US009 155703B2

(12) United States Patent (10) Patent No.: US 9,155,703 B2 Peppas (45) Date of Patent: Oct. 13, 2015

(54) METHOD AND PROCESS FOR THE 2009,0081265 A1 3/2009 Peppas et al. PRODUCTION OF MULT-COATED 2009, O232857 A1 9/2009 Peppas et al. 2009, 0232858 A1 9/2009 Peppas et al. RECOGNITIVE AND RELEASING SYSTEMS FOREIGN PATENT DOCUMENTS (75) Inventor: Nicholas A. Peppas, Austin, TX (US) EP 1664168 B1 3, 2008 (73) Assignee: Board of Regents, the University of WO O2/O71994 A1 9, 2002 WO 2005/020849 A2 3, 2005 Texas System, Austin, TX (US) WO 2006116734 A2 11/2006 WO WO2006.116734 * 11, 2006 (*) Notice: Subject to any disclaimer, the term of this WO 2008056746 A1 5, 2008 patent is extended or adjusted under 35 WO 2008112826 A1 9, 2008 U.S.C. 154(b) by 1224 days. OTHER PUBLICATIONS Appl. No.: 12/047.309 Badiger, M. V., “Porogens in the preparation of microporous (21) hydrogels based on poly(ethylene oxides).” Biomaterials (1993), 14:1059-1063. (22) Filed: Mar 12, 2008 Kabiri, K., et al., Novel approach to highly porous Superabsorbent hydrogels: Synergistic effect of porogens on porosity and Swelling (65) Prior Publication Data rate, Polymer International (2003), 52: 1158-1164. Omidian, H., et al., “Advances in Superporous hydrogels. J Con US 2008/0226684 A1 Sep. 18, 2008 trolled Release (2005), 102:3-12. Berman, H.M., et al., The Protein Data Bank. Nucleic Acids Res., 2000. 28: p. 235-242. Related U.S. Application Data Bolisay, L.D.V., et al., Separation of baculoviruses using configura tionally biomimetic imprinted polymer hydrogels. Mat. Res. Soc. (60) Provisional application No. 60/894.451, filed on Mar. Symp. Proc., 2004. 787: p. G3.1/1-G3.1/5. 12, 2007. Burt, S., Essential Oils: their antibacterial properties and potential applications in food—a review. International Journal of Food Microbiology 94 (2004) pp. 223-253. (51) Int. C. Byrne, M.E., et al., Molecular imprinting within hydrogels. Adv. A6 IK 9/6 (2006.01) Drug Deliver. Rev., 2002. 54(1): p. 149-161. A6 IK9/00 (2006.01) Byrne, M.E., et al., Biomimetic Networks for Selective Recognition A6 IK9/24 (2006.01) of Biomolecules. 2002: Materials Research Society. Cederfur, J., et al., Synthesis and Screening of a Configurationally A 6LX 9/70 (2006.01) biomimetic imprinted Polymer Library Targeted for Penicillin G. J. A6 IK3I/37 (2006.01) Comb. Chem., 2003. 5: p. 67-72. A61 K9/50 (2006.01) Chang, C.P. et al., Preparation of alginate complex capsules contain (52) U.S. C. ing eucalyptus essential oil and its controlled release. Colloids and Surfaces B: Biointerfaces 32 (2003) pp. 257-262. CPC ...... A61 K9/1676 (2013.01); A61 K9/0004 Duclairoir, C., et al., Evaluation of gliadins nanoparticles as drug (2013.01); A61 K9/209 (2013.01); A61 K delivery systems: a study of three different drugs. International Jour 9/7084 (2013.01); A61K3I/I37 (2013.01); nal of Pharmaceutics, 253 (2003) p. 133-144. A61 K9/5073 (2013.01); A61 K9/5084 Hilt, J.Z., et al., Ultrasensitive Biomems Sensors Based on (2013.01) Microcantilevers Patterned with Environmentally Responsive Field of Classification Search Hydrogels. Biomedical Microdevices, 2003. 5(3): p. 177-184. (58) Liang, C., et al., Molecular imprinting polymer coated BAW bio CPC ... A61K 31/137; A61 K9/5063; A61K 47/44; mimic sensor for direct determination of epinephrine. Anal. Chim. A61K 8/92: A61 K9/5073; C08F 20/56; Acta, 2000. 415: p. 135-141. CO8F 22/105 Mosbach, K., Toward the next generation of molecular imprinting USPC ...... 424/400, 464, 486; 526/72 with emphasis on the formation, by direct molding, of compounds See application file for complete search history. with biological activity(biomimetics). Anal. Chim. Acta, 2001. 435: p. 3-8. (56) References Cited Oral, E. et al., Responsive and recognitive hydrogels using star poly mers. J. Biomed. Mater. Res. A 2004. 68: p. 439-447. U.S. PATENT DOCUMENTS Parmpi, P. et al., Biomimetic glucose recognition using configura tionally biomimetic imprinted hydrogels. Biomaterials, 2004. 25: p. 3,538,214 A 11, 1970 Polli et al. 1969-1973. 4,228, 149 A 10, 1980 Brewer et al. 6,303,148 B1 10/2001 Hennink et al. (Continued) 6,897,271 B1 5, 2005 Domschke et al. 7,176,247 B1 2/2007 Walker, Jr. Primary Examiner — Shobha Kantamneni 7,459,316 B2 12/2008 Faid et al. (74) Attorney, Agent, or Firm — Parker Highlander PLLC 8,062,769 B2 11/2011 Kai et al. 2002fOO71877 A1 6, 2002 Mueller (57) ABSTRACT 2003/0059471 A1* 3/2003 Compton et al...... 424/489 2004/009 1541 A1 5/2004 Unger The present invention includes compositions, methods, sys 2005, OOO8686 A1 1/2005 Mannino et al. tems and kits for the controlled delivery of an active agent 2005/0249721 A1 11/2005 Houston et al. within a polymeric network upon the binding of a molecule 2005/0276781 A1* 12/2005 Ross et al...... 424/78.08 that decreases the structural integrity of the polymeric net 2007/0027213 A1 2/2007 Oberegger et al. work at one or more micro- or nanovacuoles. 2007/0134721 A1* 6/2007 Laitenberger et al...... 435.7.1 2008.0171067 A1 7/2008 Govindan et al. 5 Claims, 8 Drawing Sheets US 9,155,703 B2 Page 2

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Andersson, et al. “Mimics of the Binding Sites of Opiod Receptors Pande,et al. "Folding Thermodynamics and kinetics of imprinted Obtained by Molecular Imprinting of Enkephalin and Morphine” renaturable heteropolymers' J. Chem. Phys., 1994. 101(9): p.8246 Proc. Natl. Acad. Sci. USA, 1995.92: p. 4788-4792. 8257. Ansell, et al. “Molecularly imprinted polymers for bioanalysis: chro Pande, et al. “How to Create Polymers with Protein-Like Capabili matography, binding assays and biomimetic sensors' Current Opin ties: A Theoretical Suggestion” Physica D, 1997. 107: p. 316-321. ion in Biotechnology 1996:7:89-94. US 9,155,703 B2 Page 3

(56) References Cited Hartmans, et al. “Use of talent (carvone) as a sproutgrowth regulator of seed potatoes and the effect on stim and tuber number Potato OTHER PUBLICATIONS Research, 41 (1998) pp. 190-191. Haupt, et al. "Imprinted polymer-based enantioselective acoustic Appella, et al. “Peptide Foldamers: Robust Helix Formation in a New sensor using a quartz crystal microbalance' Anal. Commun., 1999. Family of Beta-Amino Acid Oligomers' J. Am. Chem. Soc., 1996. 36. 118: p. 13071-13072. Bartsch, et al. “Molecular and Ionic Recognition with Imprinted Herr, et al., J. Am. Chem. Soc. 89.4808-09 (1967). Polymers: A Brief Overview” Book Abstract—8 pp. Hilt, et al. “Configurational biomimesis in drug delivery: molecular Bashir, et al. “Micromechanical cantilever as an ultrasensitive pH imprinting of biologically significant molecules' Advanced Drug microsensor' Applied Physics Letters, vol. 81, No. 16, Oct. 14, 2002. Delivery Reviews 56 (2004) 1599-1620. Bergmann, et al. “Protein-Imprinted Polymeric Microparticles for Hilt, et al. “Novel Biomimetic Polymer Networks: Development and Tissue Engineering Applications' 2003 Society for Biomaterials Application as Selective Recognition Elements for Biomolecules at 29th Annual Meeting Transactions, Trans Soc. Biomat 2003:29:457. the Micro-Nanoscale” in AIChE Nanoscale Science and Engineer Breinl, et al. “Chemical Investigation of the Precipitate from Hemo ing Topical Conference Proceedings. 2003. San Francisco, CA. globin and Anti-hemoglobin Serum and Remarks on the Nature of Hilt, J. Zachary “Nanotechnology and biomimetic methods in thera Antibodies” Z. Physiol. Chem., 1930. 192: p. 45. peutics: molecular scale control with some help from nature' Bures, et al. "Surface modifications and molecular imprinting of Advanced Drug Delivery Reviews 56 (2004) 1533-1536. polymers in medical and pharmaceutical applications' Journal of Jerne, N.K. “The Generative Grammar of the Immune System, in Controlled Release 72 (2001) 25-33. Nobel Lectures” Physiology or Medicine 1981-1990, J. Lindsten, Burnet, F.M. “A Modification of Jerne's Theory of Antibody Produc Editor. 1993, World Scientific Publishing Co.: Singapore. p. 211-225. tion Using the Concept of Clonal Selection’ Aust. J. Sci., 1957. 20: p. Jerne, N.K. “The Natural Selection Theory of Antibody Formation” 67. Proc. Natl. Acad. Sci. USA, 1955. 41: p. 849. Burton, D.R. "Monoclonal Antibodies from Combinatorial Librar Kabiri, et al. “Novel approach to highly porous Superabsorbent ies' Accounts Chem. Res., 1993. 26: p. 405-411. hydrogels: Synergistic effect of porogens on porosity and Swelling Byrne, M.E. 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Chem., 1994.66: p. 2636-2639. oils” Journal of Agricultural and Food Chemistry, 45 (1997) pp. Merrifield, R.B. “Solid Phase Peptide Synthesis. II. The Synthesis of 2690-2694. Bradykinin' J. Am. Chem. Soc., 1964.86; p. 304. Gellman, S.H. "Foldamers: A Manifesto' Accounts Chem. Res., Merrifield, R.B. "Solid-Phase Peptide Synthesis, III. An Improved 1998. 31: p. 173-180. Synthesis of Bradykinin’ Biochem., 1964. 3: p. 1385-1390. Harris, et al. “Refined Structure of an Intact IgG2a Monoclonal Mosbach, et al. “The Emerging Technique of Molecular Imprinting Antibody” Biochem., 1997. 36: p. 1581-1597. and its Future Impact on Biotechnology” Biotechnol., 1996. 14: p. Hartmans, et al. “Report of the Meeting of the Section Physiology of 163-170. the EAPR, Jun. 20-24, 1994” Potato Research, 37 (1994) pp. 435 463. * cited by examiner U.S. Patent Oct. 13, 2015 Sheet 1 of 8 US 9,155,703 B2

O C ) G- OnOneS O N(\t template C ) C ) FIG. 1

SO

FIG. 2

Specific recognition site

template

FIG. 3 U.S. Patent Oct. 13, 2015 Sheet 2 of 8 US 9,155,703 B2

Penetrant Uptake vs. Time 0.6

O 10 20 30 40 50 60 70 80 Time (min) FIG. 4

Penetrant Uptake vs. Time (1/2)

O 1 2 3 4 5 6 7 8 9 Time (1/2) (min) (1/2) FIG. 5 U.S. Patent Oct. 13, 2015 Sheet 3 of 8 US 9,155,703 B2

Recognitive Ratio vs. Time

O D-glucose 100 mg/dLIDI water sample 1

O 10 20 30 40 50 60 70 80 90 Time (min) FIG. 6

- s's U.S. Patent Oct. 13, 2015 Sheet 4 of 8 US 9,155,703 B2

FIG. 9

FIG. 15 FIG. 16

U.S. Patent Oct. 13, 2015 Sheet 6 of 8 US 9,155,703 B2

1st burst 3h

IntervalterWa 12h

O O 1st burst 6h -o- O interval 12h C G)

s E.nterval 3h 1st burst 9h O -o- OO interval 12h OO

SCO 1st burst 12h O O interval 12h

FIG. 18 U.S. Patent Oct. 13, 2015 Sheet 7 of 8 US 9,155,703 B2

N LO cro LO can L2 v- Lo o cro CN v O U.S. Patent Oct. 13, 2015 Sheet 8 of 8 US 9,155,703 B2

BA released at 275 nm 120

100 F Na

C 80 Dy 60 C-SA a s 40 as 20 R k -0-BA released. O O 50 100 150 200 250 300 Time (min) FIG. 20

