFLUENZA VIRUSE DETECTION Ravil A Sitdikov and Ebtisam S Wilkins, Department of Chemical and Nuclear Engineering, University of New Mexico, Albuquerque, NM 87131; [email protected]
Avian influenza is an infectious disease of birds caused by type A strains of the influenza virus. Amperometric immunosensor for Parainfluenza (PI) Type I virus and Influenza virus Type A (IA) detection and flow-through sandwich immunoassay scheme was developed . Highly dispersed graphite microparticles offers a large surface area and at the same time functions as the working electrode because of its good conductive nature. This provides the basis for covalent immobilization of anti-influenza A or anti-parainfluenza goat antibodies on the graphite particles and amperometric detection of peroxidase enzyme label. Such a process enhances the proximity of biological components with the transducer, a very essential factor for biosensor development. Electrochemical transduction offers certain advantages with respect to ease of operation and sensitivity. The immunosorbent is deposited on the filter membrane by vacuum resulting in dispersed graphite immunosorbent forming the measuring immunoelectrode (disposable immunosensing elements). Flow cell serves as an immunoreactor as well as an electrochemical cell. The optimization of disposable immunosensing elements of the amperometric transducer is based on the principle of close attachment of the components of the ligand-receptor system to the electrode surface of the transducer. Lower detection limits for PI and IA assay are 0.5 and 0.4 ng/ml respectively with a coefficient of variation (CoV) of 0.05. The upper
260 Poster Abstracts detection limit for PI is very high as 10,000 ng/ml. The total time of the assay is about 22 minutes. Described electrochemical biosensor can provide rapid, simple and low-cost on-field detection. Electrochemical measurement protocols are also suitable for mass fabrication of miniaturized devices.
P2 CHARACTERIZATION OF REP 2006 ANTIVIRAL DRUG AND DRUG 349 DELIVERY SYSTEM FOR SMALL ANIMAL MODELS Divey Saini, Mark Buller, St. Louis University, Dept. of Molecular Microbiology and Immunology, St. Louis, MO; Pratim Biswas, Washington University in St Louis, Environment Engineering Science, St Louis, MO; and Andrew Vaillant, REPLICor Inc., 500 Cartier Blvd West, Suite 135, Laval, Quebec, Canada; [email protected]
Due to the mounting bioterrorism threats in recent years and the resulting heightened security levels there is an urgent need to stockpile vaccines and antivirals for national bio-defense. A step in this direction has been taken by REPLICor, which is developing a broad-spectrum anti-viral drug to treat at least 12 families of viruses. The drug is in its preclinical phase where it is being tested in small animal models through aerosol inoculation. The drug was aerosolized using a Aero Mist Nebulizer and delivered to mice by an eight-port nose-only aerosol chamber. Drug particle size and concentration was measured with a Scanning Mobility Particle Sizer (SMPS 3081) and an Aerodynamic Particle Sizer (Model 3321). The drug particle sizes were measured in the micron and submicron size range with the relative amounts of each particle dependant on drug concentration. For 0.1 mg/mL, 1 mg/mL, 10 mg,mL, and 100 mg/mL the geometric mean diameters were 28.93 nm, 43.80 nm, 75.77 nm, and 107.33 nm, respectively. The particle number concentrations for similar drug concentrations was measured to be 1.17e6, 2.49e6, 7.89e6, 2.88e7, and 3.29e7 particles/cc. The aerosol delivery chamber was characterizd as a function of particle size and number concentration. The drug characterization parameters were measured at the four ports of the chamber. For 10 mg/mL drug concentration the percentage standard deviation in particle size and number concentration for the four ports was 4.15% and 7.02 % respectively. This data suggests a consistant aerosol dose will be delivered to the ports of the aerosol chamber at an optimum particle size for inhalation studies with small animal models.
P2 A PRACTICAL APPROACH TO THE USE OF NANOPARTICLES FOR 350 VACCINE DELIVERY Janet R. Wendorf, Manmohan Singh, Jina Kazzaz, Padma Malyala, Elawati Soewanan, James Chesko, Mildred Ugozzoli, Derek T. O’Hagan, Novartis Vaccines & Diagnostics; 4560 Horton Street, Emervyille, California, 94608, [email protected]
Poly (DL-lactide-glycolide) (PLG) polymers were used to create nanoparticles ranging in size from 110 nm to 230 nm. Protein antigens were adsorbed to the nanoparticles for a
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vaccine delivery system. The nanoparticles were compared to alum in vivo with a recombinant menigococcal vaccine protein. The nanoparticles in comparison to alum were found to have either enhanced or comparable immunogenicity. This makes the nanoparticles an enticing candidate for a vaccine delivery system. Nanoparticles offer advantages over microparticles with ease of processing and higher surface area for protein adsorption. We found that proteins could be adsorbed to nanoparticles up to at least 5% (wt. protein/wt. PLG) while microparticles plateaued at approximately 1.5% (wt. protein/wt. PLG). However, nanoparticles do not resuspend to the same size post lyophlization without the addition of excipients. Sugars and surfactants were investigated as resuspension agents for nanoparticles. Vaccine compatible excipient combinations were tested to find conditions where initial particle size was recovered. Sterile filtration of smaller nanoparticles (<130 nm) led to minimal PLG losses and allowed the initial nanoparticle preparation to be a non-aseptic process simplifying preparation in comparison to microparticles. We found that the smaller nanoparticles of size 120 nm needed more surfactant to resuspend than slightly larger (~220 nm) nanoparticles. To resuspend 120nm nanoparticles formulations of poly (vinyl alcohol) with sucrose/mannitol or dioctyl sodium sulfosuccinate (DSS) with trehalose/mannitol were sufficient. The protein-nanoparticle resuspension with the same excipient combinations was also dependent on the protein and protein loading level. A lyophilized nanoparticle formulation with adsorbed protein antigen and minimal excipients is an effective vaccine delivery system.
P2 EVALUATION OF NANOENCAPSULATION PERFORMED BY SPRAY 351 DRYING TECHNIQUE FOR THE DELIVERY OF TUBERCULOSIS DRUGS (ATD) Hulda Shaidi Swai1*, K.T. Hillie2, N. Cingo3, L. Kalombo1, M. Legodi1, B. Semete4, and L. Shoba4, 1Council of Industrial Scientific Research (CSIR)- Polymer Ceramics and composites, P.O Box 395, Pretoria 0001, South Africa; 2Council of Industrial Scientific Research (CSIR)- National Metrology Laboratory, P.O Box 395, Pretoria 0001, South Africa; 3Departiment of Chemistry, University of South Africa-UNISA, P.O Box 392 Pretoria 0003, South Africa; 4Council of Industrial Scientific Research (CSIR)- BioSciences P.O Box 395, Pretoria 0001, South Africa; [email protected]
Tuberculosis (TB) is gaining ground: in 2001, the disease killed more people than any previous year in history. Globally, there is a 3% increase in new TB cases each year, while in Africa, the increase is 20% per year, largely due to co-infection with HIV/AIDS. Every year, eight million people worldwide develop active TB and three million die from it, while more than 400 000 new cases of multi-drug resistant TB (MDR-TB) are diagnosed. Although an effective therapeutic regimen is available, patient non-compliance results in treatment failure, while the emergence of drug resistance can lead to MDR-TB. Not a single new class of TB drug has been developed in over 40 years. The TB nano drug delivery study seeks to address patient non-compliance in TB control programmes through the development of a system whereby drugs can be administered in a single dose that maintains an active level (minimum inhibitory concentration – MIC) for a number days,
262 Poster Abstracts weeks or months. This will be done by nano-encapsulating techniques with known TB drugs, which have very poor bioavailability using an FDA approved biodegradable polymer which will allow slow steady release of the drugs for longer periods. The present work will focus on the encapsulation of the anti-tuberculosis drug (Isonaizid) into a biodegradable polymer, Poly(lactide-co-glycolide (PLG) using spray drying technique. These particles have been prepared in our lab and characterised via SEM, LLS, Zetasizer and UV-Vis spectrophotometry. It is believed that the cellular uptake of the nanocarriers is strongly dependent on their surface charge. Therefore to get positively charged nanoparticles remains an additional target for this work. Chitosan, a positively charged natural and biodegradable polymer will be used as a co-surfactant aside polyvinyl alcohol-89% partially hydrolysed, with the aim of imparting a positive charge to the nanoparticles. The process parameters of the spray drying i.e. temperatures, feeding rate and airflow rate will be optimised in terms of high incorporation efficiency of the drug, size and the morphology of the nanoparticles.
P2 DOUBLE CARRIER SYSTEMS AS NOVEL DRUG DELIVERY 352 VEHICLES Edith C. Rojas, Nurettin Sahiner, Vijay T. John, Kyriakos D. Papadopoulos. Department of Chemical and Biomolecular Engineering, Tulane University, New Orleans, LA 70118, USA; [email protected]; [email protected]
Cationic hydrogel particles were prepared as carriers in nano and micro size. It was shown that these carriers can be transported by another micro-scale delivery vehicle such as an oil-in-water (O/W) or a water-in-oil-in-water (W1/O/W2) emulsion system. The perception is that the use of a carrier inside another carrier provides the advantage of both systems. For example, while one carrier is keeping the load in perfect conditions i.e. stealth, the other carrier can afford the storage and handling for the initial shipment. Furthermore, the conjugated action of multiple emulsions and hydrogels can be designed to work sequentially, which may be useful for achieving prolonged delivery of the active substance, better-tuned release and/or higher-uptake efficiency. This system can also be used as double delivery-vehicle, each carrier carrying specific loads. The microcapillary technique developed in our laboratory was employed to closely monitor the individual multiple emulsion systems. Here, the emulsion system consists of either nanohydrogels dispersed in the internal aqueous phase (H/W1/O/W2), or microhydrogels themselves suspended in the oil phase (H/O/W). Through such observations, the selective active-substance release from the delivery vehicles was shown to be promoted by external stimuli. Since fluorescent dye was used as a model active substance, fluorescence microscopy images revealed that hydrogels (first carrier) prevent the fluorescent dye from spontaneously migrating into the external aqueous phase of the second carrier. Then, the fluorescent dye loaded into the hydrogels was shown to be released upon changing the pH of the medium.
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P2 AN INVESTIGATION INTO USING SPRAY CHILLED GELUCIRE 50/13 353 MICROSPHERES AS CARRIERS FOR POORLY SOLUBLE DRUGS Sheng Qi, Duncan Q.M. Craig, School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich NR4 7TJ, UK; and Delphine Marchaud, Gattefossé SAS, 36 Chemin de Genas, BP 603-F-69804 Saint-Priest Cedex, France; [email protected]
Spray chilled Gelucire 50/13 microspheres were used as drug carriers to improve the solubility of poorly soluble drugs. This formulation combines the advantages of solid dispersions and microparticulate dosage forms. It has the potential of not only improving the bioavailability of poorly soluble drugs in vivo, but also of reducing the risks of single dose formulations after administration. Piroxicam as the model drug was encapsulated in the Gelucire 50/13 matrix using a customized spray-chilling apparatus. The microspheres were determined to have a spherical shape with smooth surfaces and to be between 20μm and 200μm in diameter. Thermal studies revealed that there was no interaction between piroxicam and Gelucire 50/13 matrix. Hot stage microscopy (HSM) experiments in polarized mode proved that the drug particles exist as separate crystals in the microspheres. The dissolution behaviour of the microspheres in water was monitored using constant temperature stage (37˚C) HSM. The microspheres were observed to swell and lose their spherical shape on exposure to water. The dissolution results of the piroxicam loaded microspheres showed significant improvement compared to those of drug alone. This is as a result of improved wettability of the drug particles by using lipid materials with relatively high HLB value. Particle size, drug loading, and aging of Gelucire 50/13 have been shown as the main factors influencing the drug release from the microspheres. For microspheres of size between 60-200μm in diameter, slower drug release was observed compared to smaller microspheres. For particles in this size range (60-200µm) lower drug loading and a longer aging period increased the dissolution rate. However, for the microspheres with size below 60μm in diameter, the drug release rate did not vary with these factors. Further investigation is required for a fuller understanding of the drug release mechanism of spray chilled Gelucire 50/13 microspheres.
P2 LONG-TERM STUDY OF EPO-SECRETING MYOBLASTS ENTRAPPED 354 IN MICROCAPSULES ELABORATED WITH FIVE DIFFERENT COMMERCIAL ALGINATES Sara Ponce, Gorka Orive, Rosa Hernández, Alicia R. Gascón, Jose Luis Pedraz, Laboratory of Pharmacy and Pharmaceutical Technology, University of Basque Country, Vitoria, Spain; and Josep M. Canals, Jordi Alberch, Dept. Cellular Biology and Pathologic Anatomy, Faculty of Medicine, IDIBAPS, University of Barcelona, Spain; [email protected]
The alginate is the most frequently employed anionic polysaccharide for cell microencapsulation. However, to ensure the clinical application of this polymer, it is critical to prove that the alginate has a suitable and reproducible biocompatibility. Since it
264 Poster Abstracts has a natural origin, several contaminants exist in its composition. These contaminants are in general mitogenic and inflammation provoking molecules which activate host’s immune response provoking the release of cytokines such as IL-lβ, IFN-y, IL-6 and TNF-α. In the present study, five different commercial alginates were used for the elaboration of microcapsules. EPO-secreting C2C12 myoblasts from C3H mice were immobilized in the different microcapsules in order to study the impact of the alginate properties (purification degree, composition and viscosity) on the long-term efficacy of the therapeutic strategy. Myoblasts were used since the experience has shown that murine C2C12 myoblast cell line is particularly well suited for cell encapsulation technology. The elaborated microcapsules were implanted subcutaneously in Balb/c mice. High and constant hematocrit levels were maintained until day 130 after only one shot of the cell-loaded microcapsules and without implementation of immunosuppressive protocols. According to the results, all types of alginate microcapsules seemed to function in a similar manner increasing the hematocrit level in the transplanted mice including the group of mice which received microcapsules elaborated with a nonbiomedical grade LVM alginate, although the latter showed an increased fibrotic overgrowth. The potential role of EPO effects and the impact of the released EPO dose on these results are discussed in detail.