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RTV Rubber US 9,155,703 B2 1. 2 METHOD AND PROCESS FOR THE ognitive control that do not leak and that are amenable to PRODUCTION OF MULTI-COATED controlled release upon exposure to the analyte. RECOGNITIVE AND RELEASING SYSTEMS The present invention includes a molecule-imprinted poly meric network that includes a polymer or gel comprising one CROSS-REFERENCE TO RELATED or more micro- or nanovacuoles, or micro- or nanopores APPLICATIONS wherein the nanovacuoles or nanopores recognize a specific molecule while Subsequent contact with the molecule creates This application claims priority to U.S. Provisional Appli internal stresses that rupture the polymeric network at the cation Ser. No. 60/894,451, filed Mar. 12, 2007, the entire micro- or nanovacuoles or micro- or nanopores. Upon expo 10 sure of the polymeric network to the analayte, but not the contents of which are incorporated herein by reference. Solvent alone, the polymeric network can rupture due to, e.g., osmosis upon recognition and binding of the molecule lead TECHNICAL FIELD OF THE INVENTION ing to rupture due to Swelling; change of the solubility of the polymeric network leading to polymer dissolution; local tem The present invention relates in general to the field of the 15 perature changes leading to expansion of the polymeric net controlled release of agents, and more particularly, to novel work and combinations thereof. The composition may be compositions and methods for making controlled release con loaded with one or more active agents to form an active figurational biomimetic imprinting networks. agent-loaded, molecule-imprinted network. In one aspect of the invention, the polymer swells between 2-20, 4-18, 5-15, STATEMENT OF FEDERALLY FUNDED 8-12 and 10 percent of the dried polymer upon exposure to the RESEARCH Solvent alone. In one aspect, the polymeric network Swell between 5-15% of the dried polymeric network in the pres None. ence of the solvent alone. Examples of active agents for use with the present inven BACKGROUND OF THE INVENTION 25 tion include pharmaceutical and medical applications, food components, detergents, bleaches, fabric softeners, fra Without limiting the scope of the invention, its background grances, cosmetic products, air fresheners, room deodorant is described in connection with the recognition and controlled devices, perfumed Substrates, perfumed plastics and pet col release of active agents from polymers. lars. Other actives include food and cosmetic applications This invention relates generally to the field of microencap 30 that use hydrocolloids as imprinting carriers for polymers of Sulation, and, more particularly, to the development of micro high molecular weight, wherein the hydrocolloids are particles and nanoparticles with multiple recognitive layers. extracted from plants, seaweeds or animal collagen, produced In general, microencapsulation is achieved with fluidized bed by microbial synthesis, and comprise polysaccharides, pro processes. The fluidized bed coating process is a widely used teins and combinations thereof. The molecule-imprinted net technique for large particle size. Small particles coated in a 35 work may be a carbohydrate polymer of glycosidic type fluidized bed tend to agglomerate and adhere to the wall of the mono-Sugar repeative units, galactomannans, pectins, algi bed due to the electrostatic charge and capillary forces result nates, carrageenans and Xanthan gum that are linear or ing from the remaining solvent. branched, neutral oranionic and combinations thereof. Other Despite improvements in the range of particle sizes, other examples include household products selected from laundry limitations exist in optimizing microcapsules to be applied in 40 care; paper products; specialty cleaners (chlorinated cleaners, a variety of applications; in particular, the loading and deliv scouring pads, effervescent toilet bowl cleaner powders); air ery of therapeutic agents. It is therefore a need to optimize the fresheners and combinations thereof. Further examples particle coating process for the formation and drug delivery include personal care products selected from hair care (sham vehicles. It is a further need to have optimization to the poos, hair mousses, styling agents); skin care (body lotions, Wurster process to form multiple-layers for increased func 45 Vitamin, aloe Vera); bath products (moisture-triggered release tionality, especially as drug delivery systems with multiple products); body powders; toilet Soap (milled or poured, ionic recognitive layers. strength-triggered release) and combinations thereof. Also, cosmetics and treatment products selected from lipstick; eye SUMMARY OF THE INVENTION liner, foundation; base; blush; mascara; eye shadow; lip liner; 50 facial powder, consealer; facial cream; make-up remover; The needs of the invention set forth above as well as further mascara remover, make-up; skin treatments and may even and other needs and advantages of the present invention are include one or more fragrances or carriers therefore that achieved by the embodiments of the invention described include cologne; perfume; sampling; antiperspirant; deodor herein below. The present invention is based on the recogni ant; anti-dandruff shampoos, athlete foot products and com tion that, to date, imprinted or recognitive polymers are found 55 binations thereof. Other actives include a surfactant, a bleach in two forms, Solid and gel-like. Solid recognitive polymers ing agent, a corrosion inhibitor, a Sudsing modifier, a are used in a variety of applications, namely, chromatogra fluorescent whitening agent, one or more enzymes, an anti phy, filters and molecular separation. The other category of redeposition agent, a color, a fragrance, one or more additives imprinted polymers are gelatinous polymers that are loaded and combinations thereof. with a payload during the polymerization phase and are dried 60 The present invention also include the release of active until use. Upon exposing these gelatinous imprinted poly agent upon exposure to the cognate molecule or moiety that mers to a solvent, e.g., water and/or water with the analyte or ultimately causes release upon a loss of structural integrity recognitive molecules, the gelatinous polymers Swell. Unfor caused by, e.g., changes in solubility, pressure, a pH shift, a tunately, for the delivery of most payloads swelling of the change in temperature, a temperature increase, enzymatic gelatinous polymer in the presence of solvent alone leads to 65 breakdown, diffusion and combinations thereof. The skilled leakage of the payload. What are needed are compositions, artisan will also be able to be formed integrally or as a coating methods and systems for the delivery of payloads under rec for controlled release or one or more layers of the active, the US 9,155,703 B2 3 4 recognitive polymer or both. Furthermore, the polymeric rec present disclosure provides a system for forming multilayer ognitive network may beformed into one or more layers, each mimetic structures in which the molecularly imprinted poly of which recognizes a different molecule, a different active or mer layer with controllable properties for bursting and drug inert agent or both. The polymeric recognitive network is release. formed into a sphere, film, planar, semi-spherical, cylinder, According to another embodiment, the present disclosure rod, hemispheres, conical, hemi-cylinders and combination provides methods for controlling bursting and release of thereof and may also be at least partially porous. The poly molecularly imprinted polymer layers by manipulation of meric recognitive network also may be formed into one or various factors including, but not limited to, disintegration more layers, each of which recognizes a different molecule, kinetics, tensile strength of the polymers via crosslinking and and where a different active or inert agent may be contained 10 intrapolymer complexes, molecular weight, pressure differ between polymeric layers. ences by use of osmotic agents, and thickness of the layer. The present invention also includes an active agent-loaded, molecule-imprinted polymeric network that includes two or BRIEF DESCRIPTION OF THE DRAWINGS more active agent loaded, aggregated polymeric or gel nano particles or microparticles comprising micro- or nanovacu 15 For a more complete understanding of the features and oles or micro- or nanopores previously imprinted with a mol advantages of the present invention, reference is now made to ecule, wherein one or more pre-determined molecules bind the detailed description of the invention along with the specifically to the micro- or nanovacuoles or micro- or nan accompanying figures and in which: opores and contact with the molecule creates internal stresses FIG. 1 shows the monomeric mixture used to create the that rupture the network at the micro- or nanovacuoles or present invention. micro- or nanopores thereby releasing one or more active FIG. 2 shows the polymerized mixture, before the washing agents loaded into the active agent-loaded, molecule-im step. printed polymeric network. FIG. 3 shows the polymer after washing in which the The present invention also includes a method of making a template has been removed and the specific recognition site recognitive release system by selecting one or more mol 25 remains within the polymer. ecules for recognition; forming micro- or nano-vacuoles in a FIG. 4 is a graph that shows penetrant uptake of a recog polymeric recognitive network about the one or more mol nitive polymer continuous films overtime. The data points are ecules; removing the molecule from the micro- or nanovacu amount of penetrant uptake in Milli-Q deionized water (DI oles or micro- or nanopores; and coating one or more active water) and 100 mg/dL D-glucose and DI water. The films agents with a polymeric recognitive network, wherein the one 30 were cut into disks 8 mm in diameter and 0.12 mm thick; the or more active agents release upon contact by the polymeric initial weights were approximately 6 mg each. Measurements recognitive network with its cognate molecule. were taken every 10 minutes. The present invention also includes a method of making a FIG. 5 is a graph that shows penetrant uptake of a recog polymeric recognitive network that includes selecting one or nitive polymer continuous film versus the square root of time. more targets for recognition; forming micro- or nano-vacu 35 The data points are amount of penetrant uptake in Milli-Q oles in the polymeric recognitive network about the one or deionized water (DI water) and 100 mg/dL D-glucose and DI more targets; embedding within the polymeric recognitive water. The films were cut into disks 8 mm in diameter and network one or more active agents for release upon dissocia 0.12 mm thick; the weights were approximately 6 mg each. tion of the polymeric recognitive network; and removing the Measurements were taken every 10 minutes. targets from the micro- or nano-vacuoles, wherein Subse 40 FIG. 6 is a graph that shows the recognitive ratio of con quent binding of the target to the micro- or nano-vacuoles figurational biomimetic imprinted polymers (CBIP) in the causes disruption of the polymeric recognitive network and presence of 100 mg per dL deionized water compared to release of the one or more active agents. continuous films in the presence of deionized water. The films Yet another embodiment of the present invention includes were cut into squares approximately 9 mm by 9 mm and 0.22 a kit for making a polymeric recognitive network that 45 mm thick. The penetrant uptake amount was obtained from includes one or more targets for recognition by the polymeric measurements of mass every 10 minutes once the squares recognitive network; monomers for forming a polymeric rec were placed in the solutions of either deionized Water or ognitive network comprising micro- or nano-vacuoles about glucose solution with 100 mg D-glucose per dL deionized the one or more targets; a polymeric catalyst for forming the water. The ratio is the amount of penetrant uptake in the polymeric recognitive network about the targets; and instruc 50 glucose solution to amount of penetrant uptake in deionized tions for polymerizing the polymeric recognitive network and Water. removing the targets from the micro- or nano-vacuoles and FIG. 7 shows a Glucose CBIP (5/31 mixture) after exposure loading of the polymeric recognitive network with one or to 100 mg/dL glucose-water (60 seconds pass between each more active agents. frame). (50x objective). According to one embodiment, the present disclosure pro 55 FIG. 8 shows a nebula of disintegrated particles from a vides a system for forming multilayer mimetic structures that porous film exposed to 100 mg/dL glucose-water (5x objec comprise a core and coating materials, including molecularly tive). imprinted polymers, materials for use as a spacer, and mate FIG.9 shows the Stress lines seen on a polymer film30sec rials for use as a binder. A Wuester coating system for forming after addition of glucose. (5x objective). multilayer mimetic structures for using a Grow Max Spouted 60 FIG. 10 shows a section of a glucose-CBIP film immersed bed assisted with a draft tube and a bottom spray, also know in a glucose solution and seen breaking at a first time point as the Wurster method. According to another embodiment, (10x objective). the present disclosure provides a system for forming multi FIG. 11 shows a section of a glucose-CBIP film immersed layer mimetic structures wherein the core comprises spheri in a glucose solution and seen breaking at a second time point cal or non-spherical compositions, molecularly imprinted 65 (10x objective). polymers, hydroxyl propyl cellulose (HPC) as a spacer and FIG. 12 shows a Trypan Blue dyed water-front moving in mannitol as a binder. According to another embodiment, the toward a CBIP particle. US 9,155,703 B2 5 6 FIG. 13 shows a CBIP particle in glucose-water viewed Drugs having an action on the central nervous system, for through two polarized lenses. Soft image means no signifi example sedatives, hypnotics, antianxiety agents, analgesics cant stresses in the Surface of the particle (the image is mostly and anesthetics, such as, chloral, buprenorphine, naloxone, dark due to the lack of reflection). haloperidol, fluphenazine, pentobarbital, phenobarbital, FIG. 14 shows a CBIP polymer particle viewed through 5 secobarbital, amobarbital, cydobarbital, codeine, lidocaine, two polarized lenses. The reflective areas indicate changes in tetracaine, dyclonine, dibucaine, cocaine, procaine, mepiv Surface morphology due to mechanical stresses. acaine, bupivacaine, etidocaine, prilocalne, benzocaine, fen FIG. 15 shows a glucose CBIP exposed to fluorescent tanyl, nicotine, and the like. Local anesthetics such as, ben glucose; the intake of the glucose is indicated by the brightly Zocaine, procaine, dibucaine, lidocaine, and the like. 10 Antihistaminics or antiallergic agents such as, diphenhy lit areas (10x objective). dramine, dimenhydrinate, perphenazine, triprolidine, pyril FIG. 16 shows a glucose CBIP exposed to fluorescent amine, chlorcyclizine, promethazine, carbinoxamine, tripe glucose, breakage is apparent here (10x objective). lennamine, brompheniramine, hydroxyZine, cyclizine, FIG. 17 shows the optional combinations for the various meclizine, clorprenaline, terfenadine, chlorpheniramine, and embodiments of the present invention in which the optional 15 the like. Anti-allergenics such as, antazoline, methapyrilene, analyte, recognition and transduction events and payloads. chlorpheniramine, pyrilamine, pheniramine, and the like. FIG. 18 is a diagram that shows mixing multilayered Decongestants such as, phenylephrine, ephedrine, naphazo mimetic structures with different release profiles that allows line, tetrahydrozoline, and the like. the system to be tailored to fit any release profiles. Mixing Antipyretics such as, aspirin, Salicylamide, non-steroidal four different microcapsules allowed the system to rupture anti-inflammatory agents, and the like. Antimigrane agents every 3 h for 4 days instead of a system that ruptured every 12 Such as, dihydroergotamine, pizotyline, and the like. h for 4 days or a system that ruptured every 3 h for one day. Acetonide anti-inflammatory agents, such as hydrocortisone, FIG. 19 shows glucose/water uptake of a CBIP. cortisone, dexamethasone, fluocinolone, triamcinolone, FIG. 20 is a graph that shows BSA release of a multilami medrysone, prednisolone, flurandrenolide, prednisone, halci nate system. 25 nonide, methylprednisolone, fludrocortisone, corticosterone, FIG. 21 shows a diagram indicating the several layers of a paramethasone, betamethasone, ibuprophen, naproxen, feno multilaminate system. profen, fenbufen, flurbiprofen, indoprofen, ketoprofen, Suprofen, indomethacin, piroxicam, aspirin, salicylic acid, DETAILED DESCRIPTION OF THE INVENTION diflunisal, methyl salicylate, phenylbutaZone, Sulindac, mefe 30 namic acid, meclofenamate Sodium, tolmetin, and the like. While the making and using of various embodiments of the Muscle relaxants such as, tolperisone, baclofen, dantrolene present invention are discussed in detail below, it should be sodium, cyclobenzaprine. appreciated that the present invention provides many appli Steroids Such as, androgenic steriods, such as, testosterone, cable inventive concepts that can be embodied in a wide methyltestosterone, fluoxymesterone, estrogens such as, con variety of specific contexts. The specific embodiments dis 35 jugated estrogens, esterified estrogens, estropipate, 17-B cussed herein are merely illustrative of specific ways to make estradiol, 17-B estradiol Valerate, equilin, mestranol, estrone, and use the invention and do not delimit the scope of the estriol, 173 ethinyl estradiol, diethylstilbestrol, progesta invention. tional agents, such as, progesterone, 19-norprogesterone, To facilitate the understanding of this invention, a number norethindrone, norethindrone acetate, melengestrol, chlor of terms are defined below. Terms defined herein have mean 40 madinone, ethisterone, medroxyprogesterone acetate, ings as commonly understood by a person of ordinary skill in hydroxyprogesterone caproate, ethynodiol diacetate, nor the areas relevant to the present invention. Terms such as “a”, ethynodrel, 17-C. hydroxyprogesterone, dydrogesterone, “an and “the are not intended to refer to only a singular dimethisterone, ethinylestrenol, norgestrel, demegestone, entity, but include the general class of which a specific promegestone, megestrol acetate, and the like. example may be used for illustration. The terminology herein 45 Respiratory agents such as, theophilline and B2-adrenergic is used to describe specific embodiments of the invention, but agonists, such as, albuterol, terbutaline, metaproterenol, rito their usage does not delimit the invention, except as outlined drine, carbuterol, fenoterol, quinterenol, rimiterol, Solmefa in the claims. mol, Soterenol, tetroquinol, and the like. Sympathomimetics As used herein, the term “active agent(s). “active ingredi Such as, dopamine, norepinephrine, phenylpropanolamine, ent(s).” “pharmaceutical ingredient(s), and “bioactive 50 phenylephrine, pseudoephedrine, amphetamine, propyl agent(s) are defined as drugs and/or pharmaceutically active hexedrine, arecoline, and the like. ingredients. The present invention may be used to encapsu Antimicrobial agents including antibacterial agents, anti late, attach, bind or otherwise be used to affect the storage, fungal agents, antimycotic agents and antiviral agents; tetra stability, longevity and/or release of any of the following cyclines such as, oxytetracycline, penicillins, such as, ampi drugs as the pharmaceutically active agent in a composition. 55 cillin, cephalosporins such as, cefalotin, aminoglycosides, One or more of the following bioactive agents may be com Such as, kanamycin, macrollides such as, erythromycin, bined with one or more carriers and the present invention chloramphenicol, iodides, nitrofrantoin, nystatin, amphoteri (which may itself be the carrier): cin, fradiomycin, Sulfonamides, purrolnitrin, clotrimazole, Analgesic anti-inflammatory agents such as, acetami miconazole chloramphenicol, Sulfacetamide, SulfamethaZ nophen, aspirin, salicylic acid, methyl salicylate, choline sali 60 ine, Sulfadiazine, Sulfamerazine, Sulfamethizole and Sulfisox cylate, glycol salicylate, 1-menthol, camphor, mefenamic azole; antivirals, including idoxuridine, clarithromycin; and acid, fluiphenamic acid, indomethacin, diclofenac, alclofenac, other anti-infectives including nitrofuraZone, and the like. ibuprofen, ketoprofen, naproxen, pranoprofen, fenoprofen, Antihypertensive agents such as, clonidine, C.-methyldopa, Sulindac, fenbufen, clidanac, flurbiprofen, indoprofen, pro reserpine, Syrosingopine, rescinnamine, cinnarizine, hydra tizidic acid, fentiazac, tolmetin, tiaprofenic acid, bendazac, 65 Zine, prazosin, and the like. Antihypertensive diuretics Such bufexamac, piroxicam, phenylbutaZone, oxyphenbutaZone, as, chlorothiazide, hydrochlorothiazide, bendoflumethazide, clofeZone, pentazocine, mepirizole, and the like. trichloromethiazide, furosemide, tripamide, methylclothiaz US 9,155,703 B2 7 8 ide, penfluzide, hydrothiazide, Spironolactone, metolaZone, doxepin, desipramine, nortriptyline, protriptyline, amoxap and the like. Cardiotonics such as, digitalis, ubidecarenone, ine, maprotiline, traZodone, and the like. dopamine, and the like. Coronary vasodilators such as, Anti-diabetics such as, insulin, and anticancer drugs such organic nitrates such as, nitroglycerine, isosorbitol dinitrate, as, tamoxifen, methotrexate, and the like. erythritol tetranitrate, and pentaerythritol tetranitrate, dipy Anorectic drugs such as, dextroamphetamine, metham ridamole, dilaZep, trapidil, trimetazidine, and the like. Vaso phetamine, phenylpropanolamine, fenfluramine, diethylpro constrictors such as, dihydroergotamine, dihydroergotoxine, pion, mazindol, phentermine, and the like. and the like. B-blockers or antiarrhythmic agents such as, Anti-malarials such as, the 4-aminoquinolines, alphamino timolol pindolol, propranolol, and the like. Humoral agents quinolines, chloroquine, pyrimethamine, and the like. 10 Anti-ulcerative agents such as, misoprostol, omeprazole, Such as, the prostaglandins, natural and synthetic, for enprostil, and the like. Antiulcer agents such as, allantoin, example PGE1, PGE2C, and PGF2C, and the PGE1 analog aldioxa, alcloxa, N-methylscopolamine methylsuflate, and misoprostol. Antispasmodics such as, atropine, methanthe the like. Antidiabetics such as insulin, and the like. line, papaverine, cinnamedrine, methScopolamine, and the Anti-cancer agent such as, cis-platin, actinomycin D, like. 15 doxorubicin, Vincristine, vinblastine, etoposide, amsacrine, Calcium antagonists and other circulatory organ agents, mitoxantrone, tenipaside, taxol. colchicine, cyclosporin A, Such as, aptopril, diltiazem, nifedipine, nicardipine, Vera phenothiazines or thioxantheres. pamil, bencyclane, ifenprodil tartarate, molsidomine, cloni For use with vaccines, one or more antigens, such as, dine, prazosin, and the like. Anti-convulsants such as, natural, heat-killer, inactivated, synthetic, peptides and even nitrazepam, meprobamate, phenyloin, and the like. Agents T cell epitopes (e.g., GADE, DAGE, MAGE, etc.) and the for dizziness such as, isoprenaline, betahistine, Scopolamine, like. and the like. Tranquilizers such as, reserprine, chlorprom Example therapeutic or active agents also include water azine, and antianxiety benzodiazepines such as, alprazolam, soluble or poorly soluble drug of molecular weigh from 40 to chlordiazepoxide, cloraZeptate, halazepam, oxazepam, 1,100 including the following: Hydrocodone, Lexapro, Vico prazepam, clonazepam, flurazepam, triazolam, lorazepam, 25 din, Effexor, Paxil, Wellbutrin, Bextra, Neurontin, Lipitor, diazepam, and the like. Percocet, Oxycodone, Valium, Naproxen, Tramadol, Antipsychotics such as, phenothiazines including thiopro Ambien, Oxycontin, Celebrex, Prednisone, Celexa, Ultracet, pazate, chlorpromazine, triflupromazine, mesoridazine, pip Protonix, Soma, Atenolol, Lisinopril, Lortab, Darvocet, erracetazine, thioridazine, acetophenazine, fluphenazine, Cipro, Levacquin, Ativan, Nexium, Cyclobenzaprine, Ultram, perphenazine, trifluoperazine, and other major tranquilizers 30 Alprazolam, Trazodone, Norvasc, Biaxin, Codeine, Clon Such as, chlorprathixene, thiothixene, haloperidol, bromperi azepam, Toprol, Zithromax, Diovan, Skelaxin, Klonopin, dol, loxapine, and molindone, as well as, those agents used at Lorazepam, Depakote, Diazepam, Albuterol, Topamax, Sero lower doses in the treatment of nausea, vomiting, and the like. quel, Amoxicillin, Ritalin, Methadone, Augmentin, Zetia, Drugs for Parkinson's disease, spasticity, and acute muscle Cephalexin, Prevacid, Flexeril, Synthroid, Promethazine, spasms such as levodopa, carbidopa, amantadine, apomor 35 Phentermine, Metformin, Doxycycline, Aspirin, Remeron, phine, bromocriptine, selegiline (deprenyl), trihexyphenidyl Metoprolol, Amitriptyline, Advair, Ibuprofen, Hydrochlo hydrochloride, benztropine mesylate, procyclidine hydro rothiazide, Crestor, Acetaminophen, Concerta, Clonidine, chloride, baclofen, diazepam, dantrolene, and the like. Res Norco, Elavil, Abilify, Risperdal, Mobic, Ranitidine, Lasix, piratory agents such as, codeine, ephedrine, isoproterenol, Fluoxetine, Coumadin, Diclofenac, Hydroxyzine, Phener dextromethorphan, orciprenaline, ipratropium bromide, 40 gan, Lamictal, Verapamil, Guaifenesin, Aciphex. Furo cromglycic acid, and the like. Non-steroidal hormones or semide, Entex, Metronidazole, Carisoprodol, Propoxyphene, antihormones such as, corticotropin, oxytocin, vasopressin, Digoxin, Zanaflex, Clindamycin, Trileptal, Buspar, Keflex, salivary hormone, thyroid hormone, adrenal hormone, kal Bactrim, Dilantin, Flomax, Benicar, Baclofen, Endocet, likrein, insulin, oxendolone, and the like. Avelox, Lotrel, Inderal, Provigil, Zantac, Fentanyl, Premarin, Vitamins such as, vitamins A, B, C, D, E and Kand deriva 45 Penicillin, Claritin, Reglan, Enalapril, Tricor, Methotrexate, tives thereof, calciferols, mecobalamin, and the like for der Pravachol, Amiodarone, Zelnorm, Erythromycin, Tegretol, matologically use. Enzymes Such as, lysozyme, urokinaze, Omeprazole, and Meclizine. and the like. Herb medicines or crude extracts such as, Aloe The drugs mentioned above may be used in combination as Vera, and the like. required. Moreover, the above drugs may be used either in the Antitumor agents such as, 5-fluorouracil and derivatives 50 free form or, if capable of forming salts, in the form of a salt thereof, krestin, picibanil, ancitabine, cytarabine, and the with a suitable acid or base. If the drugs have a carboxyl like. Anti-estrogen oranti-hormone agents such as, tamoxifen group, their esters may be employed. or human chorionic gonadotropin, and the like. Miotics Such Examples of monomers that may be used to achieve the low as pilocarpine, and the like. or minimal Swelling include: Poly(allylamine), Acrylic acid, Cholinergic agonists such as, choline, acetylcholine, 55 Acrylamide, (Diethylamino)ethyl methacrylate, (Ethylami methacholine, carbachol, bethanechol, pilocarpine, muscar no)methacrylate, Methacrylic acid, methylmethacrylate, Tri ine, arecoline, and the like. Antimuscarinic or muscarinic azacyclononane-copper(II) complex, 2-(methacryloyXloxy) cholinergic blocking agents such as, atropine, Scopolamine, ethyl phosphate, methacrylamide, 2-(trifluoromethyl)acrylic homatropine, methScopolamine, homatropine methylbro acid, 3-aminophenylboronic acid, poly(allylamine), o-ph mide, methantheline, cyclopentolate, tropicamide, propan 60 thalic dialdehyde, oleyl phenyl hydrogen phosphate, 4-vi theline, anisotropine, dicyclomine, eucatropine, and the like. nylpyridine, vinylimidazole, 2-acryloilamido-2,2'-methop Mydriatics such as, atropine, cyclopentolate, homatropine, ropane Sulfonic acid, Silica, organic silanes, N-(4-vinyl)- Scopolamine, tropicamide, eucatropine, hydroxyamphet benzyl iminodiacetic acid, Ni(II)-nitrilotriacetic acid, amine, and the like. Psychic energizers such as 3-(2-amino N-acryloyl-alanine. These monomers may be combined with propy)indole, 3-(2-aminobutyl)indole, and the like. 65 one or more crosslinkers to achieve the desired low or mini Antidepressant drugs such as, isocarboxazid, phenelZine, mal Swelling upon exposure to solvent alone that include: tranylcypromine, imipramine, amitriptyline, trimipramine, ethylene glycol dimethacrylate, pentaerythritol triacrylate, US 9,155,703 B2 10 pentaerythritol tetraacrylate, trimethylolpropane tri The bioactive may be orally administered, for example, methacrylate, vinyl triethoxysilane, vinyl trimethoxysilane, with an inert diluent or an assimilable edible carrier. The toluene 2,4-diisocyanate, epichlorohydrin, triglycerolate dia therapeutic compound and other ingredients may also be crylate, polystyrene Surface, Propylene glycol dimethacry enclosed in a hard or soft shell gelatin capsule, compressed late, poly(ethylene glycol) in dimethacrylate, methacrylate into tablets, or incorporated directly into the subject’s diet. derived silica, acrylonitrile, N,N'-dimethylacrylamide, poly For oral therapeutic administration, the therapeutic com (ethylene glycol) diacrylate. Examples of solvents that may pound may be incorporated with excipients and used in the be used to achieve low or minimal Swelling include Acetoni form of ingestible tablets, buccal tablets, troches, capsules, trile, Acetic acid, ethanol, aqueous buffer, toluene, water, elixirs, Suspensions, syrups, wafers, and the like. The percent chloroform, hexane, methanol, tetrahydrofuran. 10 age of the therapeutic compound in the compositions and The acid mentioned above may be an organic acid, for example, methanesulfonic acid, lactic acid, tartaric acid, preparations may, of course, be varied as will be known to the fumaric acid, maleic acid, acetic acid, oran inorganic acid, for skilled artisan. The amount of the therapeutic compound in example, hydrochloric acid, hydrobromic acid, phosphoric Such therapeutically useful compositions is such that a Suit acid or Sulfuric acid. The base may be an organic base, for 15 able dosage will be obtained. example, ammonia, triethylamine, or an inorganic base, for It is especially advantageous to formulate parenteral com example, sodium hydroxide or potassium hydroxide. The positions in dosage unit form for ease of administration and esters mentioned above may be alkyl esters, aryl esters, uniformity of dosage. Dosage unit form as used herein refers aralkyl esters, and the like. Also with Sugar to release as a to physically discrete units Suited as unitary dosages for the bitterness masking agent (Sugar as the agent). Subjects to be treated; each unit containing a predetermined The bioactive may also be administered, e.g., parenterally, quantity of therapeutic compound calculated to produce the intraperitoneally, intraspinally, intravenously, intramuscu desired therapeutic effect in association with the required larly, intravaginally, Subcutaneously, or intracerebrally. Dis pharmaceutical carrier. The specification for the dosage unit persions may be prepared in glycerol, liquid polyethylene forms of the invention are dictated by and directly dependent glycols, and mixtures thereof and in oils. Under ordinary 25 on (a) the unique characteristics of the therapeutic compound conditions of storage and use, these preparations may contain and the particular therapeutic effect to be achieved, and (b) a preservative to prevent the growth of microorganisms. the limitations inherent in the art of compounding Such a Pharmaceutical compositions suitable for injectable use therapeutic compound for the treatment of a selected condi include sterile aqueous solutions (where water soluble) or tion in a subject. dispersions and sterile powders for the extemporaneous 30 Aqueous compositions of the present invention comprise preparation of sterile injectable solutions or dispersion. In all cases, the composition must be sterile and must be fluid to the an effective amount of the nanoparticle, nanofibril or extent that easy Syringability exists. It must be stable under nanoshell or chemical composition of the present invention the conditions of manufacture and storage and must be pre dissolved and/or dispersed in a pharmaceutically acceptable served against the contaminating action of microorganisms 35 carrier and/or aqueous medium. The biological material Such as bacteria and fungi. The carrier may be a solvent or should be extensively dialyzed to remove undesired small dispersion medium containing, for example, water, ethanol, molecular weight molecules and/or lyophilized for more poly-ol (for example, glycerol, propylene glycol, and liquid ready formulation into a desired vehicle, where appropriate. polyethylene glycol, and the like), Suitable mixtures thereof, The active compounds may generally be formulated for and vegetable oils. 40 parenteral administration, e.g., formulated for injection via The proper fluidity may be maintained, for example, by the the intravenous, intramuscular, Subcutaneous, intralesional, use of a coating Such as lecithin, by the maintenance of the and/or even intraperitoneal routes. The preparation of an required particle size in the case of dispersion and by the use aqueous composition that contain an effective amount of the of Surfactants. Prevention of the action of microorganisms nanoshell composition as an active component and/or ingre may be achieved by various antibacterial and antifungal 45 dient will be known to those of skill in the art in light of the agents, for example, parabens, chlorobutanol, phenol, ascor present disclosure. Typically, such compositions may be pre bic acid, thimerosal, and the like. In many cases, it will be pared as injectables, either as liquid Solutions and/or Suspen preferable to include isotonic agents, for example, Sugars, sions; Solid forms suitable for using to prepare solutions Sodium chloride, or polyalcohols such as mannitol and Sor and/or Suspensions upon the addition of a liquid prior to bitol, in the composition. Prolonged absorption of the inject 50 injection may also be prepared; and/or the preparations may able compositions may be brought about by including in the also be emulsified. composition an agent that delays absorption, for example, The present inventor has developed methods and tech aluminum monostearate or gelatin. niques to form synthetic biomimetic networks, gels or poly Sterile injectable solutions may be prepared by incorporat mers that will bind and respond to specific molecules, ana ing the therapeutic compound in the required amount in an 55 lytes or moieties. These biomimetic polymer networks, gels appropriate solvent with one or a combination of ingredients or polymers are advantageous because they can be tailored to enumerated above, as required, followed by filtered steriliza bind any molecule with controlled selectivity and affinity. tion. Generally, dispersions are prepared by incorporating the There are some significant characteristics to consider in the therapeutic compound into a sterile carrier that contains a design of a biomimetic polymer networks via a configura basic dispersion medium and the required other ingredients 60 tional biomimetic imprinting (CBIP) technique. To achieve a from those enumerated above. In the case of sterile powders relatively easy on/off binding event, a non-covalent recogni for the preparation of sterile injectable solutions, the methods tion process is favored. Therefore, Supramolecular interac of preparation may include vacuum drying, spray drying, tions, such as hydrogen bonding, electrostatic interactions, spray freezing and freeze-drying that yields a powder of the hydrophobic interactions, and van der Waals forces, are active ingredient (i.e., the therapeutic compound) plus any 65 employed to achieve recognition. For the formation of the additional desired ingredient from a previously sterile-fil network, it is imperative that the functional monomers, tered solution thereof. crosslinker, and template are mutually soluble. In addition, US 9,155,703 B2 11 12 the solvent must be chosen wisely, so that it does not interact ments and injections. Such systems while useful in treating and destabilize the self-assembled functional monomer and Some diseases have certain disadvantages: (1) they are diffi template. cult to regulate drug delivery; (2) they deliver their bioactive The ability to engineer traditional polymers with specific agent (drug) relatively fast; and (3) agent delivery is usually material properties is hampered by lack of control of molecu decreasing with time lar weight, chain configuration and polymerization kinetics. It must be noted that although the description above uses a Hybrid materials have been developed to preserve the bulk drug as an active agent, similar problems have been observed properties of traditional polymers while making their with release of delivery of other active or bioactive agents, molecular chains look more like proteins. The elusive goal of Such as (but not limited to): pesticides, herbicides, other agri molecular recognition in Synthetic polymer systems has been 10 cultural products, molluscicides, other marine biology prod reached in certain cases. Polyacrylic gels have been designed ucts, agents that kill ticks, flees, etc., essential oils, perfumes, as with recognition capabilities by incorporating non-co agents used in kitchen products, whitening agents used in valently crosslinked antibodies. These proteins couple the laundry detergents. reversible swelling character of the networks with molecular More recently, systems have been developed which allow recognition by only Swelling in the presence of a specific 15 controlled release of drugs to targeted areas of the body. Some antigen. The advantage of using synthetic polymeric materi methods used for the controlled delivery of drugs include: als based solely on proteins or peptides is the high degree of inserts and implants, transdermal systems, oral delivery sys control over properties. Peptides and proteins can be coded tems, nasal delivery systems, vaginal delivery systems, rectal for specific properties using a basic knowledge of inter and delivery systems, ocular delivery systems and bioadhesive/ intrachain interactions. The present and future of biomedical mucoadhesive systems. materials development requires a degree of control prediction Most of these systems while solving the problem of pro in design, synthesis and function of next generation materials. longed delivery of active agents, are not as efficacious in Recent work with this principle in mind has resulted in pro applications when the patient is unwilling or unable to take tein-based materials with properties analogous to more the necessary drug (payload) at a specific time or specific widely used polymers as well as new properties. These new 25 interval. More precisely, Such systems cannot control the materials have been generated with a variable degree of effi problem of patient compliance, a significant problem in this ciency and complexity industry. The development of drug delivery vehicles requires sys The use of carriers sensitive to the Surrounding environ tems that respond to a specific cue in the biological environ ment, Such as pH-sensitive or temperature-sensitive systems ment before the release of a drug payload. This is also coupled 30 have been reported in the field. Indeed, investigators have with the desire for such new devices to otherwise maintain reported methods of delivering drugs, active agents and bio structural integrity and avoid clearance from the body. We active agents in response to changes in pH or temperature of have described sensitive gels with stimuli-sensitive recogni the Surrounding fluid. tion very similar to recognition in proteins. By outlining the Clearly, such systems can improve the pattern of delivery principles developed by analyzing theoretical mechanics of 35 by being triggered to release their payload when a particular heteropolymers, the underlying memory of macromolecule pH prevails in the Surrounding fluid. For example, numerous conformation is discovered and empirically verified. Essen drug delivery systems have been patented where the passage tially, their design includes polymerizing in the presence of from the stomach (low pH) to the upper small intestine (high target molecule, functional monomer, thermo-sensitive pH) triggers the release of an active compound (drug). Often, monomer, and end shielded post-crosslinking monomer. 40 Such systems are accompanied by selective targeting to vari Some of these adsorption sites were destroyed upon gel Swell ous tissue sites. For example, the so-called mucoadhesive ing and reformed upon shrinking. Important contributions drug delivery systems are based on polymeric materials have been made describing the nature of recognition in low which adhere to the mucin layer of a biological membrane for cross-linked systems, and it is only a matter of time when some length of time. The desired drug is loaded into the intelligent gels can recognize other types of molecules. 45 polymers. Once introduced to the body, the polymer carrier The present invention includes imprinted gels or chains begins to Swell, allowing the release of the drug. Because the possessing certain macromolecular architecture with binding polymer binds to the mucin layer of the membrane, the drug abilities could be used as the sensing elements within analyte is released locally and is thought to be able to absorb more sensitive controlled release systems. Analyte sensitive poly easily across the membrane into the bloodstream. Some pos mer networks have been the focus of much research (mostly 50 sible routes of administration for mucoadhesive systems saccharide recognition) and have been designed in a number include: the nasal, ocular, buccal, gastrointestinal, vaginal of ways. and rectal areas. Balancing pharmaceutical research for new drugs to treat There are several distinct advantages in using controlled human illness and disease with economic factors to minimize release systems over other methods of drug delivery. First, the the cost of drug therapy has led to controlled and targeted drug 55 drug can be delivered at a relatively constant concentration. delivery products. The goal of controlled drug delivery is to Thus, the drug concentration can be maintained at a level that reduce the cost of treatment by allowing Smaller, yet equally is higher than the therapeutic level of the drug, but lower than effective, dosages through a regulating device. Some drugs the toxic level. In the case of tablets, the drug concentration have very short half-lives in the human body, and large doses steadily increases until all of the drug has been released. At of these drugs are metabolized rapidly, while other drugs, 60 this point the concentration of drug in the body may be above Such as many of the new protein drugs, are very fragile in the its toxic level. Once the drug has been released from the tablet harsh environment inside the body. Controlled release the concentration decreases until a Subsequent dose is taken. devices can prolong the release time for the former, allowing A second advantage of this type of drug delivery is that the effective dosages, and can protect sensitive drugs until the rate and time period of delivery can be controlled depending point where they are to be delivered. 65 on the properties of the polymer system. In the past, drug delivery devices have been limited to However, the previous systems do not possess the addi systems such as tablets, capsules, powders, droplets, oint tional advantage of intelligence of recognition of not just a US 9,155,703 B2 13 14 change in pH or temperature, but in response to a finite con glycol) (PEG), then hydrogen bonds can form between the centration of an external analyte, a compound with special chains when in the protonated state at low pH. This pH desirable or undesirable properties. dependant formation of hydrogen bonds provides another Most if not all of these systems, whether passive of pH- or means by which the network exists in a compact state at low T-sensitive have a structure that belongs to the category of 5 pH and a more open state at the elevated pH. Hydrogels that hydrogels. Hydrogels are highly biocompatible which makes exhibit this activity are termed pH-responsive complexation them appropriate for a number of pharmaceutical and medical hydrogels. (but also cosmetic, food and consumer) applications. In addi Through the use of monomers with side chains containing tion to drug delivery carriers, hydrogels are biomaterials used groups with pK values in the range desired, a pH-responsive as contact lenses and scaffolds for tissue engineering appli 10 hydrogel could be designed much in the same manner as cations to name only a few of the potential roles. The polymer enteric coatings. Hydrogels can exhibit Swelling to different network can contain homopolymers or copolymers with the degrees based on the intensity of the stimulus and this could chemical structure determining the properties of the hydro be used to target release of multiple compounds at different gel. sites. For example, if a hydrogel Swelled and increased it The network structure of the hydrogel can be characterized 15 mesh size Sufficiently to release a small compound at one pH by a number of parameters. Three parameters that I will and showed increased Swelling at a pH later in the gastrointes discuss here are the polymer volume fraction in the swollen tinal tract, like the colon as opposed to the Small intestine, it state, Va., the molecular weight between crosslinks, M, and could release a second larger agent at this location. The vari the distance between crosslinks also known as the mesh size. ability of the hydrogel delivery system in what it will respond The values for these parameters can be determined empiri to and how it will respond makes it an attractive candidate for cally or by theoretical calculations. numerous clinical applications including targeted drug deliv The polymer fraction in the swollen state is a measure of ery. how much water the hydrogel can imbibe when placed in an These hydrogels can be used for delivery of a variety of aqueous environment. The ability of hydrogels to retain large therapeutic agents. For example, previous work in our labo amounts of water makes them similar to natural tissue and 25 ratory has focused on the use of hydrophilic polymer carriers may contribute to their high biocompatibility. Both the for oral delivery of proteins such as insulin. The loading of molecular weight and distance between crosslinks give an proteins into the hydrogels was done by imbibition, where the indication of how highly crosslinked the network is. Due to polymer is Swollen in a solution containing the protein and the randomness involved with polymer formation, these collapsed at low pH to trap the protein inside. parameters can only be given as average values throughout 30 Recognitive Materials—Molecular Recognition. The rec the hydrogel. These parameters can indicate how much space ognition of a specific molecule out of a whole host of com is available for diffusion in and out of the hydrogel. This peting species is essential to all life processes. It is this ability value, along with the size of the agent to be delivered, will be that allows for the proper functioning of enzymes, antibodies, important in determining the release kinetics of the agent receptors, and signaling molecules. Ultimately, the design of from the hydrogel in drug delivery applications. The degree 35 biomaterials will include this molecular recognition ability, of swelling present in the network will affect the mesh size whether it is a Smart system that recognizes only diseased and therefore a physiologically-responsive hydrogel that cells, an implantable device with a tailored surface that does swells when presented with certain stimuli can have different not elicitan immune response, or a sensor that can track levels release kinetics at different sites in the body. of a specific compound in situ. In addition to use as bioma pH-Responsive hydrogels are composed of ionic networks 40 terials, the creation of synthetic materials with recognitive and Swell in response to pH changes. This Swelling behavior abilities will have great benefits in the areas of separations, is controlled by the ionization of the pendant groups in the assays, catalysis, and mass transport. network. Charged groups exhibit electrostatic repulsion that Synthetic Systems for Molecular Recognition. Undoubt leads to imbibition of water and increased mesh size. This edly, methods for the creation of materials with recognitive event also depends on the level of crosslinking present in the 45 abilities similar to those shown in biological molecules Such hydrogel. Highly crosslinked materials will not be able to as enzymes and antibodies have been heavily sought after. Swell to as high a degree as materials with lower crosslinking Molecular Recognition with Crosslinked Networks. By ratios due to decreased chain mobility. The degree to which a crosslinking the polymer chains, it is possible to restrict the hydrogel network will swell is also dependent upon the ability number of conformations a given chain may adopt. In the to imbibe water. Hydrogels with hydrophilic groups can 50 formation of a configurationally biomimetic imprinted poly imbibe more water than those with hydrophobic groups and mer (CBIP), interactions between a template molecule and can therefore swell to a greater extent. The hydrophobicity/ the monomer feed molecules leads to the creation of a binding hydrophilicity of the network will therefore also have an site that is Subsequently locked in by polymerization and impact on the diffusion of any compound embedded within a crosslinking. To date, imprinted structures have been Success hydrogel network. An example of a monomer that will create 55 fully used in chromatographic applications 34-39, as sen an ionic hydrogel with pH-responsive Swelling is methacrylic sors 40-42, and even as catalytic elements 43-46. acid (MAA). When the pH of the environment is greater than Wulff, et al. 34 demonstrated the first simple molecularly the pKa of the carboxylic acid groups in MAA, they become imprinted polymers (MIPs) utilizing monomers that had been ionized and cause interchain repulsion. The pKa of this group covalently linked to the functional monomers in order to in poly(methacrylic acid) is approximately 4.9 making the pH 60 establish a proper 1:1 stoichiometric ratio. After polymeriza shift from the stomach to upper small intestine (1.5-6) appro tion, the template molecule was freed through lysis. This priate to change the ionization of the carboxyl groups. The covalent technique of imprinting has several advantages charged groups are also hydrophilic and allow water to enter including efficient use of all available functional groups and a the network and continue the Swelling process. propensity to form a more uniform binding pocket, but is The process of ionization is reversible depending on the pH 65 restrictive in what monomers may be used. A technique later of the environment. If the MAA is grafted with another poly pioneered in the laboratories of Mosbach involves imprinting mer capable of forming hydrogen bonds, like poly(ethylene a freely soluble template without the use of covalent linkages US 9,155,703 B2 15 16 47, 48. This technique allows for greater flexibility in the the formation of configurationally biomimetic imprinted choice of functional monomers as well as template molecule. polymers for the separation of D.L-glyceric acid. Since then, However, reaction conditions need to be more strictly con a significant amount of work has been focused on creating trolled to maximize interaction between template and mono MIPs for separations, including enantiomers of Tryptophan mer molecules. In addition, a number of different binding derivatives by Yu and Mosbach 35, nicotine-like com sites may form leading to Some nonspecific binding. pounds by Andersson et al. 36, D- and L-phenylalanine The molecular imprinting procedure. The production of a anilide by Kriz et al. 37, penicillin G and related compounds Successfully imprinted polymer results in a material with by Cederfur et al. 38, and amino acids by Kempe and Mos recognitive properties. While many polymerization tech bach 39. niques are amenable to the imprinting procedure, most utilize 10 More recently, MIPs have been employed as sensing ele a free radical technique with either thermal, ultraviolet, or ments due to their molecular recognition abilities. Due to the redox methods providing the initiating radicals 49. Com minute amounts typically bound, a highly sensitive quartz mon monomers include the methacrylate and acrylate family crystal microbalance (QCM) is used to determine the mass of of molecules, acrylamides, and other vinyl derivatives as analyte absorbed. Detection by QCM has been used by Haupt these are readily available, polymerize easily with the free 15 et al. 40 for the detection of R- and S-propranolol hydro radical technique, and are available with a number of func chlorides, by Liang etal 41 for the detection of epinephrine, tional groups 50. In addition to monomer molecules with an and by Hilt et al. 42 for the detection of D-glucose. array of functional groups, it is crucial to have crosslinking Traditionally, the design of biomaterials has focused on agents that incorporate well into the polymerization. Usually, biocompatibility—the propensity for a material to not invoke a crosslinking agent is selected Such that it has similar reac a foreign body response upon implantation or contact in the tivity to the monomers used so as to form a network uniform body. Numerous studies have been done to tailor surface in crosslinking density 51. properties so as to not elicit an immune response. The main The prepolymerization mixture includes the functional method has been to functionalize the surface with a hydro monomers, crosslinking agent, initiating species, template, philic molecule. Such as grafted poly(ethylene glycol) chains, and solvent if desired. As free radical polymerizations are 25 to mask the foreign Surface from protein adsorption. How sensitive to the presence of radical scavengers such as dis ever, it is now being recognized that molecular recognition Solved oxygen, the mixture is first purged with an inert gas may play an important role in future biomaterials design. Such as nitrogen. Polymerization is then initiated. As Sug Materials that show good biocompatibility are being further gested by Pande et al. 28 in their work on random het enhanced to include molecular recognition. Articles by Rat eropolymers, Successful imprinting is more likely to occurat 30 ner 53 and by Peppas and Langer 54 discuss how building lower temperatures since entropic effects are lessened, which recognitive abilities into biomaterials has the potential to suggests the use of either a redox or UV initiation method. radically advance the field. Potential applications include: (1) However, many groups have Successfully used thermal ini materials that invoke healing pathways to rebuild tissue in the tiation, albeit at lower temperatures than normally seen for implantation area; (2) combined sensing element/controlled thermal polymerizations. 