P2 IN VIVO FATE OF THE MICELLE FORMING BLOCK COPOLYMER 355 POLY (ETHYLENE GLYCOL-BLOCK-CAPROLACTONE) IN MICE FOLLOWING INTRAVENOUS ADMINISTRATION Jubo Liu, Faquan Zeng, Christine Allen*, Department of Pharmaceutical Sciences, University of Toronto, 19 Russell street, Toronto, ON, Canada M5S 2S2; *[email protected], [email protected]
Poly (ethylene glycol)-b-poly (caprolactone) (PEG-b-PCL) copolymers were synthesized and characterized in terms of their CMC, thermal properties, biocompatibility and biodegradation profiles. The copolymers were employed to form micelles as potential drug delivery carriers. The biologically relevant physico-chemical properties of these micelle systems were investigated. The PEG5000-b-PCL5000 micelles were selected for evaluation of in vivo fate following i.v. administration due to their small size, superior in 3 vitro stability and slow degradation profile. Specifically, H-labeled PEG5000-b-PCL5000 micelles with an average size of 56nm were i.v. administered to Balb/C mice at doses of 250mg/kg, 1.5mg/kg and 0.1mg/kg in order to examine the distribution kinetics of 1) copolymer assembled as thermodynamically stable micelles (i.e. concentration of copolymer above CMC prior to and upon dilution following i.v. administration), 2) copolymer assembled as thermodynamically unstable micelles (i.e. concentration of copolymer above CMC prior to but not following dilution upon i.v. administration) and 3) copolymer single chains. The biodistribution of the copolymer in the major organs was investigated and the main pharmacokinetic parameters were determined by fitting the data into compartmental open models. In summary, the formation of micelles was found to effectively entrap the copolymer in the plasma as revealed by the significant difference between the distribution volume of micelles (i.e. 1.67mL and 1.49mL for doses of 250mg/kg and 2.5mg/kg, respectively) and copolymer single chains (i.e. 7.54mL). The
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moderate elimination half-life of the 1.5mg/kg (i.e. 16.7 hrs) dose group indicated a good kinetic stability for this micelle system even though the copolymer concentration fell below the CMC upon dilution following i.v. administration. The AUC values in all organs examined from 0 to 48 hours were found to decrease in the following order liver > kidney> spleen > lung > heart in all dose groups. The biodistribution profiles were found to be dose dependent as indicated by the increase in the accumulation of copolymer single chains compared to micelles in the kidney.
P2 TREHALOSE BEARING POLYMERIC PARTICLES: SORPTION 356 INDUCED HYDROLYTIC DEGRADATION OF PESTICIDES AND CHEMICAL AGENTS Yongwoo Lee, Ted, Mendum, Chunyong Wu, Nese Orbey, and John Puglia, Foster-Miller, Inc., 195 Bear Hill Road, Waltham, MA 02451-1003, USA; [email protected]
Cross-linked Polymeric Trehalose (PT) particles having trimeric alkyl spacers were synthesized, sieved to achieve a narrow particle size distribution, and employed as a physical support for enzyme catalysts. These particles were shown to be chemically inert and robust enough to endure extended exposure to highly acidic and highly alkaline media. Sequestration of methyl parathion (MPT) and its hydrolysis product p-nitrophenol (pNP) in the macroporous cavities of these particles was confirmed. Organophosphorous Hydrolase (OPH) was immobilized between molecular layers of non-covalently attached buffered polyelectrolyte within the cavities of the particles, utilizing secondary interactions such as ionic and/or hydrophobic interactions between molecular layers, rather than covalent bonding. These environmentally benign, polymeric particles were demonstrated to have high binding efficiency for the enzyme catalyst, high enzymatic activity, the capability to efficiently hydrolyze and sequester their substrates, and the ability to act as sorption-induced hydrolyzing vehicles against biological and chemical threat agents (GA, VX).
P2 BIODEGRADABLE NANOPARTICLES FOR VACCINE DELIVERY 357 SYSTEMS Ik Joong Kang and Bum Gil Kim, Department of Chemical and Bio Engineering, Kyungwon University, Sungnam 461-701, KOREA ; [email protected]
Many biological researches have demonstrated that chitosan derivatives were effective, safe absorption enhancers to improve the delivery efficiency of drug, and suitable for controlled drug release because it has stability, bio-compatibility and biodegradability. Recently, the interest in the extension of human life and the health has been increased. Many researches in a pharmacy and a medical area have studied in a sustained drug release property and stability of drug release property in a body. Hepatitis virus(Hv) is the liver
266 Poster Abstracts affinity DNA virus as a kind of hepadna virus has 42 nm diameter and no cell denaturalization phenomenon. Outer membrane of the virus consist of hepatitis surface antigen(HBsAg) and center is composed of Hv DNA and hepatitis B core antigen (HBcAg). It is very mortal that people over 8 % get infected with chronic hepatitis as increasing trend and consequently leaded to the death because of a complication arises of cirrhosis and liver cancer. All of the infants have to be prescribed by three consecutive injection of hepatitis vaccine just after his birth adults are also prescribed by an intra muscular injection every five years. In this study biodegradable polymer's derivatives was synthesized and made to nanoparticles as a supporter of vaccine. The character of vaccine delivery carrier as its size and surface polarity was analyzed by AFM, SEM, FT-IR, HPLC, ELS, and Zeta potential for the development of effective drug delivery system. As a result, biodegradable nanoparticles containing vaccine obtained the effectiveness in drug release control the immunological reaction.
P2 EFFECTIVE STABILIZATION OF BIOACTIVE INGREDIENTS FOR 358 LONG-TERM STORAGE USING W/O/W ENCAPSULATION AND BUFFERING LAYER Jae-Eun Jung, Mi-Ok Jung, Jee-Hyun Ryu and Kyung-Do Suh Department of Chemical Engineering, College of Engineering, Hanyang University, Seoul 133-791, Republic of Korea; and Jin-Woong Kim, Division of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138, USA; [email protected]; [email protected]
Beneficial materials for the human body such as enzyme or antioxidant have a drawback, which is too weak from the environmental factors to preserve their bioactivity for a long-term storage. For instance, papain which is an enzyme from a papaya fruit is extremely sensitive to the heat. One of the antioxidants, riboflavin (Vitamin B2) is also unstable in water phase especially in the sunlight. To overcome these problems, various stabilization methods, containing W/O/W emulsion, encapsulation and suspension polymerization, have been widely reported. However, complete solutions are not suggested yet. In addition, water-soluble biomaterials are generally stabilized by W/O/W evaporation method, but the initial drug loading ratio is 40% below. In this presentation, we proposed an efficient stabilization method for bioactive ingredients by introducing a buffering layer to the microcapsules. Papain and riboflavin are chosen as model drugs; these were microencapsulated with polymethylmethacrylate (PMMA) using W/O/W evaporation method. Various types of polyols, such as polypropylene glycol (PPG), polyethylene glycol (PEG) and triblock polyol (PEG-PPG-PEG), were applied to buffering layers. Because of the nature of polyols, it might be existed between the drug and polymer, isolating the drug from the surrounding. The bioactivity of the capsules was measured as a function of storage time, and the results were compared to the HLB values of polyols. The existence of drugs in the capsules was confirmed using a confocal laser scattering microscope (CLSM). For a clear analysis, the drugs and polyols labeled with fluorecien isocyanate (FITC), and rhodamin B isocyanate (RBITC), respectively. Optical microscope (OM) and scanning electron microscope (SEM) were used to observe the morphology of
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the microcapsules. The initial drug loading efficiency and the preservation of drugs in the buffering layer incorporated microcapsule were determined by UV-vis spectroscope.
P2 DEVELOPMENT OF A NEW MULTIPARTICULATE SYSTEM FOR 359 COLON CONTROLLED DRUG DELIVERY Elquio E. Oliveira, Acarília E. da Silva, Marcelo G. Silva, Toshiyuki N. Junior, E Sócrates. T. Egito, Universidade Federal do Rio Grande do Norte, Departamento de Farmácia, Laboratório de Sistemas Dispersos (LASID), Rua Gal. Gustavo Cordeiro de Farias, s/n, 59010-180, Natal/RN, Brazil. [email protected] ; and Anselmo G. Oliveira, UNESP-Araraquara, Rodovia Araraquara-Jaú, km 01, 14.801-902 Araraquara-SP – Brazil
Nowadays delivery of poorly absorbed drugs in stomach and small bowel and prevention of side effects caused by them in such sites have been particularly desirable. For those purposes, colon specific drug delivery systems have been developed for treatment of colon diseases like ulcerative colitis, Crohn’s disease and colon cancer. Those systems may be obtained by microencapsulation employing biodegradable polymers such as xylan, which is hydrolyzed by enzymes present in the colon, or pH-dependent polymers such as Eudragit® S-100. The various approaches used for producing the microcapsules include cross-linking reaction and spray drying technique. The aim of this study was to develop a novel multiparticulate system based on xylan microcapsules coated with Eudragit® S-100. The microcapsules were produced by the interfacial cross-linking process. An alkaline solution containing xylan and NaOH 0.6N was emulsified in 30mL of the chloroform:cyclohexane [1:4(v/v)] containing 5% (w/v) sorbitan triesterate. After 10 min, the interfacial cross-linking reaction was trigued by adding 40 mL of 5% (w/v) terephthaloyl chloride solution. The reaction was ended by dilution with 30 mL of cyclohexane. Subsequently, microcapsules were separated by centrifugation and washed several times. For the coating process using a spray dryer, the dispersion was prepared by using 150 mg of xylan microcapsules and 400 mg of Eudragit® S-100 in 50 mL of water and stirring for 25 min. Aqueous suspension was spray-dried at 1.2mL/min (inlet temperature 120ºC) using a Büchi Model 191 laboratory spray dryer using a 0.7 mm nozzle. The process induces the formation of microcapsules. The optical and electronic microscopy (SEM) revealed non-aggregated spherical microcapsules with a smooth surface showing a mean particle size of 10.5 ± 0.1µm. Regarding the visual aspect, the sample appeared to be a fine yellowish powder. This system represents an eligible colon-specific drug delivery system using a multiparticulate model based on xylan microcapsules coated with an enteric polymer.
P2 SODIUM DICLOFENAC INCORPORATION INTO XYLAN 360 MICROCAPSULES FOR COLONIC DRUG DELIVERY Elquio E. Oliveira, Acarília E. da Silva, Marcelo G. Silva, Toshiyuki N. Junior, E Sócrates. T. Egito, Universidade Federal do Rio Grande do Norte, Depto. de
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Farmácia, Laboratório de Sistemas Dispersos (LASID), Rua Gal. Gustavo C. Farias, s/n, 59010-180, Natal/RN, Brazil. [email protected]; and Anselmo G. Oliveira, UNESP-Araraquara, Rodovia Araraquara-Jaú, km 01, 14.801-902 Araraquara-SP – Brazil
Microparticles are drug delivery systems able to modify the drug action by changing its bioavailability. Xylan, a polymer abundantly found in nature, is enzimatically degraded in colonic environment in human body. Hence, it is an eligible material to produce colon specific drug carriers. The aim of this study was to incorporate sodium diclofenac (SD) in xylan microcapsules and evaluate the characteristics of both systems. The microcapsules were prepared by the interfacial cross-linking process. The polymer solution was obtained by dissolving 124 mg of xylan in 10mL of NaOH 0.6N. 6 mg of SD was added in 6mL of polymeric solution and emulsified in 30mL of chloroform:cyclohexane [1:4(v/v)] containing 5% (w/v) sorbitan triesterate. After 10 minutes, the interfacial cross-linking reaction was trigged by adding 40 mL of a 5% (w/v) terephthaloyl chloride solution in chloroform:cyclohexane [1:4 (v/v)]. The reaction was ended by dilution with 30 mL of cyclohexane. Then, the microcapsules were separated by centrifugation and washed several times. At last, the microcapsules were characterized regarding their morphology, homogeneity, pH and visual aspect. The drug loading efficiency was determined by spectrophotometry at 275nm. Concerning the visual aspect, both free microcapsules (FM) and sodium diclofenac microcapsules (SDM) were observed to be a yellowish dispersion. Microscopy analysis showed that not only FM but also SDM were quite spherical in shape and a little bit aggregated. They presented a mean diameter of nearly 25μm. The pH was 7.12 and 4.0 for FM and SDM, respectively. Such pH change is an indicative that reinforces SD encapsulation. Altogether, the results demonstrated the feasibility of producing xylan microcapsules with and without SD, both presenting the same aspect and homogeneity. A high encapsulation efficiency rate of 81.33% was achieved. Therefore, it may be concluded that SDM can be a new therapeutic carrier for the treatment of inflammatory diseases.