35 release device to meter and release appropriate amounts of Following polymerization, the imprinted polymer is Swol therapeutic compounds; (3) recognitive materials specific to len in solvent to facilitate the removal of the template mol toxins or deleterious signaling molecules (such as angio ecule. Often times before dialysis, the crosslinked material is tensin) for rapid detoxification in the blood stream; and (4) crushed and sieved to produce particles of a given diameter in antibody or enzyme mimics for in vivo use from Synthetic order to facilitate mass transfer. Once free of template, the 40 materials. MIPs are subjected to analysis of their binding ability through Researchers working towards these goals have focused on a variety of techniques including liquid chromatography, the creation of materials that are suitable for use in the body NMR, and microcalorimetry. yet interact with the biomolecule of interest. Byrne et al. 55, Yu and Mosbach 35 studied the influence of crosslinking 56 and used an imprinting technique to create hydrogels density on the recognitive ability of a non-covalently formed 45 capable of binding D-glucose. This technique was later MIP imprinted for the separation of Boc-D-Trp and Boc-L- coupled with sensing technologies developed by Hilt et al. Trp enantiomers. In their studies, it was shown that the ability 57 to form a microsensor capable of detecting D-glucose to separate enantiomers decreases as the crosslinking density 42. Other glucose responsive materials have been formed by is decreased. This supports work done by Wulff, et al. 52 Oral and Peppas 58 utilizing star polymers and by Parmpi with covalently imprinted materials where in addition to a 50 and Kofinas 59. In addition to small biomolecules, Bolisay decrease in recognition, there was also a minimum amount of et al. prepared a configurationally biomimetic imprinted crosslinking needed for recognition. While increases in material suitable for the detection and screening of baculovi crosslinking density are favorable to the recognitive ability of ruses 60. Molecular imprinting is well-known and can be a MIP, the resultant decrease in network mesh size may act to conducted using one or more biomolecules, e.g., Acetalde hinder diffusion through the network, especially limiting for 55 hyde (metabolism byproduct); Adenine, adenosine 5V-triph the recognition of large molecules such as proteins. osphate (ATP); Amino acid and peptide derivatives: Z-L-Tyr Applications of molecularly imprinted materials. Histori OH: Z-L-Phe-OH: Z-DL-Phe-OH: Z-L-Glu-OH; Boc-L- cally, the main application of MIPs has been in chromato Phe-Gly-Oet; Z-L-Ala-L-Ala-OMe, Z-L-Ala-Gly-L-Phe graphic uses. Crosslinked recognitive materials are formed OMe: Z-L-Phe-OH: Ampicillin (penicillin antibiotic); with through the imprinting process and are then crushed into 60 a-Amylase (enzyme); Angiotensin II (SA) (competitive particles Suitable for packing into chromatographic columns inhibitor of peptide hormone angiotensin II); Bupivacaine 49), or used in thin layer chromatography 37. The recog (anaesthetic drug); Butein (active anti-EGFR inhibitor); Caf nitive ability of the particles allows for affinity chromatogra feine (stimulant drug); Cephalexin (antibiotic drug; in a-ami phy whereby only the compound of interest is bound by the nocephalosporins class); Chlorphenamine (anti-histamine column and all other species elute freely. This has worked 65 drug); Clenbuterol (hadrenergic blocker); Cortisone (ste especially well for the separation ofenantiomers, a classically roid); Creatine (metabolite); Creatinine (metabolite); Choles difficult separation. In 1974, Wulff, et al., 34 first suggested terol (steroid); Cholic acid sodium salt (bile acid); Carbohy US 9,155,703 B2 17 18 drates: glucose; lactose, maltose, glucose: Glucose, Maltose; of peptides by Andersson etal 63 and for the recognition of lactose; cellobiose; Carbohydrate derivatives: octyl-gluco proteins by Venton and Gudipati 64. Recently, Rachkov and side; p-nitrophenyl fucoside, p-nitrophenylgalactoside; Per Minoura I65 have demonstrated a method where only a acetylated phenyla- and h-D-galactosides; Diazepam (i.e., fragment of the protein is used in the imprinting process, Valium; benzodiazepine anxiolytic drug); Enkephalin (neu leading to a site that is favorable for a portion of the whole ropeptide); Ephedrine (stimulant drug); Epinephrine (adrena protein. This so called “epitope approach’ mimics the ability line hormone); Estradiol (estrogenic steroid hormone); Ethy of antibody molecules to recognize a portion of a protein nylestradiol (estrogenic steroid hormone derivative); Structure. 9-ethyladenine (nucleotide base derivative): 9-ethyladenine Essential oils. Essential oils are complex mixtures of acetate (nucleotide base derivative); Glucose oxidase (en 10 numerous compounds, they are aromatic oily liquid and can Zyme); L-glutamine (amino acid); Histidine (N-terminal) be extracted from various parts of the plants. Some of the dipeptides; Homocysteine (non-essential amino acid); main chemical groups found in essential oils include alco Horseradish peroxidase (enzyme); Ibuprofen (non-steroidal hols, aldehydes, esters, ethers, ketones, phenols and terpenes. anti-inflammatory drug); Ketoprofen (non-steroidal anti-in Their use is mostly related to food as flavoring, to perfumes as flammatory drug); Lysozyme (enzyme); Morphine (narcotic 15 fragrances and to pharmaceuticals for their functional prop analgesic drug); Naproxen (non-steroidal anti-inflammatory erties. To date they are widely used as air freshener, several drug); Nerve agent degradation products: (S)-nilvadipine (di patents are reported dealing with their applications as deodor hydropyridine calcium antagonists); Nucleoside base deriva ant, Some are related to their use as good Smelling insect tives: tri-O-acetyl adenosine; tri-O-acetyl guanosine; di-O- repellent. As reported in the review proposed by Burt (2004), acetyl thymidine; tri-O-acetyl cytidine; tri-O-acetyluridine: antimicrobial properties of some essential oils have long been Nucleotide base derivatives: 9-ethyladenine: 1-propyl thym recognized; in particular they have been shown to exhibit ine; 1-propyl cytosine; 1-cyclohexyl uracil; Oxytocin (hor antiviral, antimycotic, antitoxigenic, antiparasitic and insec mone); Paracetamol (i.e., acetaminophen, analgesic); Pheny ticidal properties. In the literature several works reports on lalanine (amino acid); (E)-piceatannol (active anti-EGFR their use to improve the shelf-life and safety of food and inhibitor); Propanolol (h adrenergic antagonist); Quercetin 25 packaged food. (active anti-EGFR inhibitor); Ribonuclease A (enzyme): Controlled release systems. As reported by Peppas and Am Ricin A and B Chains (toxin bean lectin): (S)-ropivacaine Ende (1997), the incorporation of fragrances in polymers for (anaesthetic); Scopolamine (anti-cholinergic, anti-infective; the purpose of controlled release over a period of 12 or more and analgesic alkaloid drug); Sulfonamides (antibiotic drug); hours has been studied. Incorporation of essential oils in Testosterone (steroid hormone); Tetracycline (antibiotic 30 polymers could lead to new and innovative products for pro drug); Theophylline (Bronchodilator drug); Timolol (hadr longed delivery of these compounds. Peppas and Brannon energic blocker); Trypsin (enzyme): Tyrosine (amino acid); Peppas (1996) provided a wide view on papers dealing with Tyr-Pro-Leu-Gly-NH2 (tetrapeptide); Leu-enkephalin; Leu the use of systems in which the fragrances are released from enkephalin; Morphine; Morphine: Ampicillin; S-propra different matrices. In addition to those one, the release of nolol. D-phenylalanine; Adenine: 9-ethyladenine; 9-ethylad 35 linalool and linallyl acetate (the major components of aro enine; 9-ethyladenine acetate; Cholesterol; Homocysteine: matic lavender essential oils) from biopolymer gliadin-based Trypsin; Theophylline; see, e.g., Hilt & Byne, Configura nanoparticles (Duclairoir et al., 2002), the release of eucalyp tional biomimesis in drug delivery: molecular imprinting of tus essential oil from alginate complex capsule (Chang and biologically significant molecule, Advanced Drug Delivery Dobashi, 2003) and the release of limonene from polysaccha Reviews 56 (2004) 1599-1620, relevant portions and citations 40 rides matrices (Secourad et al., 2003) were studied. Besides, incorporated herein by reference. Nakayama et al. (2003) studied the release of orange essential Protein Imprinting. The potential applications for a recog oils from temperature responsive membranes obtained by UV nitive material capable of binding a protein are numerous, polymerization of mixture of N-isopropyl acrylamide and including diagnostic devices for protein assays, systems for Polyethyleneglycole dimethacrylate. These references pro use in immunochemistry, and separation media for extremely 45 vide interesting information on the fragrance release mecha complicated protein mixtures. Production of Such materials, nism but, due to the requirements and characteristics of essen however, is difficult for several reasons. First, it is known that tial oils, Swelling-controlled release systems are highly the presence of water reduces the interactions between tem desirable devices for Such applications, relevant portions plate and monomer since the water molecules compete for incorporated herein by reference. hydrogen bonds 33. Most imprinting, therefore, is done in 50 The preparation of the above mentioned devices requires the presence of non-aqueous media. However, peptides and the understanding of the thermodynamic of the three-compo proteins are especially sensitive to differing solvent condi nents system, consisting of the polymer, the fragrance and the tions and may denature in harsh solvents. Secondly, the large liquid which is usually in contact with the device. The release diameter of protein molecules may preclude the use of a ofa fragrance initially embedded into the polymer matrix into densely crosslinked polymeric network since the mesh size of 55 the surrounding solvent is limited by several factors: the the network is too small to allow for efficient diffusion. It is initial amount of fragrance loaded into the polymer, the solu also unclear how selective a protein imprinted material can be bility of the fragrance in the solvent, the equilibrium partition made, and whether subtle changes, such as the process of site coefficient of the fragrance between polymer and solvent and directed mutagenesis, can be differentiated by these materi the diffusional barrier. als. 60 The present disclosure generally relates to biomimetic To date, several attempts have been made at creating MIPs polymer network compositions, methods of forming Such for the recognition of oligopeptides and proteins. Shnek et al. polymer compositions, and methods of using Such composi 61 and Kempe et al. 62 focused on a surface imprinting tions. These compositions and have improved properties that approach whereby monomers capable of complexing a metal make them useful for a variety of applications; in particular, ion were polymerized into a MIP for with an affinity for 65 the loading and delivery of therapeutic agents. proteins with surface exposed histidine residues. Polymers The biomimetic polymer networks of the present disclo imprinted in the bulk have been prepared for the recognition Sure generally include a polymer network having architec US 9,155,703 B2 19 20 tures that have selective affinity for a moiety. Such biomi Sedoheptulose, octoses such as octolose, 2-keto-3-deoxy metic polymer networks may have shape specific cavities that manno-octonateand and nonoses such as Sialic acid. match the moiety, as well as chemical groups oriented to form Specific embodiments may use mucopolysaccharides. multiple complexation points with the moiety. In terms of Mucopolysaccharides are long unbranched polysaccharides selectivity, the resulting polymer networks are selective due 5 consisting of a repeating disaccharide unit. This unit consists to the particular chemistry of the binding site, the orientation of an N-acetyl-hexosamine and a hexose or hexuronic acid, and stabilization of the chemistry in a crosslinked matrix, as either or both of which may be sulfated. Members of this well as by the size and shape of the site for the template family vary in the type of hexosamine, hexose or hexuronic biomolecule. acid unit they contain e.g. glucuronic acid, iduronic acid, In some embodiments, the biomimetic polymer networks 10 galactose, galactosamine, and glucosamine. They also vary in may further comprise a moiety. Such compositions may be the geometry of the glycosidic linkage. Specific example capable of releasing the moiety in a relatively controlled polysaccharides that may be used as moieties include: chon fashion. The moiety may be present on a target compound, for droitin Sulphate, dermatan Sulphate, keratan Sulphate, hepa example, a therapeutic agent. Accordingly, the compositions ran Sulphate, heparin, Sodium heparin, hyaluronic acid and and methods of the present disclosure may be used in the 15 hyaluronan. treatment of a disease. For example, the compositions of the In other embodiments, the moiety may be a lipid or a short present disclosure may be used as a vehicle to deliver a amino acid sequence (e.g., a sequence of about twenty amino therapeutic agent to a subject (e.g., a human) in need thereof. acids in length). In particular, lectins may be used as a moiety. The compositions of the present disclosure also may be used Lectins are carbohydrate-binding proteins involved in a vari to form a medical device or an article. The present disclosure ety of recognition processes and exhibit considerable struc also provides methods of forming a biomimetic polymer net tural diversity. A large variability in quaternary association work of the present disclosure. resulting from Small alterations in essentially the same ter The moiety may be any portion of a molecule recognized tiary structure is a property exhibited specially by legume by a biomimetic polymer network of the present disclosure. lectins. The strategies used by lectins to generate carbohy The moiety may be covalently bound to a target compound, 25 drate specificity include the extensive use of water bridges, for example, a therapeutic agent. In this way, the moiety may post-translational modification and oligomerization. Other be used to associate a target compound with a biomimetic carbohydrate-based structures may be used as moieties may polymer network of the present disclosure. The moiety be located on the worldwide web at chem.qmul.ac.uk/iupac/ should either already be present on the target compound or 2carb? (accessed Apr. 27, 2006), incorporated by reference capable of being conjugated to a target compound. Conjuga 30 herein. tion of moieties to therapeutic agents is known in the art, for In general, the compositions of the present disclosure have example, as disclosed in A. Wong and I. Toth, Curr. Med. enhanced affinities (e.g., impart greater affinity, bound ratios Chem. 8:1123-36 (2001), the relevant disclosure of which is greater than 1) for a chosen moiety, among other things, incorporated by reference. Examples of suitable moieties allowing for increased loading efficiency. Accordingly, the include, but are not limited, to Sugars (e.g., glucose), carbo 35 compositions of the present disclosure also may be used to hydrates, peptides, and functional groups. A specific example increase the loading of a target compound or control the of a therapeutic agent that comprises a moiety is streptozoto release rate of a target compound or both. The compositions cin (R. R. Herr, et al., J. Am. Chem. Soc. 89:4808-09 (1967)), of the present disclosure also may be used for delivery of a which has a glucose moiety. therapeutic agent. For example, the compositions of the In certain embodiments, the moiety is a Sugar. For 40 present disclosure may be used as an excipient or as a vehicle example, the Sugar may be a monosaccharide. Monosaccha for a therapeutic agent. Specifically, higher quantities of a rides have the chemical formula (CH2O)n and the chemical therapeutic agent having a moiety can be loaded within the structure H(CHOH)nC=O(CHOH)nH. If norm is zero, it is biomimetic polymer networks of the present disclosure, an aldose, otherwise it is a ketose. Monosaccharides may therefore enabling for higher doses to be loaded. The release include aldoses, trioses (e.g., glyceraldehyde), tetroses (e.g., 45 of a moiety may be tailored to give a desired release profile, threose), pentoses (e.g., ribose, Xylose), hexoses (e.g. glu for example, for Sustained release of a therapeutic agent. cose, fructose, mannose, galactose), ketoses, trioses, tetroses, Thus, when the moiety is bound to a therapeutic agent, treat pentoses (e.g., ribulose), hexoses (e.g., fructose). Any of the L ment with the therapeutic agent may be optimized. and D isomers of a Sugar also may be used, although the D The compositions of the present disclosure may be formed isomer may be more preferred for biological applications. 50 using configurational biomimetic imprinting. Configuration Other examples of Suitable Sugars include polysaccharides. biomimetic imprinting techniques generally involve forming Polysaccharides have a general formula of C(H2O), where a prepolymerization complex between the template molecule n is usually a large number up to 500. Disaccharides, such as, (e.g., a moiety) and functional monomers or functional oli for example, Sucrose, lactose, maltose, and the like may be gomers (or polymers) with specific chemical structures used. Yet another example of Suitable Sugars includes oli 55 designed to interact with the template either by covalent gosaccharides and low molecular weight carbohydrates (e.g., chemistry or noncovalent chemistry (self-assembly) or both. having a molecular weight no greater than about 2,000 Da). Once the prepolymerization complex is formed, the polymer Further, each carbon atom that Supports a —OH group (ex ization reaction occurs in the presence of a crosslinking cept for the first and last) is chiral, giving rise to a number of monomer and an appropriate solvent, which controls the isomeric forms all with the same chemical formula. 60 overall polymer morphology and macroporous structure. Specific embodiments may use the following monosaccha Once the template is removed, the product is a heteropolymer rides as moieties: monoses, dioses, trioses, tetroses, pentoses, network with specific recognition elements for the template aldo-pentoses, including arabinose, ribose, deoxyribose and molecule. Xylose, keto-pentoses including ribulose, hexoses including The network structure depends upon the type of monomer aldo-hexoses such as: allose, altrose, galactose, glucose, 65 chemistry (i.e., anionic, cationic, neutral, amphiphilic), the mannose and talose, and keto-hexoses such as fructose, hep association strength and number of interactions between the toses including keto-heptoses such as mannoheptulose and monomers and template molecule, the association interac US 9,155,703 B2 21 22 tions between monomers and pendent groups, the Solvent template molecule. Since solvent interaction can stabilize or type and the amount of solvent in the mixture, the reactivity destabilize binding in noncovalent systems, functional mono ratios of the monomers, and the relative amounts of reacted mers may be selected based on optimizing specific noncova monomer species in the structure. Since noncovalent forces lent, self-assembly interactions (hydrogen bonding) with the are weaker than covalent bonds, an increased number of 5 template molecule within an aprotic Solvent (e.g., dimethyl interactions are needed for stable binding and recognition. On Sulfoxide). Such techniques are generally applicable to tem a per-bond basis, noncovalent bonds are 1-3 orders of mag plate molecules, in which hydrogenbonding, hydrophobic, or nitude weaker. Therefore, a greater number of noncovalent ionic contributions will direct recognition of the moiety. The bonding with matching structural orientation is needed for formation of an exemplary biomimetic polymer network of aqueous recognition. 10 the present disclosure according to the methods of the present A wide variety of polymers may be used to form the het disclosure is described below. eropolymer network. These include polymers produced by The multilayered mimetic structures may be constructed reaction of acrylamides and all their substituted structures from a variety of coating processes, including pan coating, including: methacrylamide, ethacrylamide, isopropyl acryla air-Suspension coating, centrifugal extrusion, vibrational mide, etc., acrylic acid, methacrylic acid, ethacrylic acid, all 15 nozzle coating, Supercritical fluid (SCF) based processing, alkyl acrylic acids, any dicarboxylic acid, Such as crotonic fluidization (both conventional Wurster coaters such as the acid, phthalic and terephthalic acid any tricarboxylic acid Glatt device) and rotating, or spray-drying. with itself another monomer of the above list (forming a The multilayered mimetic structures shown in FIGS. 1-2 copolymer), two other monomers from the above list (form may be constructed using a Grow Max Spouted bed assisted ing terpolymers), or three or more monomers from the above with a draft tube and a bottom spray, also known as the list forming higher order coploymers. The above may be in Wurster configuration. The height of the bed is 535 mm and linear, branched or grafted form, the grafted chains being the diameter of the bed is 160 mm at the bottom and 300 mm exclusively one polymer or copolymers of the above, ioni at the top. cally bound or complexed by hydrogen bonds. The bed is equipped with a sampling port to allow the The above may be crosslinked in the presence of crosslink 25 microcapsule to be withdrawn and analyzed during the coat ing agents to form insoluble but Swellable gels or networks, ing process. The bed is also equipped with a viewing window having the ability to absorb water, physiological fluids, buff to allow the observation of the fluidizing microcapsule during ers or salt solutions with final swelling as low as 1 weight% the coating process. The length of the draft tube to be used is of water and as high as 99.9% water. preferably 200 mm and is located 15 mm above the air dis The above crosslinking may be achieved with ethylene 30 tributor. The diameter of the draft tube is 70 mm. The pneu glycol dimethacrylate, ethylene glycol diacrylate, ethylene matic spray noZZle with a liquid caliber of 1.0 mm can be used glycol trimethacrylate, ethylene glycol diacrylate, ethylene to atomize the dispersion fed to the bed. Abag filter with 5um glycol multi methacrylate where “multi’ stands for n=4 to opening can be used to prevent the microcapsules from escap 200 units ethylene glycol multi acrylate where “multi’ stands ing through the top of the bed. The bag filter must be shaken for n=4 to 200 units same as above but propylene glycol multi 35 frequently to prevent the microcapsule from adhering to the methacrylate where “multi’ stands for n=1 to 200 units same bag filter. The schematic diagram of the Wurster bed appara as above but alkylene glycol multi methacrylate where tus is shown in FIG. 2. “multi’ stands for n=1 to 200 units. One may also use higher The advantages of the Wurster process compared to con order acrylates and methacrylates including but not limited to ventional fluidized bed process are: (i) the high inertia intro 1.1.1 trimethylolethane trimethacrylate (TrMETrMA, 40 duced in the Wurster process to circulate the particles pre Molecular Weight 324.4); 1.1.1 trimethylolpropane triacry vents the agglomeration of the particles and (ii) the recycling late (TrMPTrA, Molecular Weight 296.3); 1,1,1 trimethylol profile creates a uniform coat around the particles. The circu propane trimethacrylate (TrMPTrMA, Molecular Weight lating pattern of the particles inside a Wurster bed is made 338.4): pentaerythritol triacrylate (PETrA, Molecular Weight possible by a draft tube. The air inlet is concentrated just 298.3); glycerol propoxy triacrylate (GlyPTrA, Molecular 45 under the draft tube. This creates a high air velocity inside the Weight 428.5); pentaerythritol tetraacrylate (PETeA, draft tube and a vacuum like effect at the base of the draft tube. Molecular Weight 353.2); ethoxylated 1.1.1 trimethylolpro The particles collected under the bed are readily pulled by the pane triacrylate (ETrMPTrA, Molecular Weight 428); glyc air inlet into the draft tube and being carried to the top of the erol propoxylated triacrylate (GlyPTrA, Molecular Weight bed. At the exit of the draft tube, there is a sudden expansion, 428) and glycerol trimethacrylate (GlyTrMA, Molecular 50 from the diameter of the draft tube to the diameter of the Weight 396.3). One may also use with “star polymers' or chamber. This expansion causes the particle to lose its Veloc “dendrimers' with up to 72 independent chains ending in ity gradually and finally gravity will pull the particles back acrylates or methacrylates. down along the wall of the chamber to the base and the The initiator may be IRGACURE(R) products of the Ciba process will be repeated. Geigy company including IRGACURE 184, IRGACURE(R) 55 Generally, a Wurster bed is equipped with a bottom spray, 379, Ciba(RIRGACURERTM819, and Ciba RIRGACURER) and hence, every time the particles pass through the based of 250. Any other photoinitiator may also be used. The initiator the draft tube, latex dispersion will be sprayed on the par may also be any peroxide including but not limited to benzoyl ticles. This process repeats every time the particles pass peroxide, cumyl peroxide, etc. or AZobis isobutyronitrile. through the bottom of the draft tube, and finally, a uniform In some embodiments, the biomimetic polymer network of 60 coating can be obtained. The ability to coat such small par the present disclosure may be formed using a template mol ticles uniformly has broadened the applications of the ecule (e.g., D-glucose) and functional monomers selected to Wurster process for particulate drug delivery systems. match corresponding template molecule (e.g., glucose bind The Wurster coating process employed in the present dis ing protein residues. Such as aspartate, glutamate, and aspar closure is very versatile and can be applied for many different agines, as well as biological mechanisms of action that 65 materials. The process has been shown to work for many involve recognition The template molecule may be added in different type of cores including calcium carbonate crystal, Stoichiometric amounts in regard to the functionality of the glass beads and lactose 1-6. Many latex dispersions have US 9,155,703 B2 23 24 also been shown to be able to be coated using this process, perature, T (as is the case with the MIP particles disclosed Such as poly(ethyl acrylate/methyl methacrylate/2-hydroxy herein) and also by the addition of hydrophilic linear polymer ethyl methacrylate) (P(EA/MMA/HEMA)), composite latex chains as spacers in between the nanoparticles. On the other of core P(EA/MMA/HEMA) and P(NIPAAm) shell, hand, film formation is a necessity for the skin layer. Film crosslinked P(NIPAAm), Eudragit RS30D, L30D-55 and formation improves the rigidity and integrity of the skin layer, FS30D, and ethyl cellulose 1-6). and hence, provides the required barrier to prevent the drug The Wurster coating process is a very harsh process. The transport across the skin layer. The film formation in the skin process involves exposure to high temperature and high shear layer can be enhanced by the addition of some plasticizers. stress. The process requires high temperature to dry the The addition of plasticizer to the MIP may turn out to be coated microcapsules. The high shear stress is required to 10 effective in lowering the glass transition temperature of the atomize the latex dispersion before being sprayed on the system, and hence, enhanced the film formation of the coating circulating microcapsules. Fortunately, the MIP nanopar layer. ticles have shown promising stability at high temperatures Another problem with this multilayered mimetic system is and high shear stresses. the maximum size. The size is controlled by the size of the One problem encountered with the coating process is the 15 Syringe because the medical counter-part of this system is formation of a 'snowman-like' structure due to agglomera designed for injectable system. The size problem can be tion. These structures are produced by the fusion of two solved by mixing several different microcapsules. For microcapsules. This fused structure creates weak spot along example, the size limitation would only allow the multilay the contact point of the two microcapsules. Agglomeration ered mimetic structures to have 16 layers, which would be prevents the formation of a single core microcapsule, which is translated to 8 bursts. If a drug delivery system requires a the structure desired in this work. Several factors that affect burst every three hours, then the system can only lasts for one the degree of agglomeration using the Wurster bed include: day. However, if we modify the multilayered mimetic struc the feed rate of the latex dispersion, the flow rate of the dried tures, so that it would rupture every 12 hours and also the air to fluidize the microcapsule and the temperature of the outer most layer is modified into four different batches: (i) inlet air, the outlet air and the bed and the like. 25 one that will rupture after 3 hours; (ii) another that will rup The network structure of the layers depends upon the type ture after 6 hours; (iii) another after 9 hrs; and (iv) another of monomer chemistry (i.e., anionic, cationic, neutral, after 12 hrs. By mixing these four different batches, the sys amphiphilic), the association strength and number of interac tem can last for 4 instead of one day. This method of mixing tions between the monomers and template molecule, the asso made these multilayer mimetic structures to be very versatile ciation interactions between monomers and pendent groups, 30 and can easily be tailored to fit any release profiles. the solvent type and the amount of solvent in the mixture, the In order to show controllable properties, a mathematical reactivity ratios of the monomers, and the relative amounts of expression of the system an be used to predict the burst reacted monomer species in the structure. Since noncovalent process and when each drug release will occur. This expres forces are weaker than covalent bonds, an increased number sion is derived from the force balance between the inside and of interactions are needed for stable binding and recognition. 