P2 DRUG RELEASE FROM NANO-PARTICLES MADE OF 361 RADIATION-CROSSLINKED ELASTIN MODEL POLYPEPTIDES Masakazu Furuta1, Elena Constantinoiu1, Taeko Nakamura1, Natsuko Tanaka1, Mari Fujimoto1, Toshio Hayashi1, Masamichi Iwama2, Mituhiro Murata3, and Dan W. Urry4; 1Osaka Prefecture University, 1-2, Gakuen-cho, Sakai, Osaka 599-8570, Japan, 2Bioelastics Japan, 11-20, Midori-ku, Yokohama, Kanagawa 226-0003 Japan, 3JSR Co., Ltd. 25, Miyukigaoka, Tsukuba, Ibaraki 305, Japan 4. University of Minnesota, St. Paul, Minnesota, U.S.A., and Bioelastics Research, Ltd., Birmingham, Alabama, U.S.A., [email protected]
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Elastic protein-based polymers were prepared by recombinant DNA technology and based on the GVGVP repeat amino acid sequence of tropoelastin. Under specified conditions, such as a temperature above30oC, They can be coacervated to form nano-particles and stabilized by crosslinking with gamma-rays that pass through a 0.22 nm filter and exhibit diameters of less than ca. 150 nm and a narrow size distribution. Yield of the gamma-cross-linked nano-particles increases with increasing dose but is insensitive to dose rate. Here we report their use in drug delivery. In particular, time-release profiles for Biebrich Scarlet Red gave results equivalent to those of previously reported gamma-cross-linked discs made of the same polymer. Thus, this controlled release device can deliver drugs to sites accessible by small caliber hypodermic syringes. Some other anticancer drugs are being examined for loading and releasing to the crosslinked peptides and favorable results are being collected.
P2 MELITTIN-LIPID INTERACTIONS: A COMPARISON BETWEEN 362 LIPOSOMES AND POLYMER-STABILIZED NANODISCS. Per Wessman, Anna Lundquist, Katarina Edwards, Department of Physical and Analytical Chemistry, Uppsala University, Box 579, SE-751 23 Uppsala, Sweden; [email protected]
Liposomes have during the last decades been extensively used as model membranes in studies aiming at the investigation of peptide-lipid interactions. The results of liposome-based investigations have greatly increased our knowledge and understanding of the effects caused by various peptides on structure, permeability and other important qualities of lipid bilayers. In order to compare and evaluate data obtained under different conditions, e.g. at varying lipid composition/concentration or temperature, detailed knowledge about the partition behavior is required. Unfortunately the self-closed, and frequently multilamellar, structure of liposomes may lead to difficulties when evaluating partition data. We have recently developed a new type of nano-sized model membranes, the PEG-stabilized bilayer discs, which avoid the above problem. The discs show excellent temperature and long term stability, and their open structure ensures that peptides have full access to both leaflets of the bilayer. In the present study we used melittin as a model peptide to investigate and compare the partition behavior in systems containing liposomes and discs, respectively. For these studies, which included bilayers with several different lipid compositions, we employed a spectroscopic method based on changes in the intrinsic fluorescence of melittin. Further, the obtained partition coefficients were, for certain selected systems, used to compare and correlate the effect of melittin on properties such as permeability and structure of the model membranes.
P2 NIMODIPINE-NANOLIPOSOMES: PREPARATION, 363 CHARACTERIZATION AND PHARMACOKINETICS IN RAT Guifang Zhang, Department of Pharmaceutics, University of Minnesota, 308 Harvard St. SE, Minneapolis, MN, 55455, USA; and Jiabi Zhu, Xian Zhu and
270 Poster Abstracts
Shan Li. College of Pharmacy, China Pharmaceutical University, Nanjing, 210009, P. R. China; [email protected]
The purpose of this study was to improve the solubility, stability and enhance the brain target efficiency of nimodipine by preparing a formulation as nanoliposome. A lyophilized powder of nimodipine-nanoliposomes were prepared using the ethanol dripping-sonicating dispersion-freeze drying technique. The formulation consisted of eggPC:cholesterol:sodium deoxycholate:tween-80 = 8: 1: 4.5: 2.25 (by mass) with drug load 0.42%. Mannitol (70 % by total mass) was added as a protective agent for freeze drying. Following reconstitution in normal saline, the mean particle size was 18.9 nm, and the entrapment efficiency was 96.8 ± 1.3%. The lyophilized powder was found to be stable during one year storage in sealed ampules in dark and room temperature. No change in the particle size or entrapment efficiency was detectable for 10 hours test following reconstitution. Nimodipin-nanoliposomes were administered to rats by short IV infusion and the resulting serum and tissue concentrations were determined by HPLC and compared to the IV infusion of the commercial nimodipine solution (0.5mg/kg). The Cmax, AUC, and MRT were significantly greater and ke and CLs were smaller for nimodipine-nanoliposome compared to the nimodipine solution. For brain tissue, the targeted efficiency was 6.02 based on the AUC ratio for nimodipine-nanoliposome versus 2.80 for the solution. The results demonstrate that entrapment of nimodipine in nanoliposomes decreases the elimination and increases the tissue bioavailability of nimodipine and the brain targeted efficiency in rats. Thus, the nanoliposome formulation provides for greater stability and targeting over the existing commercially available solution.
P2 DETERMINING THE DISPERSION BEHAVIOR OF FORMULATED 364 BULK DRUG USING LIGHT DIFFRACTION ANALYSIS Maria R. Toler, Pfizer, Inc., 7000 Portage Rd., Kalamazoo, MI 49001;[email protected]
Understanding how a drug will disperse in a selected media is desirable for predicting in-vivo behavior and designing an appropriate formulation. A novel approach to particle size analysis is presented here, outlining the use of this technology for describing the dispersion behavior of formulated drug. Two case studies are presented: A quick dissolve tablet and an oral suspension. Traditional light diffraction particle size analysis (wet dispersion) involves the selection of an appropriate dispersing agent and adequate energy to the sample to provide a well dispersed system. In determining the dispersion behavior of drug particles, the media is chosen based on the question to be answered. In the case of a quick dissolve tablet, rapid disintegration and mouth feel are important to a satisfactory formulation. Dispersion behavior in artificial saliva was evaluated. In the case of an oral suspension made using a fairly insoluble micronized drug, the presence of aggregates in the suspension greatly affected bioavailability. The determination of the degree of aggregation in dissolution media was critical to predicting bioavailability and formulating an appropriate suspension. We emphasize that optical microscopy plays a critical role in
271 Poster Abstracts the evaluation of dispersion behavior. This, along with light diffraction particle size analysis can provide critical input to predicting adequate performance of a formulation.
P2 GIANT PHOSPHOLIPID VESICLES AS A POSSIBLE SYSTEM FOR 365 STUDYING INTERACTIONS OF MEMBRANES WITH ANTIBODIES Jasna Urbanija1, Blaž Rozman2, Aleš Iglič3, Veronika Kralj-Iglič2,4, 1Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia; 2Department of Rheumatology, Clinical Center, Ljubljana, Slovenia; 3Faculty of Electrical Engineering, University of Ljubljana, Ljubljana, Slovenia; [email protected]
Antiphospholipid syndrome (APS) is an autoimmune disease characterized by thrombotic events and/or pregnancy morbidity. The etiology and underlying mechanisms of APS are not yet understood, however, activation of the coagulation system is evident. Antiphospholipid antibodies are present in the sera of patients with APS. These antibodies were found to interact directly with phospholipids constituting cell membranes (e.g. cardiolipin) or to bind to an antigen beta2-glycoprotein1 (ß2GPI). Therefore giant phospholipid vesicles (GPV) were prepared with three different lipids: POPC, cholesterol and cardiolipin in proportions 2:2:3, respectively. With such selection of lipids we approach the actual constitution of biological membranes involved in APS and make the GPV stable even for days. Interactions between GPV, antibodies and ß2GPI were studied under phase contrast microscope. The change of morfology of GPV due to addition of different antibodies as well as ß2GPI, could provide us with better understanding of the effects of antibodies on biological processes in patients with APS. Strong adhesion of vesicles to each other and to the ground was observed, together with suppression of vesicles contour fluctuations, enhancement of the contrast of the vesicle contour, permeabilisation of vesicle membrane to sugar resulting in dissapearance of the contrast between the vesicle interior and exterior, lateral segregation of the membrane constituents and bursts of vesicles. Our results indicate that antibodies and antigens may have an important role in the formation of complexes composed of cells, membrane fragments and proteins - complexes which are important in blood clot formation.
272 Preregistered Conferees
Index of Speakers/Presenters (Alphabetically With Paper Number)
Akiyoshi, Kazunari – 6 Caliceti, Paolo – 273 Alasundaram, Ganesan – 277 Camata, Renato P. – 188 Allen, Christine – 84 Carbone, Roberta – 92 Almeida, José F. – 332 Carmona–Ribeiro, Ana M. – 203 Amsden, Brian G. – 18 Castro, Lizandra – 311 Arifin, Dian R. – 218 Celis, Maria–Teresa – 12, 184 Asai, Tomohiro – 347 Chakrapani, Aravind – 146 Asher, Sandy – 95 Chan, Chak K. – 47 Backer, Joseph – 177 Chen, Wei – 66 Barbé, Christophe J. – 152 Chertok, Beata – 342 Bauer, Christina A. – 333 Cho, Chong–Su – 226 Bäumler, Hans – 2 Chow, Gan–Moog – 198 Benson, James R. – 108 Clausi, Amber – 312 Berkland, Cory – 194 Cliffel, David E. – 79 Berret, Jean–François – 32 Colvin, Vicki – 59 Berret, Jean–Francois – 334 Cook, Robert – 110 Bhowmick, Tridib K. – 197 Coulter, Kent – 52 Bill Turnell – 262 Couzis, Alexander – 36 Blanchard, Jim – 109 Dai, Yujie – 154 Boch, Ron – 101 Dan, Nily – 156 Bogan, Mike – 46 Darlington, Jerald W. – 331 Bonroy, K. – 213 Darvari, Ramin – 217 Boyle, Ross W. – 250 De Geest, Bruno G. – 142, 330 Braun, Paul – 139 De Theije, Femke – 212 Brayner, Roberta –64, 340 Delair, Thierry – 204 Bringley, Joseph F. – 189 DeLong, Robert – 196 Brown, David P. – 305 Derwinska, Katarzyna – 343 Brown, Larry – 221 DeSimone, Joe – 186 Bumiller, Mark – 107 Dihang, Hélène – 42 Bunjes, Heike – 17 Driscoll, David – 11 Burgess, Diane – 90 Drummond, Daryl – 37 Burkhard, Peter – 229 Duarte, Ana Rita C. – 269, 314 Burnett, Daniel J. – 278, 279, 324, Duguet, Etienne – 335 325 Durant, Yvon – 211 Caffrey, Martin – 35 Dybiec, Maciej – 344 Caldwell, Karin – 33
273 Speaker/Presenter Index
Dziewiszek, Krzysztof J. – 219 Haas, Heinrich – 170 Ebert, Katrin – 149 Hain, Jessica – 327 Edsman, Katarina – 19 Happawana, Gemunu – 246 Egito, E. Sócrates. T. – 296, 297, Harms, Meike – 266, 326 359, 360 Harms, Meike – 326 Enriquez, Iris V. – 114 Hedderich, Regine – 317 Etzler, Frank M. – 43 Heitfeld, Kevin A. – 276 Euliss, Larken E. – 328 Herold, Marc – 29, 301, 302 Eversdijk, Jacco – 158 Hino, Tamoaki – 56 Fahr, Alfred – 245 Hjelm, Rex P. – 82 Farnsworth, Stan – 193 Hoerr, Robert – 15 Fischbach, Ingrid – 180 Hofmann, Heinrich – 185 Fischer, Sabine – 22 Hook, Sarah – 309 Florence, A.T. (Sandy) – 173 Horozov, Tommy – 148 Fonnum, Geir – 165 Howdle, Steve – 133 Forsayeth, John – 31 Hu, Min – 67 Foster, Neil – 267 Hu, Jianhua – 290 Frank, Matthias – 172 Huettmann, Gereon – 68 Fransén, Nelly – 329 Hurt, Robert – 199 Friggeri, Arianna – 5 Hütten, Andreas – 123 Frimpong, Reynolds A. – 235 Hwang, Sung–Joo – 138 Fritzsche, Wolfgang – 168, 310 Hyeon, Taeghwan – 119 Fujii, Gary – 201 Ichikawa, Hideki – 159 Furuta, Masakazu – 361 Ivkov, Robert – 70 Galperin, Anna – 295 Iwamoto, S. – 50 Gamazo, Carlos – 58 Jacobs, Irwin C. – 23 Gao, Xiaohu – 253 Jans, Hilde – 256 Gardner, David L. – 237 Jia, Xinqiao – 98 Giersig, Michael – 7 Jin, Yiguang – 190 Goia, Dan V. – 4 Johansson, Christian – 183 Goins, Beth – 214 Johnston, Keith – 132 Gold, Lynn – 13 Jørgensen, Kent – 86 Goldberg, Eugene – 144 Josefsen, Leanne B. – 72 Golomb, Gershon – 100 Joshi, Hemant – 182 Gower, Laurie – 97 Juillerat–Jeanneret, L. – 232 Grant, Gilbert – 127 Jung, Jae–Eun – 358 Green, Jennifer A. – 134 Kamau, Sarah W. – 223 Gruverman, Irwin – 10 Kamps, Jan A.A.M. – 38 Gupta, Ram B. – 136 Kaneda, Shinichi – 39
274 Preregistered Conferees
Kauppinen, Esko – 53 Lu, Yi – 207 Kell, Arnold J. – 300 Luby, Thomas – 202 Kim, Bum Gil – 357 Luo, Dan – 40 Kim, Eun–Jung – 288 Ma, Dongling – 247 Kim, Gloria J. – 248 MacCuspie, Robert I. – 208 Kollar, Csilla – 16 Maeda, Mizuo – 87 Kompella, Uday – 105 Mailänder, Volker – 80 Kono, Kenji – 129 Mak, Wing Cheung – 167 Koynova, Rumiana – 89 Margel, Shlomo – 26 Krauss, Todd D. – 77 Markland, Peter – 161 Kuhn, Liisa T. – 289 Martin, Andrew – 238 Kumar, Venisetty R. – 274, 275 Martins, Sofia – 339 Lau, Kenneth K. S. – 24 Masuda, Yoshitake – 287 Lavasanifar, Afsaneh – 83 Matsunaga, Terry – 28 Leblanc, Roger – 73 Mattoussi, Hedi – 74 Lee, Bob (RJ) – 117 McGrath, James L. – 200 Lee, Jim – 187 Miyata, Takashi – 140 Lee, Jonghwi – 141 Morimoto, Nobuyuki – 286 Lee, Robert W. – 263 Morrison, Shannon – 192 Lee, Sang–Kyung – 225 Moudgil, Brij – 236 Lee, Yongwoo – 356 Muhrer, Gerhard – 271 Legastelois, Stephane – 257 Müller, Rainer H. – 179 Lerche, Dietmar – 49, 151, 308 Müller, Angelika – 341 Leubner, Ingo – 181 Müller–Schulte, Detlef – 75, 160, Leuenberger, Hans – 244 298, 299 Leupold, Eik – 323 Mulvaney, Shawn P. – 164 Li, Chun – 265 Murtomaa, Matti – 241 Li, Pei (Pauline) – 94 Musyanovych, Anna – 99 Li, Yuzhuo – 27 Nakamura, Hiroyuki – 220 Li, Zhengmao – 103 Nakamura, Michihiro – 285 Lich, Ben – 307 Nandi, Indranil – 128 Lin, Victor – 176 Narasimhan, Balaji – 205 Lipp, Michael – 240 Nath, Sudip – 291 Liu, Jubo – 355 Navailles, Laurence – 131 Liu, Tao – 255 Needham, David – 215 Low, P. – 115 Ng, Boon Sing – 283 Lowe, Tao L. – 178 Nilsen, Thor – 175 Loxley, Andrew – 55 Nisisako, Takasi – 14 Lu, Wei – 153 Nooney, Robert – 254
275 Speaker/Presenter Index
Odukale, Anika A. – 284 Shao, Jun – 251 Oku, Naoto – 69 Sharma, Jaswinder – 294, 338 Oreskovic, Tammy – 316 Sheff, David – 216 Otake, Katsuto – 270 Shekunov, Boris – 135 Ozer, Ozgen – 147 Sievers, Bob – 137 Paciotti, Giulio – 3 Sitdikov, Ravil A. – 348 Panchapakesan, Balaji – 116 Smaïhi, Monique – 234 Parak, Wolfgang – 96 Smirnov, Alex I. – 322 Park, Chul Ho – 282 Sokolov, Igor – 57, 293 Park, Sang–Eun – 337 Somasundaran, Ponisseril – 20 Park, Sang Im – 346 Starostina, Natasha – 45 Parker, Andrew – 306 Stone, Vicki – 195 Pelton, Robert – 228 Straub, Julie – 8 Perez, J. Manuel – 252 Sullivan, Sean – 222 Petri, Dense F.S. – 169 Svenson, Sonke – 174 Petry, Harald – 93 Swai, Hulda Shaidi – 351 Pirollo, Kathleen F. – 113 Szebeni, Janos – 62 Pishko, Michael – 155 Szoka, Francis – 1 Ponce, Sara – 354 Talton, James – 143 Porter, Marc – 122 Tan, Wee Beng – 91, 292 Preece, Jon – 88 Taubert, Andreas – 51 Qi, Sheng – 157, 353 Thanh, Nguyen TK – 126 Raula, Janne – 303 Thomas, Dave – 125, 259 Ravenelle, Francois – 85 Thompson, David – 34, 224 Rehrig, Rick – 162 Tinke, A.P. – 44 Rogueda, Philippe – 104, 239, 304 Tofoli, Giovana R. – 315 Rojas, Edith C. – 352 Toler, Maria R. – 364 Rosenthal, Sandy – 102 Torchilin, Vladimir – 81 Rotello, Vince – 25 Trau, Dieter – 21 Rothrock, Ginger Denison – 280 Tsikhudo, Robert M. – 78 Russo, Paul S. – 120 Tung, Hsien–Hsin – 233 Saez–Martinez, Virginia – 281 Uchegbu, Ijeoma F. – 261 Saini, Divey – 349 Uddin, Mohammad S. – 121 Sampaio de Sousa, A.R. – 313 Urbanija, Jasna – 365 Sandler, Niklas – 242 Vail, Neal K. – 191 Santra, Swadeshmukul – 171 van den Bergh, Hubert – 65 Sawafta, Reyad – 30 Van der Wood, Tim B. – 48 Scott, Terrence – 130 van Nostrum, Cornelus F. – 71 Seino, Satoshi – 9 Van Thienen, Tinneke – 163
276 Preregistered Conferees van Veggel, Frank – 209 Varaksa, Natalia – 63 Vasseur, Sebastien – 249 Veiseh, Omid – 231 Velev, Orlin D. – 206 Velikov, Krassimir P. – 321 Verreck, Geert – 272 von Rechenberg, Brigitte – 118 Vu, Tania Q. – 210 Waligorski, Amy M. – 106 Wang, Yongxian – 76 Wang, Chang Chun – 145 Watts, Daniel J. – 61 Wegner, Karsten – 345 Wendorf, Janet R. – 350 Wessman, Per – 362 Williams, P. Stephen – 41 Wirix–Speetjens, Roel – 166 Woodle, Martin – 264 Worden, James – 230 Wu, X.Y. (Shirley) – 112 Wuang, Shy Chyi – 124, 320 Yamasaki, Yuichi – 54 Yang, Hong – 60 Yates, Matt – 268 Yoshizawa, Hidekazu – 150 Yu, Ji–Hun – 336 Yu, Kui – 260 Yun, Jimmy – 111, 243 Zhang, Guifang – 363 Zhang, Jin – 258 Zumstein, Lou – 227
277 Speaker/Presenter Index
Preregistered Conferees
Tomohiro Asai Dean Abadzic University of Shizuoka LUM Corporation 52-1, Yada, Suruga-ku 200 Boston Avenue, Suite 2900 422-8526 Shizuoka, Japan Medford, MA 02155 [email protected] [email protected] Sanford Asher Kazunari Akiyoshi University of Pittsburgh Institute of Biomaterials & Bioengineering 701 Chevron Science Center Tokyo Medical and Dental University 219 Parkman Avenue 2-3-10 Kanda-Surugadai, Chiyoda-ku Pittsburgh, PA 15260 Tokyo 101-0062, Japan [email protected] [email protected] Joseph Backer Buket Aksu SibTech, Inc. EGE University 705 North Mountain Road Okmeydani-Borucicegi-20 Newington, CT 06111 34382 Sisli, Istanbul, Turkey [email protected] [email protected] Ganesan Balasundaram Christine Allen Brown University University of Toronto Division of Engineering 19 Russell Street 182 Hope Street Toronto, Ontario, M5S 2S2, Canada Providence, RI 02912 [email protected] José Filipe da Silva Lapas Almeida University of Coimbra-ITQB-UNL Rebanta Bandyopadhyay Pinhal de Marrocos-Polo II Dr. Reddy’s Laboratories Limited 3030-290 Coimbra, Portugal Bollaram Road, Miyapur [email protected] Hyderabad, Andhra Pradesh 500 049, India Brian Amsden [email protected] Queen’s University Dupuis Hall Christophe Barbé 19 Division Street Australian Nuclear Science & Kingston, Ontario, K7L 3N6, Canada Technology Organisation [email protected] ANSTO-CeramiSphere, Bldg 3, PMB 1 Menai, NSW, 2234, Australia Marc Anton [email protected] INRA Rue de la Géraudière Stan Barnett BP 71627 Merck & Co., Inc. Nantes 44316, France 201 Hemlock Circle [email protected] North Wales, PA 19454 [email protected] Dian Arifin Monash University Christina Bauer Dept of Mechanical Engineering Sandia National Laboratories Clayton Campus, Wellington Road 7011 East Avenue Clayton, VIC 3800, Australia Bldg. 916, MS-9161 [email protected] Livermore, CA 94550 [email protected] Agu Artma Solis BioDyne Pikk 14 51013 Tartu, Estonia [email protected]
278 Preregistered Conferees
Hans Bäumler Ron Boch Charité–Universitätsmedizin Berlin QLT Inc. Schumannstrasse, 20/21 887 Great Northern Way D-10117 Berlin, Germany Vancouver, British Columbia V5T 4T5 [email protected] Canada [email protected] Rina Ben Shabat Bar-Ilan University Michael Bogan Chemistry Dept Lawrence Livermore National Lab Ramat-Gan, 52900, Israel 7000 East Avenue, L-211 [email protected] Livermore, CA 94550 [email protected] James Benson Polygenetics, Inc. Kristien Bonroy PO Box 33115 Interuniversitair Micro-Electronica Los Gatos, CA 95031 Centrum (IMEC) [email protected] Kapeldreef 75 Heverlee 3001, Belgium Cory Berkland [email protected] University of Kansas 2030 Becker Drive Ross Boyle Lawrence, KS 66047 University of Hull [email protected] Chemistry & Clinical Biosciences Institute Cottingham Road Jean-Francois Berret Kingston-upon-Hull, East Yorkshire, HU6 7RX CNRS-Université Paris 7 United Kingdom Matière et Système Complexes [email protected] 140 rue de Lourmel Paris, 75015, France Paul Braun [email protected] University of Illinois at Urbana-Champaign 204A MSEB, MC 246 Tridib Bhowmick 1304 West Green Street Indian Institute of Technology Bombay Urbana, IL 61801 Chemical Engineering [email protected] Powai, Maharashtra Mumbai-400076, India Roberta Brayner [email protected] Interfaces, Traitements, Organisation de Dynamique des Systèmes (ITODYS) John Bishop UMR-CNRS 7086 JB Particle Technology Université Paris 7-Denis Diderot 2251-4 East Main Street case 7090, 2 place Jussieu Rochester, NY 14609 75251 Paris Cedex 05, France [email protected] [email protected]
Jason Bjork Joseph Bringley 3M Company Eastman Kodak Company 3M Center, Bldg 270-2N-03 Research Laboratories St. Paul, MN 55144 Rochester, NY 14650-02002 [email protected] [email protected]
James Blanchard Beth A.-S. Brown Aradigm Corp. Cima Labs 3929 Pt. Eden Way 10000 Valley View Road Hayward, CA 94545 Eden Prairie, MN 55344 [email protected] [email protected]
279 Preregistered Conferees
David Brown Martin Caffrey Helsinki University of Technology University of Limerick Nanomaterials Group Plassey Drive Biologinkuja 7 Limerick, Ireland 02044 VTT Espoo, Finland [email protected] [email protected] Karin Caldwell Larry Brown Uppsala University Epic Therapeutics, Inc. Physical and Analytical Chemistry 220 Norwood Park South Box 577, BMC Norwood, MA 02062 Uppsala, 75123, Sweden [email protected] [email protected]
Michael Brown Paolo Caliceti CPS Instruments, Inc. University of Padua 3755 Green Ridge Road Via F. Marzolo 5 Furlong, PA 18925 Padova, 35131, Italy [email protected] [email protected]
Mark Bumiller Renato Camata Malvern Instruments University of Alabama at Birmingham 10 Southville Road 1530 3rd Avenue S, CH310 Southboro, MA 01772 Birmingham, AL 35294 [email protected] [email protected]
Heike Bunjes Raffaele Cammarano Jena University iCeutica Pharmaceutical Technology 33 Montreal Street Lessingstrasse 8 Fremantle, WA, 6160, Australia 07743 Jena, Germany [email protected] [email protected] John Caola Christopher Burcham FEI Company Eli Lilly and Company 5350 NE Dawson Creek Drive Chemical Process R&D Hillsboro, OR 97124 Indianapolis, IN 46285 [email protected] [email protected] Roberta Carbone Diane Burgess European Institute of Oncology University of Connecticut Dept Experimental Oncology School of Pharmacy via Ripamonti 435 69 North Eagleville Road 20141 Milan, Italy Storrs, CT 06269 [email protected] [email protected] Ana Maria Carmona-Ribeiro Peter Burkhard Instituto de Química-Universidade de São Paulo University of Connecticut Caixa Postal 26077 97 North Eagleville Road, Unit 3136 Av Prof Lineu Prestes 748 Storrs, CT 06269 São Paulo,CEP 05513-970, Brazil [email protected] [email protected]
Daniel Burnett Lizandra Belmonte Rodrigues de Castro Surface Measurement Systems Instituto de Química-Universidade de São Paulo 2222 South 12th Street, Suite D Av Prof Lineu Prestes 748, bloco03 Allentown, PA 18103 São Paulo,05508-900, Brazil [email protected] [email protected]
280 Preregistered Conferees
Maria Teresa Celis Gan-Moog Chow University of The Andes National University of Singapore Lab. FIRP and Lab. POLYCOL Materials Science & Engineering Facultad de Inginiería Kent Ridge, 119260, Singapore Mérida, 5101, Venezuela [email protected] [email protected] Amber Clausi Aravind Chakrapani University of Colorado at Boulder The Ohio State University Chemical & Biological Engineering 270 Bevis Hall 1111 Engineering Drive 1080 Carmack Road Boulder, CO 80309 Columbus, OH 43210 [email protected] [email protected] David Cliffel Chak K. Chan Vanderbilt University Hong Kong University of Science & Technology VU Station B 351822 Chemical Engineering Nashville, TN 37235 Clear Water Bay [email protected] Hong Kong SAR, China [email protected] Vicki Colvin Rice University Hui Chen Center for Biological & Environmental University of Central Florida Nanotechnology Nanoscience Technology Center 6100 Mail Street, MS-63 12424 Research Parkway, Suite 400 Houston, TX 77005 Orlando, FL 32826 [email protected] [email protected] Robert Cook Tongqian Chen MAP Pharmaceuticals Sirna Therapeutics, Inc. 2400 Bayshore Parkway 2950 Wilderness Place Mountain View, CA 94403 Boulder, CO 80301 [email protected] [email protected] John Corte Wei Chen Merck & Co., Inc. Nomadics, Inc. PO Box 2000 1024 South Innovation Way RY 814-S200 Stillwater, OK 74074 Rahway, NJ 07065 [email protected] [email protected]