35 outside of the seal coat layer. The microcapsule is assumed to On a per-bond basis, noncovalent bonds are 1-3 orders of be a perfect sphere with finite coating thickness. The MIP coat magnitude weaker. Therefore, a greater number of noncova has a tensile strength, which is dependent on the molecular lent bonding with matching structural orientation is needed weight of the material. The force balance can be simplified by for aqueous recognition. dividing the microcapsule into two equal hemisphere, and A wide variety of polymers may be used to form multilay 40 thus, canceling the X-component of the pressure. The pressure ered mimetic structures. These include polymers produced by inside is larger than the pressure outside due to the osmotic reaction of acrylamides and all their substituted structures pressure and the Swelling of the hydrogel nanoparticles. including: methacrylamide, ethacrylamide, isopropyl acryla The y-component of this pressure will effectively act on the mide, etc., acrylic acid, methacrylic acid, ethacrylic acid, all projected area of the hemisphere as represented by the left alkyl acrylic acids, any dicarboxylic acid, Such as crotonic 45 hand side of Equation 1. The tensile strength of the seal coat acid, phthalic and terephthalic acid any tricarboxylic acid layer on the other hand will oppose this force along the rim with itself another monomer of the above list (forming a microcapsule as represented by the right side of Equation 1. copolymer), two other monomers from the above list (form ing terpolymers), or three or more monomers from the above list forming higher order coploymers. The above may be in 50 linear, branched or grafted form, the grafted chains being P and P are the pressures outside and inside of the exclusively one polymer or copolymers of the above, ioni hemisphere, respectively, t is the tensile strength of the MIP cally bound or complexed by hydrogen bonds. For applica coating, ris the radius of the hemisphere, and 1 is the thickness tion in organisms, such as humans, the polymers that will of the seal coat layer. This equation assumes the radius of the have most application are those that are biocompatible, and in 55 hemisphere is much larger than the thickness of the MIP coat. Some cases, also biodegradable. Simplifying Equation (1), the tensile strength can be The above may be crosslinked in the presence of crosslink expressed as a function of the net pressure, the radius of the ing agents to form insoluble but Swell-able gels or networks, hemisphere, and the thickness of the MIP coat layer as shown having the ability to absorb water, physiological fluids, buff in Equation (2). ers or salt solutions with final swelling as low as 1 weight% 60 of water and as high as 99.9% water. Typically, highly crosslinked nanoparticles with high T. cannot fuse together AP* (2) and form films because the mobility of the polymer chain is of highly restricted. This restriction on the mobility of the poly merchains prevents the polymer chains to entangle. The film 65 The tensile strength of polymers increases with increased formation of the hydrogel layer can be prevented by using crosslinking and ultimately reaches an asymptotic value. This highly crosslinked particles with high glass transition tem can be represented as Equation (3), where A and B are con US 9,155,703 B2 25 26 stants 7.8. The A and B constants depended on the degree of The MIP Hydrogel Coating. A CBIP coating was prepared crosslinking and the intrapolymer interactions of the poly by reacting 20 ml of acrylamide (Aam), 1.7 g of 2.2- C. dimethoxy-2-phenyl acetophenone (DMPA) as an initiator, 18 ml of dimethylsulfoxide (DMSO) as a solvent, and 8 ml of ethylene glycol dimethacrylate (EGDMA) as a crosslinking B (3) agent. The system was reacted in the presence of 10 g D-glu cose. The reaction was carried out in a 100 ml glass reactor placed under a UV source (Dymax Ultraviolet Flood Cure From Equations (3) and (2) we obtain the molecular weight System) and exposed to UV light with an intensity of 6.0-12.0 (crosslinking) as a function of the net pressure, the radius of 10 mW/cm for 30 minutes to initiate the free-radical polymer the hemisphere, the thickness of the MIP coat and the con ization. The ensuing particles, typically of 800 nm to 20 stants of the material as shown in Equation (4). micron size were washed repeatedly in deionized water to eliminate any unreacted monomers and to extract all the 15 glucose. This process was completed in 4 days at 25-30 C. (4) The Wurster Bed Coating Process. In a suggested, typical coating study with a Wurster bed, 10 g of the core material was charged into the bed. A 5% w/w dispersion of the CBIP particles prepared before in the amount of about 40 g (2 g of Since the coating material of interest comes apart with CBIP solids) at a feed rate of 2.4 min/min can be sprayed on Swelling and particle separation, M, decreases with time. The the core particle to form the 6 um thick hydrogel layer. Then, swelling disintegration of first order kinetics as described by 1 g of insulin was sprayed on the system at a feed rate of 2.4 Equation (5), where k is a rate constant. min/min. This process created a 2 um thickness coating. The factors governing the flow rate of the dispersion were M(t)=Mek (5) 25 the solid content of the dispersion and the stickiness of the Inserting Equation (5) into Equation (4) and Solving fort, material. The lower the solid content, the lower the feed rate we may calculate the estimated lag time before the rupture to the nozzle, because it took more time to dry the system. The process. dispersions can be fed to the spray nozzle by a peristaltic pump and atomized with a dried compressed air. The disper 30 sion was continuously stirred during the coating process. (6) During all these processes, heated air (40°C.) was fed into the chamber at a flow rate of 0.25-0.75 m/minto fluidize the particles. The thickness of each layer was predetermined before the coating process. The calculation was based on the 35 average size of the core and the thickness of the CBIP nano This time parameter is an important parameter that can be particles and insulin layers. The Volume of each layer was tailored by several approaches: (1) select materials with dif calculated and adjusted by assuming 23% void fraction for ferent disintegration kinetics resulted in different lag time by spherical systems. The dry weight of the dispersion required controlling the k parameter; (2) select materials with different to form the layers for a particles was calculated from the degree of crosslinking and intrapolymer complexes thus 40 known densities of the dispersion. The number of the particles affecting the lag time in term of the A and B parameters; (3) could be calculated by dividing the total weight of the core select material with high starting concentration and molecu charged, which was 10g by the weight of a single core. Based lar weight that can prolong the lag time; (4) increase the on these calculations, the total dry weight of each dispersion pressure difference between the inside and outside of the could be calculated. multilayered mimetic structure by adding osmotic agents, 45 Once the coating process was finished, the multilayered Such as Salt and high molecular weight hydrophilic polymers mimetic structure was collected and weighed to determine the that could decrease the lag time; and, (5) increase the thick yield. The multilayered mimetic structure was then sieved ness of the seal coat layer could prolong the lag time. and the particle size distribution was analyzed. The multilay There are several important assumptions in this system. ered mimetic structure was then dried under vacuum for 24hr. The net pressure difference between the inside and outside of 50 The feed rate of the latex dispersion depended on the prop the microcapsule is assumed to be constant throughout the erties of the latex itself. Tacky dispersions, with low T. degradation process. The net pressure difference is assumed required a lower feed rate. Dispersions with low solid content constant. The thickness of the coating layer is assumed to be also required a low feed rate to allow the microcapsule to dry. constant throughout the process. The flow rate of the dried air used to fluidize the microcapsule 55 controls the inertia of the microcapsule. High inertia could Example 1 prevent the microcapsule from agglomerating. However, high inertia could also break fragile microcapsule. An example of the present invention was constructed for The temperature of the dried air at the inlet and outlet glucose recognition followed by insulin release. The system governs the temperature of the bed. These temperatures con was formed by using a core, followed by a coating of the 60 trol the rate of the drying of the microcapsule. High tempera CBIP polymer in multiple layers. The core used was glass ture allows the microcapsule to dry faster, and hence, allow a beads (size range 63-75um, Sigma, St Louis, Mo.) although faster process. However, high temperature cannot be applied potato starch or other materials such as calcium carbonate for a dispersion system with low T. Based on my past expe crystals (size range 63-75 um) could be used. The MIP or rience on the Wurster coating process with other materials, recognitive layer consisted of a CBIP hydroxy propyl cellu 65 the operating conditions for the preparation of multilayer lose(HPC) as a spacer and mannitol as a binder. Triacetin was microcapsule in this work must be around (i) feed rate of used as a plasticizer to enhance the film formation. 2.4-3.0 min/min for MIP nanoparticle dispersion; (ii) feed US 9,155,703 B2 27 28 rate of 2.4-4.0 min/min for insulin dispersion; (iii) air flow Example 4 rate of 0.25-0.75 m/min: and (iv) air inlet temperature of 40° C. Recognitive and Release-Triggering Laminates with Multiple Recognitive Layers for Drug or Other Example 2 Active Delivery A second prototype of the present invention was con A system can be developed based on the same principles as structed for glucose recognition followed by insulin release. in Formulation No. 2, but in the form of thin films (laminates) The system was formed by using a core, followed by a coating stacked on top of each other. Thus, a multilayered device can of the CBIP polymer in multiple layers. The core used was 10 be prepared where each film contains a recognitive hydrogel calcium carbonate crystals (size range 63-75um). The MIP or is prepared and applied to a core of drug by casting technique. recognitive layer consisted of a CBIP hydroxy propyl cellu In between each coating there are gaps containing finely lose (HPC) as a spacer and mannitol as a binder. encapsulated drug particles. Each laminate expands because A CBIP coating was prepared by reacting 14 ml of acrylic of the osmotic effect described before. Each hydrogel lami 15 nate (film) is designed to rupture at preset times, thus releas acid (AA), 1.3 g of IRGACURE(R) 184, 1-hydroxycyclohexyl ing the Subsequent layer of encapsulated drug particles. phenyl ketone as an initiator, 18 ml of dimethylsulfoxide Using the above technology an active agent can be released (DMSO) as a solvent, and 7 g of poly(ethylene glycol in: multiple doses of the same drug at exactly the same time dimethacrylate) (PEGDMA, of PEG molecular weight of intervals; multiple doses of the same drug at varying intervals 200, 400 or 1000) as a crosslinking agent. The system was (e.g., first at 10 minutes, second at 15 minutes, third at 30 reacted in the presence of 10 g D-glucose. minutes, fourth at 5 minutes, etc)... multiple doses of two The reaction was carried out in a 100 ml glass reactor different drugs at the same or varying time intervals. These placed under a UV source (Dymax Ultraviolet Flood Cure drugs maybe Smaller molecular weight actives, peptides, pro System) and exposed to UV light with an intensity of 8.0-14.0 teins and others; multiple doses of several drugs or peptides or mW/cm for 30 minutes to initiate the free-radical polymer 25 proteins at equal or varying intervals and/or release of an ization. The ensuing particles, typically of 200 nm to 10 active followed by a second film containing a Sweetener to micron size were washed repeatedly in deionized water to cover (mask) the bitter after taste of the first drug. eliminate any unreacted monomers and to extract all the glucose. This process was completed in 4 days at 25-30 C. Example 5 In a typical coating study with a Wurster bed, 10 g of the 30 core material was charged into the bed. A 5% w/w dispersion Externally-Triggered Multilayered Skin-Adherent of the CBIP particles prepared before in the amount of about Films for Delivery of Essential Oils or Cosmetic 40 g (2 g of CBIP solids) at a feed rate of 2.4 min/min can be Actives or Bactericides or Topical Treatment sprayed on the core particle to form the 6 um thick hydrogel Over-the-Counter Agents layer. Then, 1 g of insulin was sprayed on the system at a feed 35 rate of 2.4 min/min. This process created a 2 um thickness A system can be developed based on the same principles as coating. in Examples 2 and 3, but in the form of thin films (laminates) stacked on top of each other. In this case, however, the mul Example 3 tilayered device can contain also a final thin layer of a skin 40 adhesive polymer (e.g., pressure sensitive adhesive or poly (acrylic acid) or similar that can be used to attach the whole Formulation No. 2 system to the skin. The remaining part of the device is pre pared with each film containing a recognitive hydrogel pre Recognitive and Release-Triggering Systems with pared and applied to a core of active by a casting technique. In Multiple Recognitive Coatings for Drug Delivery 45 between each coating there are gaps containing finely encap Sulated particles of essential oils, cosmetic actives, bacteri The system in Example 2 can be developed into a multi cides or over-the-counter agents that can be used in local skin layered or multicoated device where each layer contains a treatment. Each laminate expands because of the osmotic recognitive hydrogels prepared and applied to a core of drug effect described before. Each hydrogel laminate (film) is by a spray coating technique (e.g., a Glatt GPCG, fluid bed 50 designed to rupture at preset times, thus releasing the Subse coater, Glatt Air Techniques, Inc., Ramsey, N.J.). In between quent layer of encapsulated agent particles. each coating there are annular pouches containing finely Using the above technology an active agent can release: encapsulated drug particles. Each coating (layer) expands multiple doses of the same agent at exactly the same time because of the osmotic effect described before. Each hydro intervals; multiple doses of the same agent at varying inter gel coating layer is designed to rupture at preset times, thus 55 vals (e.g., first at 10 minutes, second at 15 minutes, third at 30 releasing the Subsequent layer of encapsulated drug particles. minutes, fourth at 5 minutes, etc)... multiple doses of two Using the above technology an active agent can be released different agents (e.g., a perfume compound and a bactericide) in: multiple doses of the same drug at exactly the same time at the same or varying time intervals; multiple doses of sev intervals; multiple doses of the same drug at varying intervals eral drugs agents at equal or varying intervals. (e.g., first at 10 minutes, second at 15 minutes, third at 30 60 minutes, fourth at 5 minutes, etc)... multiple doses of two Example 6 different drugs at the same or varying time intervals. These drugs maybe Smaller molecular weight actives, peptides, pro Sweat-Triggered Multilayered Skin-Adherent Films teins and others; multiple doses of several drugs or peptides or for Delivery of Essential Oils or Cosmetic Actives proteins at equal or varying intervals and/or release of an 65 active followed by a second layer containing a Sweetener to A system developed on principles similar to those of cover (mask) the bitter after taste of the first drug Examples 2-4, but in the form of multiple thin films (lami US 9,155,703 B2 29 30 nates) triggered by actives produced by the human Sweating (film) is designed to rupture at preset times, thus releasing the process and releasing actives. Such triggering molecules will Subsequent layer of encapsulated agent particles. be epinephrine and related compounds. The multilayered The remaining part of the device is prepared with each film device will also contain a final thin layer of a skin adhesive containing an epinephrine-imprinted, recognitive hydrogel polymer (e.g., pressure sensitive adhesive or poly(acrylic 5 prepared and applied to a core of active by a casting tech acid) or similar) that can be used to attach the whole system to nique. In between each coating there are gaps containing the skin. finely encapsulated particles of creams that can be used in The remaining part of the device is prepared with each film local skin treatment. Each laminate expands because of the containing an epinephrine-imprinted, recognitive hydrogel osmotic effect described before. Each hydrogel laminate prepared and applied to a core of active by a casting tech- 10 (film) is designed to rupture at preset times, thus releasing the nique. In between each coating there are gaps containing Subsequent layer of encapsulated agent particles. finely encapsulated particles of essential oils, cosmetic actives, bactericides or over-the-counter agents that can be Example 9 used in local skin treatment. Each laminate expands because of the osmotic effect described before. Each hydrogel lami- 15 Preparation of Recognitive Systems nate (film) is designed to rupture at preset times, thus releas ing the Subsequent layer of encapsulated agent particles. Preparation of Films from Recognitive Polymers. The con Using the above technology we can release: multiple doses figurational biomimetic imprinted polymer (CBIP) recogni of the same agent at exactly the same time intervals; multiple tive films prepared in this work included a crosslinked doses of the same agent at varying intervals (e.g., first at 10 20 copolymer of methacrylic acid (MAA 99%, inhibited with minutes, second at 15 minutes, third at 30 minutes, fourth at 100-250 ppm HQ, Sigma-Aldrich, St. Louis, Mo.) and ethyl 5 minutes, etc); multiple doses of two different agents (e.g., a ene glycol dimethacrylate (EGDMA, stabilized, 98%. Acros perfume compound and a bactericide) at the same or varying Organics, N.J.). The reaction mixture was imprinted with time intervals; and multiple doses of several drugs agents at D-glucose (A.C.S. reagent, Aldrich Chemical Co., Milwau equal or varying intervals. 25 kee, Wis.). The monomers were photopolymerized in the presence of UV light with 2,2-dimethoxy-2-phenylacetophe Example 7 none (DMPA, 99%, Acros Organics) used as the free-radical initiator. All solvents were of analytical grade. Sweat-Triggered Multilayered Nanoparticles for MAA (10% w/w) and D-glucose (5% w/w) were added to Delivery of Essential Oils or Cosmetic Actives 30 a solution comprised of a 3:1 ratio of ethanol to water (by volume) and were sonicated for 20 minutes to evenly disperse A system developed on principles similar to those of the components. To this mixture, EGDMA (84% w/w) and Examples 2-4, but in the form of multiple nanoparticles in the DMPA (1% w/w) were added and mixed by mechanical shak form of devices triggered by actives produced by the human ing. This mixture was then purged with N in an oxygen-free Sweating process and releasing actives. Such triggering mol- 35 environment (e.g., a sealed glove box) for 30 minutes. After ecules will be epinephrine and related compounds. Such sys purging, the mixture was loaded into a polymerization cham tems can be applied as a dry powder and can release at various ber (made from two 75x50x1 mm microscope slides with a times, as late as 8 hours after application. They may contain 200L Teflon spacer placed in between, bound with binder expensive perfume, essential oils, bactericides, etc. clips around the edge) and polymerized under UV light (at 15 40 mW/cm2) for 15-20 minutes. The resulting films were then Example 8 removed from the glove box and washed with Milli-Q DI water for 24-48 hours (with the water being changed every 24 Sweat-Triggered Multilayered Skin-Adherent Films hours), after which they were removed and dried in a drying for Delivery of Oils, Lipids or Creams oven (with desiccant) for 24 hours. The resulting films were 45 then stored at room temperature for observation. Delivery of highly hydrophobic active agents using the Preparation of Control Samples. The control samples (non MS/UT technology (after initial recognition) is extremely imprinted polymer films) were prepared using precisely the difficult as the lipidic structures are extremely difficult to same protocol as above, except for the absence of D-glucose penetrate through hydrophilic gels. However, with the present from the mixture (i.e., no glucose was added). technology a multivariate system can be prepared, where the 50 Configurational Biomimetic Imprinting in the Presence of encapsulated nanoparticles are wholly covered by hydropho Glucose. The biomedical applications for the novel systems bic coatings and reside in the space between consecutive are far reaching due to a unique development in the field layers of epinephrine-recognitive hydrogel films. A system known as molecular imprinting, a concept upon which our was in the form of multiple thin films (laminates) triggered by CBIP systems are based. Molecular recognition via imprint actives produced by the human Sweating process and releas- 55 ing is accomplished by having a certain template molecule (in ing actives. Such triggering molecules will be epinephrine our case, glucose) dispersed between the various monomers and related compounds. The multilayered device may also during the process of polymerization (FIG.1). The monomers contain a final thin layer of a skin adhesive polymer (e.g., proceed to polymerize as usual, while the specific template is pressure sensitive adhesive or poly(acrylic acid) or similar) imprinted directly within the polymer network (FIG. 2). The that can be used to attach the whole system to the skin. 60 polymer films are then washed, thus removing the template The remaining part of the device is prepared with each film molecule (e.g., glucose) and leaving behind a chemically containing an epinephrine-imprinted, recognitive hydrogel Stereospecific site where the template molecule was once part prepared and applied to a core of active by a casting tech of the network (FIG.3). When the film is subsequently dried, nique. In between each coating there are gaps containing nanovacuoles are present, while the polymer’s physical char finely encapsulated particles of creams that can be used in 65 acteristics change (notably a decrease in overall size). When local skin treatment. Each laminate expands because of the glucose is reintroduced to the system, the attachment of the osmotic effect described before. Each hydrogel laminate molecule to the Stereospecific site causes a mechanical stress US 9,155,703 B2 31 32 in the local region that will eventually lead to rupturing; it is samples exposed to DI water should not Swell as much as the this template-specific recognition/rupture/delivery that we samples placed in a glucose solution. seek to exploit in the form of novel drug delivery systems. The Every ten minutes, Samples were removed by tweezers, templates used in this type of recognition can be extended to blotted gently with a wipe, and weighed. The total time that almost any molecule; further investigation will reveal new each sample was out of solution was roughly one minute. All applications for this technique. studies were done at 37 C. In addition, the pH of each solution Sample Preparation. After numerous different procedures was measured. or “recipes' were performed, a general procedure was In all studies, the amount of penetrant uptake was calcu selected. There were many variations to the results of each lated by Subtracting the dry weight of sample (polymer) from 10 a later weight and then dividing by the dry weight of sample. recipe and method. At present time, not all factors have been These values are a clear indication of a fast uptake by the tested. Samples prepared with low amounts of EGDMA as a recognitive system. These values were plotted as a function of crosslinker showed significant differences from those films time and as a function of square root of time (see FIGS. 4-7). made with at least 25 wt % crosslinker (the preferred ones). These Figures show that glucose is recognized by the recog The crosslinker must be present in a high enough amount to 15 nitive samples, leading to fastbinding. A very fast recognition hold the copolymer together. This was noticed from the films process is observed with glucose (100 mg/dl solution) with which remained fluid or tacky and glutinous after the poly Subsequent Saturation of the binding sites and reduction of the merization time ended. Different procedures were also fol water uptake until a constant value is obtained, almost the lowed with different amounts of ethanol and water. Adding same as for pure water absorption. Scientifically, this phe more ethanol to the recipe seemed to help the films polymer nomenon can be interpreted in a similar way as the action of ize more but also sometimes made the films turn out white. Solid catalysts. Once the active sites are occupied, no further It must be noted though, that it is necessary to do also reaction can take place. studies at lower crosslinking ratios because of the ability to Numerous studies of recognition at higher glucose levels imprint larger proteins in these systems. It is particularly (e.g., 150 mg/dL and 200 mg/dL) indicated that these samples necessary to appreciate that such systems will be important 25 fractured within a few minutes (typically 13-20 minutes for for consumer applications where added lipids will be neces thin films) after exposure to the solutions. Indeed, graphs of Sary. penetrant uptake (1) versus time cannot be presented as the The polymerization time (under the UV lamp in the glove weight decreased after being the samples were placed in box) made a difference as to how rigid and continuous each Solution because of the rupture or cracking. film was. The first samples were prepared by polymerization 30 FIG. 4 is a graph that shows the penetrant uptake of recog for approximately 15 to 20 minutes according to the protocol nitive polymer continuous films overtime. The data points are described in Example 2. But many of the recognitive and the amount of penetrant uptake in Milli-Q deionized water non-recognitive films polymerized under these conditions (DI water) versus 100 mg/dL D-glucose in DI water. The were still “sticky'. Some films were polymerized for longer films were cut into disks 8 mm in diameter and 0.12 mm thick; amounts of time (up to 60 minutes). 35 the initial weights were approximately 6 mg each. Measure There were other laboratory procedures that were followed ments were taken every 10 minutes. before complete polymerization of the films. First, the mono FIG. 5 is a graph that shows the penetrant uptake of a mer solution was sonicated for at least 20 minutes before recognitive polymer continuous film versus the square root of adding EGDMA and DMPA to ensure a mixture. Once the time. The data points are amount of penetrant uptake in EGDMA and DMPA were added and sonicated again for a 40 Milli-Q deionized water (DI water) versus 100 mg/dL D-glu few seconds. cose in DI water. The films were cut into disks 8 mm in Characterization. Characterization of the prepared films diameter and 0.12 mm thick; the weights were approximately was done by Swelling studies, optical and electron micros 6 mg each. Measurements were taken every 10 minutes. copy, FTIR analysis and differential scanning calorimetry. FIG. 6 is a graph that shows the recognitive ratio of con We will report here only what has been completed already. 45 figurational biomimetic imprinted polymers (CBIP) in the Microparticles and continuous films were observed under presence of 100 mg per dL deionized water compared to polarized light, normal light, and with fluorescein isothiocy continuous films in the presence of deionized water. The films anate-(FITC)-glucose under fluorescent light. The swelling were cut into squares approximately 9 mm by 9 mm and 0.22 of the films was studied in solutions of different glucose mm thick. The penetrant uptake amount was obtained from concentrations and DI water. 50 measurements of mass every 10 minutes once the squares Swelling Studies. The purpose of these studies was to were placed in the solutions of either deionized Water or examine the recognitive and Swelling characteristics of all glucose solution with 100 mg D-glucose per dL deionized recognitive samples. This was done because of numerous water. The ratio is amount of penetrant uptake in the glucose questions received by Mimetic Solutions as to the ability of Solution to amount of penetrant uptake in deionized water. the recognitive samples to recognize their templates fast and 55 Observation of Swelling and Recognitive Processes. A rupture as a result of the stresses created. large number of the recognitive films were crushed using For each Swelling study, the recognitive continuous films mortar and pestle or cut into 8 mm disks using a cork borer in were cut into disks or squares between 0.13 mm and 0.22 mm order to observe their response to glucose solutions under the thick. The disks were cut with a cork-borer with a radius of microscope. The swelling behavior of different CBIP and NIP approximately 8 mm; the squares were cut with a razorblade 60 (control) films was observed in DI water, and solutions of 100 to approximately 9x9 mm. Before a swelling study, the mg/dL glucose, 150 mg/dL glucose, 200 mg/dL glucose, 300 weight, thickness, and dimensions of each sample were mg/dL, FITC-glucose, and trypan blue. The samples were recorded. The disks or squares were then immersed into bea tested under normal light, polarized light, and fluorescent kers with either DI water, 100 mg/dL glucose solution, 150 light (when using FITC-glucose). The particles (approxi mg/dL glucose solution, or 200 mg/dL glucose solution. The 65 mately 100-300 micrometers) were placed on a microscope DI water was used as a control to see if the disks and squares slide and pictures were taken before any solution was added. would swell without glucose present. It was found that the Then using either a spatula or a pipette, a drop or two of US 9,155,703 B2 33 34 solution were placed on the samples. Effort was made to the change of the polymer film thickness as a function of time. avoid capillarity effects. Still photographs were taken in Suc This technique is also limited to those polymers that give a cession to form videos. The purpose of using a trypan blue negligible gel layer because good interference cannot be solution was to observe the liquid front movement into the obtained if the gel layer thickness is significant. particles or films. Differential refractometry can also be used to measure the Observations Without Polarization. Swelling of particles polymer dissolution rate. In this study, a polymer sample is and films was difficult to observe without polarized light. In immersed in a glucose solution in a special container, general, the particles tended to agglomerate together when in equipped with a differential refractometer and an agitator. Solution, which could sometimes be mistaken for Swelling. Polymer cracking and dissolution are followed by measuring FIG. 8 shows typical recognitive response and Swelling 10 the refractive index of the solution. Using this method, it is behavior of particles within a few seconds from exposure to also possible to measure induction times, which are the times the glucose-containing Solution. After one minute, the par necessary for a build-up of a swollen surface layer. The dif ticles have started recognizing glucose, which creates internal ferential refractometry technique can be used even in the stresses. The third panel of FIG. 8 clearly shows the swollen presence of gel layers. However the thickness of the gel layer particles that have formed from the larger particles due to 15 cannot be measured simultaneously. rupture. To measure both the recognition and CBIP polymer disso FIG.9 shows a very large number of particles produced by lution rate and the gel layer thickness as well as to investigate the same recognitive process but using films containing a polymer morphology during dissolution, techniques using porosigen (see below) to produce large pores within the rec optical microscopy have been used. The apparatus consists of ognitive polymer. an optical microscope and a sample cell containing a CBIP FIG. 10 show stress lines formed during the first few sec polymer sample engulfed by a matrix inert to the solvent and onds after addition of a glucose solution to a recognitive film sandwiched between two glass slides. As the contrast produced as described above. Clearly, these observed stress between the different layers was usually poor, dyes are lines indicate the effect of glucose on the CBIP system. These resorted to in the glucose solution. lines cannot be observed in similar films exposed just to water 25 To improve the contrast between the different layers, we or in NIP films (neat films, not imprinted). designed an optical microscopy apparatus with modifications FIG. 11 shows a typical sequence of stills from a video of in the sample cell and the optical design, which obviated the the recognition/swelling and rupture of a thin film of a glu need for the dye tracer. By changing the angle of illumination cose-recognitive CBIP film exposed to glucose. Clearly, the of the sample, we found that the contrast achieved between film ruptures with continuous extended cracks. This is a fun 30 the different layers in the recognitive, cracking/rupturing or damental difference over the rupture of CBIP particles (FIG. dissolving polymer was sufficient for their resolution. The 8) that occurs in the form on numerous irregularly shaped optimum angle of illumination depended on the refractive particles. indices of the glucose solution and the CBIP polymer, and Observations of Moving Fronts with Dyes. Trypan blue also on the sample thickness. The sample cell design was also was used in the recognitive solution to better delineate the 35 modified to improve the flow rate control and to allow greater fronts moving into all the particles and to help us observe the precision in measuring the motions of the boundaries of the recognition due to the motion of the glucose Solution and different layers. associated Swelling of particles and continuous films. FIG. 12 Thus, techniques using optical microscopy are good tools shows one such process with a clear indication of the position for observing the different layers as well as the possible of glucose fronts (darker area) as they penetrate in the micro 40 crazing or cracking at the interface of a dissolving CBIP particles. This is a rather simple technique to verify front polymer. Underpolarized light, glucose-sensitive stress lines positions although it is less useful to identify stress lines (the and fractures appear in the presence of a glucose solution. clear indication of glucose action on the samples). This was When the polarizer and analyzer of the polarized microscope achieved with the techniques described below. are crossed, the only light that can be seen is from birefrin Observation of Recognitive Processes Using Polarized 45 gence (as shown in FIG. 13 from a single particle before it has Light. started rupturing). This birefringence is the result of stress Polymer stresses as a result of recognition processes, poly lines caused by glucose expanding the polymer particles. A mer crazing, cracking or dissolution involve glucose or Sol typical sample showing birefringence is seen in FIG. 14 that vent transport into the polymer followed by actual rupture and presents a CBIP particle as it has started to rupture and crack. perhaps dissolution of the latter. Numerous study techniques 50 In this particular study, the ruptured particles appeared after 2 have been reported to study polymer stresses and dissolution. min and 55 sec. The simplest method is a gravimetric technique where poly Observation of Recognitive Processes Using Fluorescent mer specimens are immersed in a solvent and removed after Markers. FITC-glucose provided one of the best ways to fixed time intervals. These specimens are then dried to observe the recognitive process as well as the swelling of the remove residual solvent and the thickness of the remaining 55 particles and continuous films, because fluorescence can be film is measured. Thus, the temporal evolution of the thick readily detected. We have not completed studies in fluores ness is also obtained. Though simple, this method is tedious cent light, but FIGS. 15 and 16 show some of the results. The and almost impossible to use here. brightness of the particles corresponds to the stresses and Laser interferometry has been used to measure the polymer swelling caused by the FITC-glucose (FIG. 15). The swelling dissolution rate of thin films. The technique has found appli 60 generally occurred within 3-5 minutes after contact with the cability especially in following the dissolution of microlitho glucose solution. Recognition and stress development was graphic masking layers. In this study, a recognitive polymer very much dependant on particle size. Particle of 10-15 coated on a silicon wafer is placed in a solvent. When a laser microns were recognitive within 10 seconds! beam impinges upon the polymer Surface, it splits into two Particles appeared to burst in the presence of FITC-glucose beams: one is reflected by the surface and the other penetrates 65 (FIG.16). These bursts could probably be FITC-glucose solu the thin polymer film and is reflected by the silicon wafer. The tion “bubbles' bursting within the particles after they have two beams interfere with each other, providing a measure of had enough stress from Surrounding solution. This proves that US 9,155,703 B2 35 36 the recognition of glucose does in fact lead to enough tion of the swelling process we have identified a “swelling mechanical stress to cause the recognitive particles to break front” which clearly separates the rubbery region (region of apart. This is exactly what the ultimate goal of these studies is: swollen CBIP/HPMC with enough water to have its Tg below to have a recognitive layer with a glucose template that will the experimental temperature) and the glassy region (region burst upon recognition of an abnormal level of glucose and 5 where the CBIP/HPMC has a Tg above the experimental release insulin. temperature). A second front is the “erosion front” which FIG. 17 shows, at a high level, the basic combinations of separates the matrix from the solvent (glucose solution or the present invention. As the skilled artisan will appreciate, water). the molecule that is used to form the micro or nanovacuoles The rupture lines due to the recognition process as well as (shown here as the analyte) can be any of a wide variety of 10 the gel layer formed on the glassy core of a Swellable matrix molecules (or combinations thereof) that can be recognized are considered to be controlling elements of drug release by the polymeric network. Upon exposure the analyte (i.e., kinetics, although other mechanisms may be functional in the recognition event), a polymeric transduction event occurs Such systems. The gel layer structure changes during tablet in which one or more of the listed forces (or even additional Swelling, due to the molecular extension of the Solvated poly forces) trigger a dissociation, degradation, decomposition or 15 meric chains. The diffusion front position in the gel phase otherwise reduce the structural integrity of the polymeric during drug release is usually dependent on drug solubility network, thereby triggering the delivery of a payload. One and loading. In fact, the diffusion front movement can be example of a payload may even be a lower or Subsequent layer related to drug dissolution rate. of polymeric network, which may also include a core on Preparation of Particles from Recognitive Polymers. The which the layers may be disposed. typical procedure of creating the particles from recognitive FIG. 18 is a diagram that shows mixing multilayered polymers includes MAA crosslinked with EGDMA around mimetic structures with different release profiles that allows D-glucose, combined with deionized water, ethanol, and the system to be tailored to fit any release profiles. Mixing DMPA. Generally, the percentages were MAA: 3.25 wt %, four different microcapsules allowed the system to rupture D-glucose: 1.5 wt %, DI water: 31 wt %, Ethanol: 36 wt %, every 3 h for 4 days instead of a system that ruptured every 12 25 EGDMA: 28 wt %, DMPA: <1 wt %. Some solutions con h for 4 days or a system that ruptured every 3 h for one day. tained more ethanol or less EGDMA in order to obtain a more continuous film. Example 10 After the recognitive films were polymerized for 30 min utes in the UV lamp box, they were washed in deionized water Limited Swelling of the Recognitive Polymeric 30 (DI water) for 24-48 hours on average. Once taken out of the Network in Solvent Alone water, the films were placed in ventilated containers (two weigh-boats taped together with holes along top) and placed The configurational biomimetic imprinted polymer in a vacuum oven set at 30 C for at least 24 hours. After the (CBIP) recognitive films prepared in this work included a films were completely dry, they were taken out of the oven crosslinked copolymer of 4.0 ml water, 4.5 ml ethanol, 60 mg 35 and then crushed using a mortar and pestle until a very fine glucose, 0.42 g MAA, 3.1 g TEGDMA, optionally a few powder was produced; the goal was to try and get all the drops of ethanol. Next, 50 mg DMPA was added. The solution crushed particles to be the same size (roughly 100 microme is degassed for 4 minutes and loaded. The polymer is formed ters). by UV irradiation for 5 minutes. The polymer opaque while Preparation of Control Samples. All control samples were film is washed with water and removed from the slide. Wash 40 prepared the exact same way as recognitive polymers, except ing is as described hereinabove. without adding D-glucose to the monomer Solution. The same FIG. 19 shows glucose/water uptake in which glucose procedures were used minus the D-glucose amounts. Solution caused the samples to fall apart quite quickly. The Preparation of Tablets by Compression. There were vari graph shows that the polymer swelled between 5-15 percent ous procedures for forming the mixture used in tablet com (+/-3%) in the presence of a solvent (water) alone. Upon 45 pression. The first few procedures did not form durable, last exposure to the solvent and the analyte the polymeric network ing tablets because the amounts of binders and fillers were too burst to release the payload. At lower glucose concentrations Small or were not in the correct ratio. However, once a pro it is expected that the release can run the time course to cedure produced tablets that remained intact, similar formu equilibrium. las and methods were used. 50 According to the first protocol developed, we produced Example 11 reliable tablets using 53.9 wt % CBIP microparticles, 10.9 wt % microcrystalline cellulose, 34.4 wt % Poly(N-vinyl-2-pyr Recognitive/Responsive Tablet Systems rolidone) (PNVP) K-25, a binder, and less than 1 wt % mag nesium stearate, a lubricant (Table 1). The total weight was Recognitive systems that show diffusional times that are 55 582 milligrams. First, the microparticles of recognitive poly much shorter than for films were development by using mul mers were weighed and mixed with microcrystalline cellu tilayered tablets produced from recognitive particles that had lose in a weigh-boat using a spatula. A solution of 20% PNVP been compressed by 'standard pharmaceutical techniques. was made by combining 4 mg. PNVP in 20 ml deionized Swelling of hydrophilic polymeric tablets has been the sub water. A sample of 1 ml of 20% PNVP solution was added to ject of significant research in the last few years. Of particular 60 weigh-boat drop-by-drop using a 1000-ml micropipette. The interest are studies on the Swelling and Subsequent dissolu mixture was then stirred with the spatula and placed on a tion of hydroxypropyl methyl cellulose (HPMC) tablets. hotplate at a very low temperature to allow liquid to evapo Molecularly, individual chains absorb water so that their rate. Before the mixture was completely dry, it was pushed end-to-end distance and radius of gyration expand to the new through a metal sieve into granules. These granules were then Solvated State. This expansion (Swelling) is observed macro- 65 placed in a ventilated container like before in a vacuum oven scopically by the formation of distinct fronts separating to completely dry. Once the granules were completely dry, the unswollen and Swollen regions. In the macroscopic observa magnesium Stearate was added to the mixture and stirred with US 9,155,703 B2 37 38 a spatula to get a homogenous mixture. This microparticle TABLE 3 mixture was then weighed into 20-23 mg samples that would be the recognitive layers of each tablet. Bovine serum albu Multilayered Tablet Sample No 3 min was weighed into 5-6 mg samples to be used in between Percent the recognitive layers (instead of insulin). 5 Mass (g) (%) The tablets were compressed using a Corning tablet press Microparticles (BLE48) O.03428 86.17396 and a 6.33 mm diameter punch with flat edges; the pressure PNVP (K-25) O.OOSO 12.S6913 was 6000 N/inch. The first recognitive layer was a 20-23 mg Magnesium Stearate O.OOOS 1.256913 sample of microparticle mixture placed inside the lower 10 Total mass O.O3978 100 punch area and compressed; it was then left in the bottom as the additional layers were added. A 5-6 mg sample of bovine serum albumin (BSA) was then poured on top of this first recognitive layer. A Smaller punch was used lightly with a TABLE 4 hand to even out this layer so it is as flat as possible. It was then 15 Multilayered Tablet Sample No 4 compressed with the top punch so that both layers become one unit. This procedure was repeated until 5 recognitive Percent layers and 4 BSA layers were pressed all together (creating a Mass (g) (%) Microparticles O.30096 74.29644 9-layer tablet). Cellulose Microcrystalline O.O2O18 4.98.1732 Similar mixtures were made in Smaller amounts using 66.0 PNVP (K-25) O.08 19.74919 wt % microparticles, 5.9 wt % cellulose microcrystalline, Magnesium Stearate O.OO394 O.972647 27.3 wt % PNVP K-25, and <1 wt % magnesium stearate Total mass O.40508 100 (Table 2). Another mixture was made using 86.2 wt % micro particles, 12.6 wt % PNVP K-25, and 1 wt % magnesium 25 Summary of Prepared Samples. The first set of experimen stearate (Table 3). tal studies produced useful tablets in the form of thick multi From the previous data, a new mixture was made in order layer tablets (minitablets). The total weights and thickness to produce thinner tablets without using a wider punch. This were 134.23 mg, 3.11 mm; 137.2 mg, 3.42 mm; 132.31 mg, mixture included 74.3 wt % recognitive microparticles, 5.0 3.4 mm. For single-layer tablets, the thickness was 0.58 mm, wt % cellulose microcrystalline, 19.7 wt % PNVP K-25, and 30 0.63 mm, 0.68 mm, and 0.65 mm. It was possible to observe <1 wt % Magnesium Stearate (Table 4). The total weight was the different layers and even particulate aggregates in these 405 mg. This mixture followed the same procedures as tablets. These tablets were very sturdy and did not break apart before, except it was completely dry before pushed through when handled. The second and third tablet recipes used the sieve in order to recover more particles/granules. This higher amount of CBIP microparticles. One recipe without time only 7-layer tablets were pressed. In the Bovine Serum 35 microcrystalline cellulose showed promising results using Albumin layers, a 1:1 mixture of Bovine Serum Albumin: less of the mixture. Hydroxypropylmethylcellulose (HPMC) was used. The rec Single-layer tablets made without microcrystalline cellu ognitive layers were between 10-12 mg and the BSA layers lose were thicker than the others because cellulose has a good between 5-6 mg. compressibility. These tablets crumbled faster than other tab 40 lets, possibly because the PNVP was not a sufficient binder to keep all the particles together as one unit. TABLE 1. The tablet recipe with 5 wt % cellulose and 20 wt % PNVP Multilayered Tablet Sample No 1. produced much thinner disks. The total weights and thickness were 58.25 mg, 1.55 mm:59.79 mg, 1.46mm; 58.47 mg, 1.55 Mass Percent 45 mm, 59.27 mg, 1.63 mm, 60.4 mg, 1.73 mm. For single-layer (g) (%) tablets, the thickness was 0.39 mm and 0.34 mm. Microparticle O.31364 53.8771 Most tablets made from the above recipes were sturdy and Cellulose, O.O6345 10.8994 did not crumble when handled; only the verythin single-layer microcrystalline tablets seemed to fall apart when stress was applied. They all PNVP (K-25) O.2OOOO 34.3560 50 had a light-brownish hint to them, probably from the PNVP in Magnesium Stearate O.OOSOS O.86749 the mixture. All edges were distinct and made perfect circles from the punch. Total Mass (g) O.S8214 100 Observation of Swelling Process. One set of experiments was run, in which two single-layer tablets with 10 wt % 55 cellulose and 34 wt % PNVP (first recipe) were put into TABLE 2 separate beakers, one with DI water and the other with 100 mg/dL D-glucose in DI water. These tablets were both Multilayered Tablet Sample No 2 approximately 0.66 mm thick. Both floated on the top of the liquids until rupture and/or swelling. After 20 minutes, the Mass Percent tablets in DI water started crumbling and breaking apart. (g) (%) 60 After 30 minutes, the tablets in 100 mg/dL glucose cracked Microparticles (BLE36) O.O7970 65.96O4 down the middle, but did not crumble and fall to the bottom of Cellulose, microcrystalline O.OO708 5.85947 the beaker. These tablets were still floating and did not crack PNVP (K-25) O.O33OO 27.3110 significantly more after 60 minutes. After 18 hours, both Magnesium stearate O.OO 105 O.86898 tablets had crumbled and landed in big clumps in the bottom Total mass O.12083 100 65 of each beaker. Another set of studies used a 9-layer tablet coated with PVA along the lateral area. This tablet was placed in a 50 ml US 9,155,703 B2 39 40 beaker with 40 ml of 100 mg/dL glucose solution. The tablet TABLE 7 was dropped into the beaker so that one of the recognitive sides was touching the bottom. After about 6 minutes, the first Data for mixture BLE18, A, B, and C. recognitive layer absorbed glucose and started responding by cracking and Swelling along top half began Swelling. Tiny 5 % mol bubbles were observed on the tablet, but it is not clear whether # moles (chemicals) % Wiw these were from the inside of tablet or formed after the tablet was dropped into the solution. After 13 minutes, a large piece Added to A (g) crumbled off the top and landed on the bottom. After several hours, the tablet had disintegrated. There were little pieces MAA O431 O.OOSOO8 42.18806139 3.95790588 10 D-glucose O.1847 O.OO1025 1.696.11419 scattered all along the bottom. DI water 4.8OO38 O.266,392 44.08.225575 TABLE 5 ethanol 4.1998.47 O.O91162 38.5675154 EGDMA 1.235 O.OO6712 S6. S4O90759 11.34109922 Below are the data regarding the novel multi-layer recognitive Systems: DMPA O.O3867 O.OOO151 1.271031026 O3SS10956 Multilayer Multilayer Multilayer 15 total mass: 10.889597 Tablet 1 Tablet 2 Tablet 3 Laver Mass (mg) Mass (mg) Mass (mg) moles: O.O11871 y 9. 9. 9. Added to B (g) Disk 1 21.6 23.45 22.54 Bovine Album in 1 5.8 6.84 6.25 MAA O438 O.OOSO89 27.72S81374 3.6254.11552