Beata Chertok Kent Coulter University of Michigan Southwest Research Institute College of Pharmacy 6220 Culebra Road (78238-5166) 428 Church Street PO Drawer 28510 Ann Arbor, MI 48109 San Antonio, TX 78228-0510 [email protected] [email protected]
Chong-Su Cho Alexander Couzis Seoul National University The City College of New York School of Agricultural Biotechnology Chemical Engineering San 56-1, Sillim-dong, Kwanak-gu 140th Street at Convent Avenue Seoul, 151-742, South Korea New York, NY 10031 [email protected] [email protected]
Hoo-Kyun Choi Qiu Dai Chosun University University of Central Florida College of Pharmacy Nanoscience Technology Center 375 Seosuk-dong 12424 Research Parkway, Suite 400 Gwangju, 501-759, Korea Orlando, FL 32826 [email protected] [email protected]
281 Preregistered Conferees
Yujie Dai Thierry Delair Tianjin University of Science & Technology UMR CNRS-bioMérieux Hexi Qu, Dagu Nanlu, 1038 ENS-Lyon Tianjin, 300222, China 46, allée d’Italie [email protected] Lyon, 69007, France [email protected] Nily Dan Drexel University Rob DeLong Chemical & Biological Engineering Louisburg College 3141 Chestnut Street 501 North Main Street Philadelphia, PA 19104 Louisburg, NC 27549 [email protected] [email protected]
Jerry Darlington Ron Demers AMCOL International Merck Eprova 1500 West Shure Drive 5628 Ptarmigan Place Arlington Heights, IL 60004 North Vancouver, British Columbia, V7R 4S3 [email protected] Canada [email protected] Ramin Darvari Epic Therapeutics, Inc. Katarzyna Derwinska 220 Norwood Park South ARC Seibersdorf Research GmbH Norwood, MA 02062 Seibersdorf, 2444, Austria [email protected] [email protected]
Sridhar Dasaratha Tejas Desai 3M Company Vasogen Inc. 3M Center, 270-2N-03 2155 Dunwin Drive St. Paul, MN 55144-1000 Mississauga, Ontario, L5L 4M1, Canada [email protected] [email protected]
Allan David Joseph DeSimone ISTN, Inc. University of North Carolina at Chapel Hill 2101 Pennsylvania Avenue Dept of Chemistry York, PA 17404 CB #3290 Venable Hall [email protected] Chapel Hill, NC 27599 [email protected] Bruno De Geest Ghent University J. M. Devoisselle Dept of Pharmaceutics UMR CNRS/ENSCM/UM1 5618 Harelbekestraat 72 8, rue d el’école normale Ghent, 9000, Belgium Montpellier, 34296, France [email protected] [email protected]
Femke de Theije Helene Dihang Philips Research Formulaction High Tech Campus 4 (WAG02) 10 impasse Borde Basse Eindhoven, 5656 AE, The Netherlands L’Union, 31240, France [email protected] [email protected]
Martyn Deal Kenneth Dormer GlaxoSmithKline Hough Ear Institute New Frontiers Science Park University of Oklahoma Health Sciences Center 3rd Avenue 3400 NW 56th Street Harlow, Essex, CM19 5AW Oklahoma City, OK 73112 United Kingdom [email protected] [email protected]
282 Preregistered Conferees
Joe Domingue Yvon Durant Surface Measurement Systems University of New Hampshire 2222 South 12th Street G101 Parsons Hall Allentown, PA 18103 Durham, NH 03824 [email protected] [email protected]
Jakob Kisbye Dreyer Maciej Dybiec Novo Nordisk A/S Moffitt Cancer Center Device Chemistry and Biology 12902 Magnolia Drive Brennum Park MRC-Cancont Hillerød, DK-3400, Denmark Tampa, FL 33612 [email protected] [email protected]
David Driscoll Krzysztof Dziewiszek BIDMC/Harvard Medical School Antigenics Inc. Baker Bldg, Suite 605 3 Forbes Road 185 Pilgrim Road Lexington, MA 02421 Boston, MA 02215 [email protected] [email protected] Katrin Ebert Daryl Drummond GKSS Research Centre Geesthacht GmbH Hermes Biosciences, Inc. Institute of Chemistry 61 Airport Blvd, Suite D Max-Planck-Strasse 1 South San Francisco, CA 94080 Geesthacht, 21502, Germany [email protected] [email protected]
Ana Rita Duarte Katarina Edsman IBET Uppsala University Av. da Republica, EAN, qta do Merque Dept of Pharmacy Oeiras, 2781-901, Portugal Box 580 [email protected] Uppsala, SE-751 23, Sweden [email protected] Etienne Duguet ICMCB-CNRS/University of Bordeaux Marc Egen 87 ave du Dr. Albert Schweitzer Boehringer Ingelheim Pharma GmbH & Co. KG Pessac, 33608, France Binger Strasse 173 [email protected] 55216 Ingelheim, Germany [email protected] Dave Duncalf Iota NanoSolutions Ltd E. Sócrates Egito MerseyBIO Federal University of Rio Grande do Norta Crown Street Rua Praia de Areia Branca, 8948 Liverpool, L69 7ZB, United Kingdom Natal-RN, 59094-450, Brazil [email protected] [email protected]
Andrew Dunham Iris Enriquez Baxter Healthcare University of Florida Route 120 & Wilson Road 5035 SW 78th Way Round Lake, IL 60073 Gainesville, FL 32608 [email protected] [email protected]
David Duracher Frank Etzler Flamel Technologies Boehringer-Ingelheim Pharmaceuticals, Inc. Parc Club du Moulin à Vent 900 Ridgebury Road 33 avenue du Dr. Georges Levy Ridgefield, CT 06877 Venissieux, 69693, France [email protected] [email protected]
283 Preregistered Conferees
Larken Euliss University of North Carolina Alison Foster Dept of Chemistry, CB 3290 Iota NanoSolutions Chapel Hill, NC 27517 Merseybio [email protected] Crown Street Liverpool, L69 7ZB, United Kingdom Jacco Eversdijk [email protected] TNO Science and Industry De Rondom 1 Neil Russell Foster Eindhoven, 5512AP, The Netherlands University of New South Wales [email protected] School of Chemical Sciences & Engineering Sydney, NSW, 2052, Australia Alfred Fahr [email protected] FSU Jena Lessingstrasse 8 Matthias Frank Jena, Thüringen, 07743, Germany Lawrence Livermore National Laboratory [email protected] 7000 East Avenue Mail Stop L-211 Stan Farnsworth Livermore, CA 94551 Nanotechnologies, Inc. [email protected] 1908 Kramer Lane Austin, TX 78758 Nelly Fransén [email protected] Uppsala University Dept of Pharmacy, Box 580 Ingrid Fischbach Husargatan 3 BASF AG Uppsala, 751 23, Sweden GVC/F, J550 [email protected] Ludwigshafen, 67056, Germany [email protected] Arianna Friggeri Biomade Technology Foundation Sabine Fischer Nijenborgh 4 TNO Science and Industry Groningen, 9747 AG, The Netherlands De Rondom 1 [email protected] PO Bus 6235 Eindhoven, 5600 HE, The Netherlands Reynolds Frimpong [email protected] University of Kentucky Chemical & Materials Engineering Alexander Florence 177 F. Paul Anderson Tower University of London Lexington, KY 40506 The School of Pharmacy [email protected] 29/39 Brunswick Square London, WC1N 1AX, United Kingdom Wolfgang Fritzsche [email protected] Institute of Physical High Technology PO Box 100239 Geir Fonnum Jena, 07702, Germany Dynal Invitrogen Corporation [email protected] Svelleveien 29 Lillestrom, 2004, Norway Gary Fujii [email protected] Molecular Express, Inc. 13310 South Figueroa Street John Forsayeth Los Angeles, CA 90061 University of California at San Francisco [email protected] MCB226, 1855 Folsom Street San Francisco, CA 94103 Mari Fujimoto [email protected] Osaka Prefecture University Gakuen-cho 1-2, Naka-ku Sara Forssén Sakai, Osaka, 599-8570, Japan AstraZeneca R&D [email protected] Pepparredsleden 1 Mölndal, 43183, Sweden [email protected]
284 Preregistered Conferees
Masakazu Furuta Osaka Prefecture University Lynn Gold Graduate School of Sciences Sonus Pharmaceuticals. Inc. 1-2, Gakuen-cho 22026 20th Avenue SE Sakai, Osaka, 599-8570, Japan Bothell, WA 98021 [email protected] [email protected]
Anna Galperin Eugene Goldberg Bar-Ilan University University of Florida Chemistry Dept 317A MAE, Box 116400 Ramat-Gan, 52900, Israel Gainesville, FL 32611 [email protected] [email protected]
Carlos Gamazo Gershon Golomb University of Navarra The Hebrew University Irunlarrea, 1 School of Pharmacy, Faculty of Medicine Pamplona, 31008, Spain 12065 Ein Kerem [email protected] Jerusalem, 91120, Israel [email protected] Ying Gao Enzon Pharmaceuticals, Inc. Laurie Gower 20 Kingsbridge Road University of Florida Piscataway, NJ 08854 112 Rhines Hall [email protected] Gainesville, FL 32611 [email protected] David Gardner Respirics, Inc. Gilbert Grant 6008 Triangle Drive, Suite 101 New York University School of Medicine Raleigh, NC 27617 Dept of Anesthesiology [email protected] 550 First Avenue New York, NY 10016 Michael Giersig [email protected] caesar Research Center Ludwig-Erhard-Allee 2 Jennifer Green 53175 Bonn, Germany Nektar Therapeutics UK Ltd [email protected] Unit 69 Listerhills Science Park Campus Road Thierry Glauser Bradford, West Yorkshire, BD7 1HR Guidant England Lakeside Drive, S210 [email protected] Santa Clara, CA 95054 [email protected] Irwin Gruverman MFIC Corp. (Microfluidics Div.) William Glover 30 Ossipee Road University of Sydney Newton, MA 02464 Sydney, NSW, Australia [email protected] [email protected] Florence Guimberteau Dan Goia Flamel Technologies Clarkson University Parc Club du Moulin a Vent CAMP 344, Box 5814 33 Avenue du Dr. G. Levy Potsdam, NY 13699-5814 Venissieux, 69693, France [email protected] [email protected]
Beth Goins Yang Guo University of Texas Health Science Center Schering Plough Research Institute at San Antonio 2000 Galloping Hill Road Radiology Dept MSC 7800 Kenilworth, NJ 07033 7703 Floyd Curl Drive [email protected] San Antonio, TX 78229-3900 [email protected]
285 Preregistered Conferees
Ram Gupta J. Zach Hilt Auburn University University of Kentucky 161 Prather Lake Drive Chemical & Materials Engineering Auburn, AL 36830 177 F. Paul Anderson Tower [email protected] Lexington, KY 40506 [email protected] Heinrich Haas MediGene AG Tomoaki Hino Lochhamer Strasse 11 Kinjo Gakuin University Martinsried, 82152, Germany The College of Pharmacy [email protected] 2-1723 Ohmori Nagoya, Aichi, 463-8521, Japan Jessica Hain [email protected] Dresden University of Technology Mommsenstrasse 4 Rex Hjelm 01069 Dresden, Germany Los Alamos National Laboratory [email protected] MS H-805 Los Alamos, NM 87545 Gemunu Happawana [email protected] Southern Methodist University Mechanical Engineering Robert Hoerr 5990 Airline Road Nanocopoeia Inc. Dallas, TX 75275 1246 West University Avenue [email protected] St. Paul, MN 55104 [email protected] Meike Harms TU Braunschweig Heinrich Hofmann Institute for Pharmaceutical Technology Ecole Polytechnique Fédéral Lausanne (EPFL) Mendelssohnstrasse 1 Powder Technology Laboratory, Station 12 38106 Braunschweig, Germany Lausanne, CH-1015, Switzerland [email protected] [email protected]
Regine Hedderich Nick Holerca Forschungszentrum Karlsruhe/NanoMat Colgate-Palmolive Company PO Box 3640 909 River Road D-76021 Karlsruhe, Germany Piscataway, NJ 08855 [email protected] [email protected]
Kevin Heitfeld Sarah Hook University of Cincinnati University of Otago 3822 Kenker Place PO Box 913 Cincinnati, OH 45211 Dunedin, 9001, New Zealand [email protected] [email protected]
Marc Herold Peyton Hopson Fraunhofer IGB Vistakon, J&J Vision Care, Inc. Nobelstrasse 12 7500 Centurion Parkway 70569 Stuttgart, Germany Jacksonville, FL 32256 [email protected] [email protected]
Rupert Herrmann Tommy Horozov Roche Diagnostics GmbH University of Hull Nonnenwald 2 Cottingham Road 82372 Penzberg, Germany Hull, HU6 7RX, United Kingdom [email protected] [email protected]
286 Preregistered Conferees
Steve Howdle Sung Joo Hwang University of Nottingham Chungnam National University School of Chemistry College of Pharmacy University Park 220 Gung-dong, Yuseong-gu Nottingham, NG7 2RD, Great Britain Daejeon, 305 764, Korea [email protected] [email protected]
Jianhua Hu Taeghwan Hyeon Fudan University Seoul National University Macromolecular Science School of Chemical and Biological Engineering 220 Handan Road Seoul, 151-744, South Korea Shanghai, 200433, China [email protected] Hideki Ichikawa Kobe Gakuin University Min Hu Arise 518, Ikawadani-cho, Nishi-ku University of Washington Kobe, 651-2180, Japan Dept of Chemistry [email protected] Box 351700 Seattle, WA 98195-1700 Robert Ivkov [email protected] Triton BioSystems, Inc. 200 Turnpike Road Benjamin Hudson Chelmsford, MA 01824 University of Central Florida [email protected] Nanoscience Technology Center 12424 Research Parkway Satoshi Iwamoto Orlando, FL 32826 Gifu University [email protected] 1-1, Yanagido Gifu, 501-1193, Japan John Hughes [email protected] AMCOL International 1500 West Shure Drive Victor Jackson Arlington Heights, IL 60004 University of Florida [email protected] 205 Particle Science & Tech Bldg PO Box 116135 Qun Huo Gainesville, FL 32611 Nanoscience Technology Center of UCF [email protected] 12424 Research Parkway, Suite 422 Orlando, FL 32826 Irwin Jacobs [email protected] Particle and Coating Technologies, Inc. 5445 Highland Park Drive Robert Hurt St. Louis, MO 63110 Brown University [email protected] Pathology and Laboratory Medicine Providence, RI 02912 Gunilla Jacobson [email protected] Stanford University 333 Campus Drive, #121 Andreas Hütten Stanford, CA 94305-5080 Forschungszentrum Karlsruhe GmbH [email protected] Hermann-von-Helmholtz-Platz 1 PO Box 3640 Hilde Jans D-76021 Karlsruhe, Germany IMEC [email protected] Kapeldreef 75 Leuven 3001, Belgium Gereon Hüttmann [email protected] University of Lübeck Institute for Biomedical Optics Daniel Jarmer Peter-Monnik-Weg 4 Merck & Co., Inc. 23562 Lübeck, SH, Germany RY818-C314, PO Box 2000 [email protected] Rahway, NJ 07065 [email protected]
287 Preregistered Conferees
Xinqiao Jia Charalambos Kaittanis University of Delaware University of Central Florida Materials Science & Engineering Nanoscience Technology Center 201 DuPont Hall Orlando, FL 32826 Newark, DE 19716-3106 [email protected] [email protected] Sarah Kamau Christian Johansson University of Zurich Uppsala University Institute of Veterinary Biochemistry and Dept of Pharmacy Molecular Biology Box 580 Winterthurerstrasse 190 Uppsala, 751 23, Sweden CH-8057 Zurich, Switzerland [email protected] [email protected]
Keith Johnston Jan A. A. M. Kamps University of Texas University Medical Center Groningen Chemical Engineering, CO400 Medical Biology Austin, TX 78704 Hanzeplein 1 [email protected] 9713 GZ Groningen, The Netherlands [email protected] Kent Jørgensen LiPlasome Pharma A/S Shinichi Kaneda Technical University of Denmark Terumo Corporation R&D Center Bldg 207 1500 Inokuchi, Nakai-machi Lyngby, DK-2800, Denmark Ashigarakami-gun, Knagawa, 259-0151, Japan [email protected] [email protected]
Leanne Josefsen Joseph Kaparakis University of Hull University of Connecticut Cottingham Road 539 Main Street Kingston-upon-Hull, East Yorkshire, HU6 7RX Torrington, CT 06790 United Kingdom [email protected] [email protected] Esko Kauppinen Hemant Joshi Helsinki University of Technology Spectrum Pharmaceuticals Nanomaterials Group 157 Technology Drive Biologinkuja 7 Irvine, CA 92618 Espoo, 02044 VTT, Finland [email protected] [email protected]
Lucienne Juillerat Arnold Kell University Institute of Pathology National Research Council of Canada 25 rue du Bugnon 100 Sussex Drive, Room 1047 CH1011 Lausanne, Switzerland Ottawa, Ontario, K1A 0R6, Canada [email protected] [email protected]
Jae Eun Jung Lars Kilaas Hanyang University SINTEF Materials & Chemistry 17, Haengdang-dong Sem Sælandsv. 2A Seongdong-gu Trondheim, 7465, Norway Seoul, 133791, South Korea [email protected] [email protected] Bum Gil Kim Olev Kahre Kyungwon University Solis BioDyne OU San 65 Bokjeong-dong Pikk 14 Sungnam-city, Kyunggi-do, 461-701 Tartu, 51013, Estonia Korea [email protected] [email protected]
288 Preregistered Conferees
Eun-Jung Kim Rumiana Koynova Chosun University BMBCB Dept, Northwestern University College of Pharmacy 2205 Tech Drive 375 Seoseok-dong, Dong-gu Evanston, IL 60208 Gwangju, 501-759, Korea [email protected] [email protected] Andree Kraker Gloria Kim FEI Company Emory University and 5350 NE Dawson Creek Drive Georgia Institute of Technology Hillsboro, OR 97124 Dept of Biomedical Engineering [email protected] 101 Woodruff Circle, Suite 2001 Atlanta, GA 30322 Todd Krauss [email protected] University of Rochester 120 Trustee Road Jeong Geun Kim Rochester, NY 14627-0216 Korea Kumho Petrochemical Co Ltd. [email protected] 57-1, Hwaam-dong, Yuseong-gu Deajeon, 305-348, Korea Liisa Kuhn [email protected] University of Connecticut Health Center 263 Farmington Avenue, MC-1715 Nini Hofslokken Kjus Farmington, CT 06030 Dynal Bead Based Separations [email protected] Invitrogen Corporation Svelleveien 29 Mukesh Kumar Lillestrom, 2004, Norway Dabur Research Foundation [email protected] 22, Site IV, Sahibabad Ghaziabad, U.P., 201010, India Klemen Kocevar [email protected] Lek d.d. Verovskova 57 Priti Kumar 1526 Ljubljana, Slovenia CBR Institute of Biomedical Research, Harvard [email protected] 800 Huntington Avenue, Room 213 Boston, MA 02115 Csilla Kollar [email protected] Dow Corning Corp. 2200 West Salzburg Road Venisetty Raj Kumar Midland, MI 48686 Vaagdevi College of Pharmacy [email protected] H.No. 8-10-5, J.P.N. Road Warangal, Andhra Pradesh, 506002, India Uday Kompella [email protected] University of Nebraska Medical Center 985840 Nebraska Medical Center Janel Lanphere Omaha, NE 68198-5840 Boston Scientific Corporation [email protected] 100 Boston Scientific Way Marlborough, MA 01752 Kenji Kono [email protected] Osaka Prefecture University 1-1, Gakuen-cho Kenneth Lau Sakai, Osaka, 599-8531, Japan Massachusetts Institute of Technology [email protected] 77 Massachusetts Avenue, Room 66-259 Cambridge, MA 02139 Andreas Kordikowski [email protected] Nektar Therapeutics UK Ltd. Unit 69, Listerhills Science Park Afsaneh Lavasanifar Campus Road University of Alberta Bradford, West Yorkshire, BD7 1HR 4119 Faculty of Pharmacy & England Pharmaceutical Sciences [email protected] Edmonton, Alberta, T6G 2N8, Canada [email protected]
289 Preregistered Conferees
Roger Leblanc Stephane Legastelois University of Miami Indicia Biotechnology Dept of Chemistry 33 avenue de la California 1301 Memorial Drive Oullins, 69600, France Cox Science Center [email protected] Coral Gables, FL 33146 [email protected] Jeffrey Leon Eastman Kodak Company Hanchen P. Lee 1999 Lake Avenue Novartis Pharmaceuticals Corp. Rochester, NY 14650-2116 One Health Plaza, Bldg 401 [email protected] East Hanover, NJ 07936 [email protected] Dietmar Lerche Dr. Lerche KG Jaebeom Lee Rudower Chaussee 29 University of Michigan 12489, Berlin, Germany 1930 Lindsay Lane [email protected] Ann Arbor, MI 48104 [email protected] Ingo Leubner Crystallization Consulting Jonghwi Lee 35 Hillcrest Drive Chung-Ang University Penfield, NY 14526-2411 221 Heukseok-dong, Dongjak-gu [email protected] Seoul, 156-756, Korea [email protected] Hans Leuenberger Priscilla Lee Institute of Pharmaceutical Technology Oregon Health & Science University Pharmacenter, Klingelbergstrasse 50 20000 NW Walker Road 4056, Basel, Switzerland Beaverton, OR 97006 [email protected] [email protected] Eik Leupold Robert J. Lee Leibniz Institute of Molecular Pharmacology The Ohio State University Robert Rössle Strasse 10 College of Pharmacy 13125 Berlin, Germany 542 LM Parks Hall [email protected] 500 West 12th Avenue Columbus, OH 43210 Chun Li [email protected] U.T.M.D. Anderson Cancer Center Dept of Experimental Diagnostic Imaging Robert W. Lee 1515 Holcombe Blvd Novavax, Inc. Houston, TX 77030 508 Lapp Road [email protected] Malvern, PA 19355 [email protected] Pauline Pei Li The Hong Kong Polytechnic University Sang-Kyung Lee Applied Biology & Chemical Technology Hanyang University Hung Hom Dept of Bioengineering Kowloon, Hong Kong, 0000, China 17 Haendang-dong, Seongdong-gu [email protected] Seoul, 133-791, Korea [email protected] Yongcheng Li Vistakon, JNJ Vision Care Yongwoo Lee 7500 Centurion Parkway Foster-Miller, Inc. R&D Bldg, W1A 195 Bear Hill Jacksonville, FL 32256 Waltham, MA 02451 [email protected] [email protected]
290 Preregistered Conferees
Yuzhuo Li Philip Low Clarkson University Purdue University Dept of Chemistry Dept of Chemistry 8 Clarkson Avenue 560 Oval Drive Potsdam, NY 13699 West Lafayette, IN 47907 [email protected] [email protected]
Zhengmao Li Tao Lowe Bentley Pharmaceuticals, Inc. Penn State University 2 Holland Way, Bentley Park The Milton S. Hershey Medical Center Exeter, NH 03833 500 University Drive [email protected] Hershey, PA 17033 [email protected] Ben Lich FEI Company Andrew Loxley 5 Achtsteweg Noord Particle Sciences Eindhoven, 5600 KA, The Netherlands 3894 Courtney Street, Suite 180 [email protected] Bethlehem, PA 18017 [email protected] Victor S.-Y. Lin Iowa State University Wei Lu Dept of Chemistry Fudan University 1710 Gilman Hall School of Pharmacy Ames, IA 50011 138 Yi Xue Yuan Road, PO Box 130 [email protected] Shanghai, 200032, China [email protected] Michael Lipp Alkermes Inc. Yi Lu 88 Sidney Street University of Illinois at Urbana-Champaign Cambridge, MA 02139 Dept of Chemistry, MC-712, Box 8-6 [email protected] 600 South Mathews Avenue Urbana, IL 61801 Jubo Liu [email protected] University of Toronto 19 Russell Street Thomas Luby Toronto, Ontario, M5S 2S2, Canada MGI PHARMA Biologics [email protected] 44 Hartwell Avenue Lexington, MA 02421 Tao Liu [email protected] Chinese Academy of Sciences Institute of Chemistry Erik Lüddecke No. 2, Bei Yi Jie, Zhong Guan Cun BASF AG Beijing, He Bei, 100080, China Carl Bosch Strasse [email protected]; [email protected] 67056 Ludwigshafen, Rheinland-Pfalz, Germany [email protected] Xiong Liu University of Central Florida Ananda Lugade Nanoscience Technology Center Luminex Corporation 12424 Research Parkway, Suite 400 12212 Technology Blvd Orlando, FL 32826 Austin, TX 78727 [email protected] [email protected]
Yan Liu Dan Luo Arizona State University Cornell University Biodesign Institute, Box 875601 226 Riley Robb Tempe, AZ 85287 Ithaca, NY 14853 [email protected] [email protected]
291 Preregistered Conferees
Dongling Ma National Research Council of Canada Sofia de Medina Aires Martins 100 Sussex Drive, Room 1045 Instituto Superior Técnico Ottawa, Ontario, K1A 0R6, Canada Av. Rovisco Pais [email protected] Lisbon 1049-001, Portugal [email protected] Sarina Harris Ma Alza Corporation (J&J) Yoshitake Masuda 1010 Joaquin Road Nagoya University Mountain View, CA 94043 Furo-cho, Chikusa-ku [email protected] Nagoya, Aichi, 464-8603, Japan [email protected] Robert MacCuspie Hunter College Terry Matsunaga Chemistry Dept ImaRx Therapeutics, Inc. 695 Park Avenue 1635 East 18th Street New York, NY 10021 Tucson, AZ 85719 [email protected] [email protected]
Mizuo Maeda Hedi Mattoussi Riken US Naval Research Laboratory 2-1, Hirosawa Optical Sciences Division Wako, 351-0198, Japan 4555 Overlook Avenue, SW [email protected] Washington, DC 20375 [email protected] Volker Mailänder University of Ulm Shawn Mehrens Helmholtzstrasse 11 Pfizer Global R&D 89075 Ulm, Germany 2800 Plymouth Road [email protected] Ann Arbor, MI 48105 [email protected] Wing Cheung Mak National University of Singapore Anthony McGoron Division of Bioengineering Florida International University 9 Engineering Drive 1 10555 West Flagler Street Singapore, 117576, Singapore Miami, FL 33174 [email protected] [email protected]