E.OWile Albuminill 2 2. 2. 2. 20 D-glucose O.1909S O.OO106 1.58053O447 Disk 3 21.74 22.7 20.67 DI water 4.8OO38 O.266,392 39.7336829 Bovine Albumin 3 6.22 6.33 5.83 ethanol 4.1998.47 O.O91162 34.76295396 Disk 4 2212 21.46 21.68 EGDMA 2.4129 O.O13114 71.4386.1612, 19.972O446 Bovine Albumin 4 6.8 5.64 5.95 DMPA O.O3931 O.OOO153 0.835570147 0.32S376548 Disk 5 2281 22.63 21.04 25 o

Total mass (mg) 134.23 137.2 132.31 totalO88SS : 12081387 Thickness (mm) 3.11 3.42 3.4 Ole:S O.O18356 chemicals: Added to C (g) 30 TABLE 6 MAA O4236 O.004922, 19.94.8681.58 3.1950O216 D-glucose O.18901 O.OO1049 14256O755 Formula for each disk DI water 4.80O38 0.266392 36.206.85663 Mass (g) Percent ethanol 4.1998.47 O.O91162 31.67733,767 EGDMA 3.6063 O.O19599 79.4334.8508. 27.2005106 CBIP microparticle O31364 53.877O7 3 DMPA O.O3907 O.OOO152 0.617833.338 0.294685398 cellulose, microcrystalline O.O6345 10.89944 o PNVP (K-25) O.2 34.356 Magnesium Stearate O.OOSOS O.86.7489 totalO88SS : 13.2582O7 o moles O.O24674 Total Mass (g) O.S8214 chemicals: 40