Shlomo Margel James McGrath Bar-Ilan University University of Rochester Dept of Chemistry Biomedical Engineering Ramat-Gan, 52900, Israel 601 Elmwood Avenue [email protected] PO Box 639 Rochester, NY 14642 Peter Markland [email protected] Brookwood Pharmaceuticals 756 Tom Martin Drive Georges Meseguer Birmingham, AL 35211 Swissmedic [email protected] Hallerstrasse 7 3000 Berne, Switzerland Andrew Martin [email protected] University of Alberta 2-9 Mechanical Engineering Bldg Mary Miller Edmonton, Alberta, T6G 2G8, Canada MVA Scientific Consultants [email protected] 3300 Breckinridge Blvd, Suite 400 Duluth, GA 30096 Melanie Martin [email protected] Particles Conference 265 Clover Street Rochester, NY 14610-2246 [email protected]
292 Preregistered Conferees
David Mines Rainer Müller Inotech Biosystems International PharmaSol GmbH 15713 Crabbs Branch Way Blohmstrasse 66A Rockville, MD 20855-2607 12307 Berlin, Germany [email protected] [email protected]
Takashi Miyata Detlef Müller-Schulte Kansai University MagnaMedics GmbH Faculty of Engineering Martelenberger Weg 8 3-3-35, Yamate-cho D-52066 Aachen, Germany Suita, Osaka 564-8680, Japan [email protected] [email protected] Shawn Mulvaney Naomi Moldavski Naval Research Laboratory Teva Pharmaceuticals Industries NRL – Code 6177 R&D Initiative Dept 4555 Overlook Avenue SW 2 Hamerpe Street Washington, DC 20375 Jerusalem 91010, Israel [email protected] [email protected] Matti Murtomaa Mike Morgen University of Turku Bend Research Dept of Physics 64550 Research Road 20014 Turku, Finland Bend, OR 97701 [email protected] [email protected] Anna Musyanovych Nobuyuki Morimoto University of Ulm Tokyo Medical and Dental University Albert-Einstein-Allee 11 2-3-10 Kanda-surugadai 89081 Ulm, Germany Chiyoda-ku, Tokyo 101-0062, Japan [email protected] [email protected] Maryanne Naath Shannon Morrison Pfizer Inc. Virginia Commonwealth University 295 Bud Lane Chemistry Dept Northville, MI 48167 1001 West Main Street [email protected] Richmond, VA 23298 [email protected] Hiroyuki Nakamura Gakushuin University Brij Moudgil 1-5-1, Mejiro, Toshima-ku University of Florida Tokyo, 171-8588, Japan 205 PS&T Bldg [email protected] Gainesville, FL 32611 [email protected] Michihiro Nakamura The University of Tokushima Graduate School Gerhard Muhrer 3-18-15 Kuramoto-cho Novartis Pharmaceuticals Corp. Tokushima, 770-8503, Japan WSJ-145.11.51 [email protected] 4002 Basel, Switzerland [email protected] Yoshio Nakano NOF Corporation Angelika Müller 20-3, Ebisu 4-chome Charité Universitätsmedizin Berlin Shibuya-ku Institut für Transfusionsmedizin Tokyo, 150-6019, Japan Schumannstrasse 20/21 [email protected] 10117 Berlin, Germany [email protected] Indranil Nandi Sandoz 2400 Route 130 North Dayton, NJ 08810 [email protected]
293 Preregistered Conferees
Anika Odukale Balaji Narasimhan University of Florida Iowa State University 314 Materials Engineering Bldg Dept of Chemical and Biological Engineering PO Box 116400 2035 Sweeney Hall Gainesville, FL 32611-6400 Ames, IA 50011 [email protected] [email protected] Teppei Ogata Sudip Nath Ono Pharmaceutical Corporation Ltd University of Central Florida 3-1-1 Sakurai Shimamoto-cho Nanoscience Technology Center Mishima-gun 12424 Research Parkway Osaka, 618-8585, Japan Orlando, FL 32826 [email protected] [email protected] Patrick O’Hagan Laurence Navailles Particle Sizing Systems, Inc. Centre de Recherche Paul Pascal-CNRS 8203 Kristel Circle avenue A. Schweitzer Port Richey, FL 34668 Pessac 33600, France [email protected] [email protected] Yosuke Okamura David Needham Waseda University Duke University Graduate School of Science & Engineering CIEMAS, Room 3391 3-4-1 Ohkubo, Shinjuku-ku Durham, NC 27708 Tokyo, 169-8555, Japan [email protected] [email protected]
Boon Sing Ng Naoto Oku Stockholm University University of Shizuoka Dept of Inorganic Chemistry 52-1, Yada, Suruga-ku Arrhenius Laboratory Shizuoka, 422-8526, Japan Stockholm 10691, Sweden [email protected] [email protected] Monika Oli Thor Nilsen Banyan Biomarkers Genisphere 12085 Research Drive 2801 Sterling Drive Alachua, FL 32615 Hatfield, PA 19440 [email protected] [email protected] Anselmo Gomes de Oliveira Takasi Nisisako State of São Paulo University The University of Tokyo Pharmacy School, Campus Universitario Graduate School of Engineering Rodovia Araraquara-Jau km, 01 7-3-1 Hongo, Bunkyo-ku Araraquara, São Paulo, 14801-902, Brazil Tokyo, 113-8656, Japan [email protected] [email protected] Tammy Oreskovic Robert Nooney National Institute of Standards and Technology Dublin City University 325 Broadway, MS 853 Biomedical Diagnostics Institute Boulder, CO 80305 Collins Avenue [email protected] Glasnevin, Dublin 9, Ireland [email protected] Sergei Ostapenko Tim Norman University of South Florida UCB Celltech 4202 East Fowler Avenue 216 Bath Road Tampa, FL 33620 Slough, Berkshire, SL1 4EN, United Kingdom [email protected] [email protected]
294 Preregistered Conferees
Katsuto Otake Andrew Parker National Institute of Advanced Industrial Science Molecular Profiles Ltd Higashi 1-1-1, Tsukuba Central 5 Nottingham Business Park Tsukuba, Ibaraki 305-8565, Japan 8 Orchard Place [email protected] Nottingham, NG8 6PX, United Kingdom [email protected] John Otte Novartis Pharmaceutical Corp. Robert Paull One Health Plaza Lux Capital Bldg 401, Room B184D 140 East 45th Street, 30th floor East Hanover, NJ 07936 New York, NY 10017 [email protected] [email protected]
Jean Owens Robert Pelton Horiba Instruments, Inc. McMaster University 1720 Peachtree Street, NW 1280 Main Street West Peachtree 25th Bldg, Suite 601 Hamilton, Ontario, L8S 4L7, Canada Atlanta, GA 30309 [email protected] [email protected] J. Manuel Perez Giulio Paciotti University of Central Florida Cytimmune Sciences, Inc. 12424 Research Parkway, Suite 400 9640 Medical Center Drive Orlando, FL 32826 Rockville, MD 20850 [email protected] [email protected] Denise F. S. Petri Balaji Panchapakesan Universidade de São Paulo University of Delaware Av. Prof. Lineu Prestes 748 140 Evans Hall São Paulo, 05508-900, Brazil Newark, DE 19716 [email protected] [email protected] Harald Petry Wolfgang Parak Berlex Biosciences Ludwig Maximilians University-Munich 2600 Hilltop Drive Center for Nanoscience Richmond, CA 94804 Amalienstrasse 54 [email protected] 80799 Munich, Germany [email protected] Catherine Phelan Moffitt Cancer Center Chul Ho Park 12902 Magnolia Drive Chung-Ang University MRC-CANCONT 221 Heukseok-dong, Dongjak-gu Tampa, FL 33612 Seoul, 156-756, Korea [email protected] [email protected] Kathleen Pirollo Sang-Eun Park Georgetown University Medical Center Kyungwon University 3970 Reservoir Road NW, TRB/E420 San 65 Bockjung-dong, Soojung-gu Washington, DC 20057-1469 Seongnam-shi, Gyeonggi-do, 461-701, Korea [email protected] [email protected] Michael Pishko Sang-Im Park Penn State University Chungnam National University Dept of Chemical Engineering 220 Gung-dong, Yuseong-gu 204 Fenske Laboratory Daejeon, 305-764, Korea University Park, PA 16802-4400 [email protected] [email protected]
295 Preregistered Conferees
Michael Platscher Janne Raula Merck Eprova AG Helsinki University of Technology Im Laternenacker 5 Biologinkuja 7 8200 Schaffhausen, Switzerland Espoo, 02044, Finland [email protected] [email protected]
Sara Ponce Marquez François Ravenelle University of Basque Country Labopharm Inc. Pharmacy and Pharmaceutical Technology 480 Armand Frappier Paseo de la Universidad, 6 Laval, Quebec City, H7V 4B4, Canada Vitoria, 01006, Spain [email protected] [email protected]; [email protected] Rick Rehrig Marc Porter Harper International Iowa State University West Drullard Avenue 42 Spedding Hall Lancaster, NY 14086 Ames, IA 50011-3020 [email protected] [email protected] Andrew Riederer Jon Preece Battelle Memorial Institute University of Birmingham 505 King Avenue, 7-1-078 School of Chemistry, Haworth Bldg Columbus, OH 43201 Edgbaston, Birmingham, B15 2TT [email protected] United Kingdom [email protected] Preston Rodenbaugh CeloNova BioSciences, Inc. Sheng Qi 49 Spring Street University of East Anglia Newnan, GA 30263 School of Chemical Sciences & Pharmacy [email protected] Norwich, Norfolk, NR4 7TJ, United Kingdom [email protected] Philippe Rogueda AstraZeneca R&D Charnwood Haoyu Qin Bakewell Road Intel Corporation Loughborough, Leicestershire, LE11 5RH 2200 Mission College Blvd United Kingdom Santa Clara, CA 95054 [email protected] [email protected] Edith Carolina Rojas Huihe Qiu Tulane University Hong Kong University of Science & Technology 300 Lindy Boggs Dept Mechanical Engineering New Orleans, LA 70118 Clear Water Bay [email protected] Kowloon, Hong Kong, China [email protected] Sandra Rosenthal Vanderbilt University Steve Rannard Box 1822, Station B Iota NanoSolutions Nashville, TN 37235 Merseybio, Crown Street [email protected] Liverpool, Merseyside, L69 7ZB United Kingdom Vincent Rotello [email protected] University of Massachusetts Dept Chemistry Natalya Rapoport Amherst, MA 01003 University of Utah [email protected] 20 South 2030 East, Room 108 Salt Lake City, UT 84112 Ginger Denison Rothrock [email protected] Liquidia Technologies PO Box 110085 Research Triangle Park, NC 27709 [email protected]
296 Preregistered Conferees
Luke Roush Isabella Sasso Liquidia Technologies, Inc. Ryland Corporate Solutions PO Box 110085 PO Box 2825 Research Triangle Park, NC 27709 New York, NY 10017 [email protected] [email protected]
Paul Russo Reyad Sawafta Louisiana State University QuarTek Corporation 1222 Louray Drive 4180 Piedmont Parkway Baton Rouge, LA 70808 Greensboro, NC 27410 [email protected] [email protected]
Kevin Ryan Curtis Sayre Dow Corning Corporation Kimberly-Clark 2200 West Salzburg Road 1400 Holcomb Bridge Road Midland, MI 48686 Roswell, GA 30076 [email protected] [email protected]
Subir Sabui Jay Schild University of Central Florida Microtrac Inc. Nanoscience Technology Center 12501-A 62nd Street North 12424 Research Parkway Largo, FL 33773 Orlando, FL 23826 [email protected] [email protected] Terrence Scott Virginia Saez-Martinez Epic Therapeutics, Inc. University of the Basque Country 220 Norwood Park South Bo. Sarriena s/n Norwood, MA 02062 Leioa, Vizcaya, 48940, Spain [email protected] [email protected] Satoshi Seino Divey Saini Osaka University St. Louis University 2-1 Yamadaoka 3555 Vista Avenue, R405 Suita, Osaka, 565-0871, Japan St. Louis, MO 63111 [email protected] [email protected] Noboru Sekiya Jarno Salonen Ono Pharmaceutical Corporation Ltd University of Turku 3-1-1 Sakurai Shimamoto-cho Dept Physics Mishima-gun Vesilinnantie Osaka, 618 8585, Japan Turku, 20014, Finland [email protected] [email protected] Boitumelo Semete Niklas Sandler CSIR University of Otago Meiring Naude Road, Bldg 14C School of Pharmacy Lynwood 18 Fredrick Street Pretoria, Gauteng, 0002, South Africa Dunedin 9001, New Zealand [email protected] [email protected] Kyung Do Seo Swadeshmukul Santra Hanyang University University of Central Florida 17, Haengdang-dong, Seongdong-gu Nanoscience Technology Center Seoul, 133791, Korea 12424 Research Parkway, #400 [email protected] Orlando, FL 32826 [email protected] Jun Shao St. John’s University 8000 Utopia Parkway Jamaica, NY 11439 [email protected]
297 Preregistered Conferees
Kenneth Smith Jaswinder Sharma GlaxoSmithKline Pharmaceuticals Arizona State University NFSP South Site Biodesign Institute, Box 875601 Third Avenue Tempe, AZ 85287 Harlow, Essex, CM195AW [email protected] United Kingdom [email protected] David Sheff University of Iowa Herman Snyder 2-570-BSB Nektar Therapeutics 51 Newton Road 150 Industrial Road Iowa City, IA 52242 San Carlos, CA 94070 [email protected] [email protected]
Boris Shekunov Igor Sokolov Ferro Pfanstiehl Labs Clarkson University 7500 E. Pleasant Valley Road 6 Wellings Drive Independence, OH 44131 Potsdam, NY 13676 [email protected] [email protected]
Robert Sievers Ponisseril Somasundaran University of Colorado Columbia University Aktiv-Dry LLC Henry Krumb School 6060 Spine Road 500 West 120th Street Boulder, CO 80301 New York, NY 10027 [email protected] [email protected]