TABLE 8 Data Recipes for other various mixtures: Added (g) Added (ml) # moles % mol (chemicals) % Wiw MW g/mol BLE4 recipe from 051707 (Zach Hilt's original recipe)

MAA O415 O.OO4822 2O.O3793222 3.SS11364 86.06 D-glucose O.1796 O.OOO997 15368291 18O16 DI water 3.992 4 O.221532 34.15936S 18.02 ethanol 3.5505 4.5 O.O77O68 30.381469 46.07 EGDMA 3.519 O.O1912S 79.4708O132 30.111925 184 DMPA O.O303 O.OOO118 O491.266461 0.2592757 256.29

total mass: 11.6864 moles chemicals: O.O2406S BLE36 CBIP made 061207

MAA O.41635 O.OO4838 20.1081.614 3.5629108 D-glucose O.18132 O.OO1 OO6 1551644 DI water 3.992 4 O.221532 34.161499 ethanol 3.5505 4.5 O.O77O68 30.383367 EGDMA 3.S145 O.O19101 79.3890966 30.075.297 DMPA O.O31 O.OOO121 O.SO2741999 O.2652822

total mass: 11.68567 moles chemicals: O.O24059 US 9,155,703 B2 41 42 TABLE 8-continued Data/Recipes for other various mixtures: Added (g) Added (ml) # moles % mol (chemicals) % ww MW g/mol BLE48 CBIP made 062007

MAA O.836 O.OO9714 20.OS1271S2 3.568.8992 D-glucose 0.35971 O.OO1997 1.53S6085 DI water 7.984 8 O.443063 34.083841 ethanol 7.101 9 O.15413S 30.314298 EGDMA 7.0832 O.O38496 79.46OO1886 30.238.309 DMPA O.O6068 O.OOO237 O4887.09626 O.259044 total mass: 23.42459 moles chemicals: O.048.447 Recipe for BLE62 CBIP made 070507

MAA O4495 O.OOS223 22.7023S1 3.SS44049 D-glucose 0.1898 O.OO 1054 1. SOO8366 DI water 3.992 4 O.221532 31.566595 ethanol 4.734 6 0.102757 37.433.933 EGDMA 3.2499 O.O17663 76.770SSO44 25.6984.66 DMPA O.O3108 O.OOO121 0.527098.559 O.245,764 total mass: 12.64628 moles chemicals: O.O23007

25 Preparation of Samples. The control samples for these TABLE 9-continued studies with multilaminate systems involved the creation of a 7- or 9-layer “sandwich' similar in composition to the mul- Layer Amount tilayer tablet Systems using continuous films or discs (i.e., Disk 2 0.00659 g recognitive layer, followed by the bovine serum albumin, 30 BSA 2 0.00294g followed by another recognitive layer, etc. . . . ). In this case, Disk3 0.00626g however, instead of the recognitive layers, non-imprinted Rs. 2 8. polymer film layers were used (thus preventing any specific BSA 4 0.00512g recognition of glucose). Top Disk 5 0.00627g Preparation of Multilaminate Systems. The multilaminate 35 systems were developed in the same way as the multilayer tablets, with alternating layers of recognitive film and bovine TABLE 10 serum albumin (used in our experiments in the place of a drug Such as insulin). Layer Amount The just-washed polymer films (created using the protocol 40 discussed in Section I of this report) were cut into 8 mm Bottom Disk 1 0.00502 g (diameter)diameter) circcircles using9. a cork borer- 0 and were SubsequentlC y DiskBSA 21 0.00634g0.00500 g dried in the vacuum oven. The dried circles were then stacked BSA 2 0.00504g together (in a dry environment at room temperature) with a Disk3 0.00673 g layer of bovine albumin serum in between each one (used in 45 BSA 3 0.00396 g our experiment in the place of a drug such as insulin).- 0 Once DiskBSA 4 0.002530.00679g g the 7- or 9-layer stack was formed, room temperature Vulca- Top Disk 5 0.00659 g nized (RTV) rubber was applied along the circumference of the stack using a toothpick, after which the system was left to Each of the BSA layers was evenly dispersed upon the dry for at least 24 hours. 50 Parameters and Layers. The structure of the multilaminate polymer disk below it using a spatula. The RTV rubber was system. In these studies, Small amounts of bovine serum then applied as needed. The multilaminate systems that we albumin were used sufficient to fully cover the discs of the created were visibly much bulkier than we would prefer; the recognitive films below them. The amount of BSA/drug to be difficulty of applying such a course material as RTV rubber placed between recognitive layers will depend on the rate of 55 makes the tablet appear messy and mechanically rigid. Swell diffusion of water inward and the rate of disintegration of the ing Studies and Release Studies. Studies were done on the recognitive layers (among many other things), but for now our Swelling and release of these multilaminate systems. These emphasis was on validating the proof of concept of a multi studies show the release of bovine serum albumin from glu layered system comprised of continuous films. cose-recognitive planar multilayered systems. FIG. 20 shows The following amounts were used: 60 the behavior of such systems in a 100 mg/dL solution of glucose. Clearly, all incorporated BSA was released in two TABLE 9 pulses. The recognitive layers correspond to regions AB and CD while the releasing layers correspond to regions BC and Layer Amount DE. FIG. 21 shows on embodiment of a multilayer tablet. Bottom Disk 1 0.00728 g 65 It is contemplated that any embodiment discussed in this BSA 1 0.00061 g specification can be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. US 9,155,703 B2 43 44 Furthermore, compositions of the invention can be used to 2. F. Breinl and F. Haurowitz, Chemical Investigation of the achieve methods of the invention. Precipitate from Hemoglobin and Anti-hemoglobin Serum It will be understood that particular embodiments and Remarks on the Nature of Antibodies. Z. Physiol. described herein are shown by way of illustration and not as Chem., 1930. 192: p. 45. limitations of the invention. The principal features of this 5 3. L. Pauling, A Theory of the Structure and Process of For invention can be employed in various embodiments without mation of Antibodies. J. Am. Chem. Soc., 1940. 62: p. departing from the scope of the invention. Those skilled in the 2643-2657. art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific 4. N. K. Jerne. The Natural Selection Theory of Antibody procedures described herein. Such equivalents are considered Formation. Proc. Natl. Acad. Sci. USA, 1955. 41: p. 849. to be within the scope of this invention and are covered by the 10 5. D. W. Talmage, Allergy and Immunology. Ann. Rev. Med., claims. 1957.8: p. 239. All publications and patent applications mentioned in the 6. F. M. Burnet, A Modification of Jerne's Theory of Anti specification are indicative of the level of skill of those skilled body Production Using the Concept of Clonal Selection. in the art to which this invention pertains. All publications and Aust. J. Sci., 1957. 20: p. 67. patent applications are herein incorporated by reference to the 15 7. D. R. Davies and S. Chacko, Antibody Structure. Accounts same extent as if each individual publication or patent appli Chem. Res., 1993. 26: p. 421-427. cation was specifically and individually indicated to be incor 8. N. K. Jerne, The Generative Grammar of the Immune porated by reference. System, in Nobel Lectures, Physiology or Medicine 1981 The use of the word “a” or “an' when used in conjunction 1990, J. Lindsten, Editor. 1993, World Scientific Publish with the term “comprising in the claims and/or the specifi ing Co.: Singapore. p. 211-225. cation may mean "one.” but it is also consistent with the 9. G. Köhler and C. Milstein, Continuous cultures of fused meaning of “one or more.” “at least one.” and “one or more cells secreting antibody of predefined specificity. Nature, than one.” The use of the term 'or' in the claims is used to 1975. 256: p. 495-497. mean “and/or unless explicitly indicated to refer to alterna 10. G. J. F. Köhler, Derivation and Diversification of Mono tives only or the alternatives are mutually exclusive, although 25 clonal Antibodies, in Nobel Lectures, Physiology or Medi the disclosure supports a definition that refers to only alter cine 1981-1990, J. Lindsten, Editor. 1993, World Scientific natives and “and/or.” Throughout this application, the term Publishing Co.: Singapore. p. 228-243. “about is used to indicate that a value includes the inherent variation of error for the device, the method being employed 11. C. 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Soc., 1964.86: p. cited elements or method steps. 3O4. The term “or combinations thereofas used herein refers to 14. R. B. Merrifield, Solid-Phase Peptide Synthesis, III. An all permutations and combinations of the listed items preced 40 Improved Synthesis of Bradykinin. Biochem., 1964. 3: p. ing the term. For example, A, B, C, or combinations thereof 1385-1390. is intended to include at least one of A, B, C, AB, AC, BC, or 15. B. Merrifield, Solid Phase Synthesis, in Nobel Lectures, ABC, and if order is important in a particular context, also Chemistry 1981-1990, T. Frängsmyr, Editor. 1992, World BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing Scientific Publishing Co.: Singapore. p. 149-175. with this example, expressly included are combinations that 45 16. K. Kirshenbaum, A. E. Barron, R. A. Goldsmith, P. contain repeats of one or more item or term, such as BB, Armand, E. K. Bradley, K. T. V. Truong, K. A. Dill, F. E. AAA, MB, BBC, AAABCCCC, CBBAAA, CABABB, and Cohen, and R. N. 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