Ravil Sitdikov Ana Raquel Sousa University of New Mexico IBET Chemical & Nuclear Engineering av da Republica, EAN, qta do Marque MSC01-1120, 1 UNM Oeiras, 2781-901, Portugal Albuquerque, NM 87131-0001 [email protected] [email protected] Howard Stamato Gary Slater Bristol-Myers Squibb Eastman Kodak Company PO Box 191 Lake Avenue New Brunswick, NJ 08903-0191 Rochester, NY 14650 [email protected] [email protected] Natasha Starostina Lisa Slater Pacific Nanotechnology Inc. Eastman Kodak Company 17981 Sky Park Circle, Suite J Lake Avenue Irvine, CA 92614 Rochester, NY 14650 [email protected] [email protected] Stefan Steigerwald Monique Smaïhi Sympatec Inc. CNRS-IEM UMR 5635 9 Princess Road, Suite A 1919 route de Mende Lawrenceville, NJ 08648 34293 Montpellier Cedex 5, France [email protected] [email protected] Vicki Stone Alex Smirnov Napier University North Carolina State University School of Life Sciences Dept Chemistry Merchiston Campus Campus Box 8204 Edinburgh, EH10 5DT, United Kingdom Raleigh, NC 27695-8204 [email protected] [email protected]
298 Preregistered Conferees
Julie Straub Jim Talton Acusphere, Inc. Nanotherapeutics, Inc. 500 Arsenal Street 12085 Research Drive Watertown, MA 02472 Alachua, FL 32615 [email protected] [email protected]
Sean Sullivan Wee Beng Tan University of Florida National University of Singapore Pharmaceutics Dept Blk 723 Jurong West Avenue 5, #10-140 Box 100494, JHMHC 640723 Singapore 1600 SW Archer Road [email protected] Gainesville, FL 32610 [email protected] Andreas Taubert University of Basel Besik Surguladze Dept Chemistry Center of Clinical Medicine Klingelbergstrasse 80 6/10 Baratashvili Str. 4056 Basel, Switzerland Tbilisi 0105, Georgia [email protected] [email protected] Wyatt Tenhaeff Sonke Svenson Massachusetts Institute of Technology Dendritic NanoTechnologies Chemical Engineering 2625 Denison Drive 25 Ames Street, Room 66-419 Mount Pleasant, MI 48858 Cambridge, MA 02129 [email protected] [email protected]
Hulda Swai John Texter CSIR Eastern Michigan University Meiring Naude Road, Bldg 14C Coatings Research Institute Lynwood 430 West Forest Avenue Pretoria, Gauteng, 0002, South Africa Ypsilanti, MI 48197 [email protected] [email protected]
Janos Szebeni Nguyen Thi Kim Thanh Walter Reed Army Institute of Research University of Liverpool 1600 East Gude Drive Dept Chemistry Rockville, MD 20850 Crown Street [email protected] Liverpool, L69 7ZD, United Kingdom [email protected] Francis Szoka University of California at San Francisco Dave Thomas Depts Biopharmaceutical Sciences & Brightwell Technologies Inc. Pharmaceutical Chemistry 195 Stafford Road West San Francisco, CA 94143-0446 Ottawa, Ontario, K2H 9C1, Canada [email protected] [email protected]
Eriko Takeko David Thompson Sumitomo Chemical Co., Ltd. Purdue University 2-1, Takatsukasa 4-chome Dept Chemistry Takarzuka, Hyogo, 665-8555, Japan 560 Oval Drive [email protected] West Lafayette, IN 47907 [email protected] Yashushi Takemura Yokohama National University Arjen Tinke 79-5 Tokiwadai, Hodogaya Johnson & Johnson Pharmaceutical R&D Yokohama, 240-8501, Japan Turnhoutseweg 30 [email protected] Beerse, 2340, Belgium [email protected]
299 Preregistered Conferees
Giovana Radomille Tofoli Ijeoma Uchegbu State University o fCampinas University of Strathclyde Dept Biochemistry Pharmaceutical Science Cidade Universitária Zeferino Vaz s/n 27 Taylor Street Campinas, SP, 13083-970, CP6109, Brazil Glasgow, Scotland, G4 0NR, United Kingdom [email protected] [email protected]
Maria Toler Mohammad Shahab Uddin Pfizer Inc. National University of Singapore 7000 Portage Road Chemical & Biomolecular Engineering PORT-259-277 4 Engineering Drive 4 Kalamazoo, MI 49001 117576 Singapore [email protected] [email protected]
Vladimir Torchilin Jasna Urbanija Northeastern University University of Ljubljana 360 Huntington Avenue, Mugar R312 Faculty of Medicine Boston, MA 02115 Lipiceva 2 [email protected] 1000 Ljubljana, Slovenia [email protected] Dieter Trau National University of Singapore Martine Usdin 9 Engineering Drive 1 Médecins Sans Frontières 117576 Singapore 78, rue de Lausanne [email protected] PO Box 116 1211 Geneva 21, Switzerland Chi-Ming Tseng [email protected] 3M Company 236-3A-01 3M Center Neal Vail St. Paul, MN 55144 Southwest Research Institute [email protected] 6220 Culebra San Antonio, TX 78238 Robert Tshikhudo [email protected] University of Liverpool Nanoscale Science Centre Hubert van den Bergh Chemistry Dept, Oxford Street Swiss Federal Institute of Technology (EPFL) Liverpool, NW, L73BX, United Kingdom LPAS – Station 6 [email protected] 1015 Lausanne, Switzerland [email protected] Bill Turnell MediVas, LLC Cornelus van Nostrum 6275 Nancy Ridge Drive Utrecht University San Diego, CA 92121 Dept Pharmaceutics [email protected] Sorbonnelaan 16 3584 CA Utrecht, The Netherlands Hsien-Hsin Tung [email protected] Merck Company PO Box 2000 Tinneke Van Thienen Rahway, NJ 07065 University Ghent [email protected] Lab General Biochemistry & Physical Pharmacy Charlotta Turner Harelbekestraat 72 Uppsala University B-9000 Ghent, Belgium Physical & Analytical Chemistry [email protected] PO Box 599 Uppsala, SE-75124, Sweden Frank van Veggel [email protected] University of Victoria Dept Chemistry PO Box 3065 Victoria, BC, V8W 3V6, Canada [email protected]
300 Preregistered Conferees
Tania Vu Tim Vander Wood Oregon Health & Sciences University MVA Scientific Consultants Biomedical Engineering 3300 Breckinridge Blvd, #400 20000 NW Walker Road Duluth, GA 30096 Beaverton, OR 97006-8921 [email protected] [email protected]
Natalia Varaksa Amy Waligorski National Institute of Standards Chemimage and Technology 7301 Penn Avenue 325 Broadway Street Pittsburgh, PA 15208 Boulder, CO 80305 [email protected] [email protected] Jinping Wan Sebastein Vasseur Guidant Corp. University of Bordeaux 3200 Lakeside Drive Institute of Condensed Matter Santa Clara, CA 95054 Chemistry (ICMCB) [email protected] 87 ave Dr. Schweitzer 33608 Pessac Cedex, France Qiangbin Wang [email protected] Arizona State University Chemistry & Biochemistry Omid Veiseh Tempe, AZ 85287-1604 University of Washington [email protected] Materials Science & Engineering Box 352120 Changchun Wang Kirkland, WA 98195-2120 Fudan University [email protected] Micromolecular Science Shanghai 200433, China Orlin Velev [email protected] North Carolina State University Chemical & Biomolecular Engineering Weimin Wang EB 1.911 Partners Way Sirna Therapeutics, Inc. Raleigh, NC 27695-7905 2950 Wilderness Place [email protected] Boulder, CO 80301 [email protected] Krassimir Velikov Unilever R&D Vlaardingen Yongxian Wang Olivier van Noortlaan 120 Chinese Academy of Sciences Vlaardingen, 3133 AT, The Netherlands Shanghai Institute of Applied Physics [email protected] No. 2019 Jialuo Road Jiading, Shanghai 201800, China Geert Verreck [email protected] Johnson & Johnson Pharmaceutical R&D Kevin Ward Turnhoutseweg 30 Alkermes, Inc. 2340 Beerse, Belgium 88 Sidney Street [email protected] Cambridge, MA 02139 [email protected] Brigitte von Rechenberg University of Zurich Clive Washington Equine Hospital AstraZeneca Musculoskeletal Research Unit PU6, Lab Block Winterthurerstrasse 260 Silk Road Business Park 8057 Zurich, Switzerland Maclesfield, Cheshire, SK10 2NA [email protected] United Kingdom [email protected]
301 Preregistered Conferees
Drew Wassel Roel Wirix-Speetjens Hough Ear Institute IMEC 3400 NW 56 Street MCP NEXT-ART Oklahoma City, OK 73112 Kapeldreef 75 [email protected] 3001 Heverlee-Leuven, Belgium [email protected] Daniel Watts New Jersey Institute of Technology Martin Woodle Otto York Center for Environmental Intradigm Corp. Engineering & Science 12115 K Parklawn Drive 323 Martin Luther King Blvd Rockville, MD 20852 Newark, NJ 07102 [email protected] [email protected] James Worden Wolfgang Weber University of Central Florida LUM Corporation Nanoscience Technology Center 200 Boston Avenue, Suite 2900 12424 Research Parkway, Suite 400 Medford, MA 02155 Orlando, FL 32826 [email protected] [email protected]
Karsten Wegner Shirley X.Y. Wu Swiss Federal Institute of Technology University of Toronto ETH Zentrum ML F22 19 Russell Street Sonneggstrasse 3 Toronto, Ontario, M5S 2S2, Canada CH-8092 Zurich, Switzerland [email protected] [email protected] Yi Wu Janet Wendorf Pfizer Inc. Novartis Vaccines & Diagnostics 10646 Science Center Drive (CB4) 4560 Horton Street, M/S 4.3 San Diego, CA 92121 Emeryville, CA 94530 [email protected] [email protected] Zemin Wu Per Wessman Starpharma Uppsala University 75 Commercial Road Physical & Analytical Chemistry Melbourne, Victoria, 3004, Australia Box 579 [email protected] SE-75123 Uppsala, Sweden [email protected] Shy Chyi Wuang National University of Singapore Anthony West Chemical & Biomolecular Engineering Innovata plc 4 Engineering Drive 4, E5 04-07 One Mere Way 117576 Singapore Ruddington, Nottinghamshire NG11 6JS [email protected] United Kingdom [email protected] Tian-Xiang Xiang University of Kentucky Nicola Whitfield College of Pharmacy Innovata plc 725 Rose Street, Room 501 One Mere Way Lexington, KY 40536-0082 Ruddington, Nottinghamshire NG11 6JS [email protected] United Kingdom [email protected] Hiroshi Yamakose Sankyo Co., Ltd. P. Stephen Williams 1-2-58 Hiromachi, Shinagawa-ku Cleveland Clinic Tokyo 140-8710, Japan Biomedical Engineering [email protected] 9500 Euclid Avenue Cleveland, OH 44195 [email protected]
302 Preregistered Conferees
Yuichi Yamasaki The University of Tokyo Guifang Zhang 7-3-1 Hongo University of Minnesota Bunkyo, Tokyo 113-8656, Japan 308 Harvard St SE, 9-177 WDH [email protected] Minneapolis, MN 55455 [email protected] Hong Yang University of Rochester Huixiang Zhang Chemical Engineering Alcon Research Ltd 206 Gavett Hall 6201 South Freeway R1-26 Rochester, NY 14627 Fort Worth, TX 76134 [email protected] [email protected]
Xiqiang Yang Jin Zhang Eastman Kodak Company University of California at Santa Cruz Lake Avenue Dept Chemistry Rochester, NY 14650 156 High Street [email protected] Santa Cruz, CA 95064 [email protected] Matthew Yates University of Rochester Shi-Wei Zhang Chemical Engineering Saoirse Corporation Rochester, NY 14627 300 Technology Square [email protected] Cambridge, MA 02139 [email protected] Ji Youn Yoo Chung-ang University Shiying Zheng 221 Heukseok-dong, Dongjak-gu Eastman Kodak Company Seoul 156-756, Korea 1999 Lake Avenue [email protected] Rochester, NY 14530 [email protected] Hidekazu Yoshizawa Okayama University Tao Zheng 3-1-1 Tsushima-Higashi Avon Products, Inc. Okayama 700-8530, Japan One Avon Place [email protected] Suffern, NY 10901 [email protected] Ji-Hun Yu Korea Institute of Machinery & Materials Tatyana Zhukov 66 Sangnam Moffitt Cancer Center Changwon, Gyeongnam 641-010, Korea 12902 Magnolia Drive [email protected] MRC-Cancont Tampa, FL 33612 Kui Yu [email protected] National Research Council of Canada 100 Sussex Drive Jianhua Zou Ottawa, Ontario, K1A 0R6, Canada University of Central Florida [email protected] Nanoscience Technology Center 12424 Research Parkway, 4th fl. Jimmy Yun Orlando, FL 32826 NanoMaterials Technology Pte Ltd [email protected] 28 Ayer Rajah Crescent #03-03 139959 Singapore Louis Zumstein [email protected] Introgen Therapeutics 2250 Holcombe Blvd Aleksandra Zajak Houston, TX 77030 Moffitt Cancer Center [email protected] 12902 Magnolia Drive MRC-Cancont Tampa, FL 33612 [email protected]
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Receipt
This receipt acknowledges payment of ______from ______by
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On behalf of: Particles Conference 265 Clover Street Rochester, NY 14610-2246, USA EIN No. 16-1565388 Tel: 1-585-288-5913 Fax: 1-585-482-7795
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