Development of Novel PLGA Contrast Agents for Use As Ultrasound
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Development of Novel PLGA Contrast Agents for Use as Ultrasound Targeted Drug Delivery Vehicles A Thesis Submitted to the Faculty of Drexel University by Dalia El-Sherif in partial fulfillment of the requirements of the degree of Doctor of Philosophy May 2003 ÓCopyright 2003 Dalia El-Sherif. All Rights Reserved ii Dedications I dedicate this Ph.D. thesis to my parents, Drs Mahmoud El-Sherif and Jeylan El- Mansoury, and to my husband, Saif Khalil, who have been supportive, motivating, and encouraging throughout the course of my Ph.D. work. iii Acknowledgements The list of those that I would like to acknowledge seems to be endless; however there are those who provided support, without which this PhD thesis would not have been possible: Dr. Margaret Wheatley and my committee members, Drs. Peter Lewin, Flemming Forsberg, Fred Allen, and Ronald Tallarida for their guidance, advice and support The Thomas Jefferson University team, especially Dr. Flemming Forsberg, Dr. Bill Shi, and Raymond Ro, for their help with all the in vivo, attenuation and pulse length studies of this research. Their input has been enriching and has added great value to my thesis Dr Peter Lewin and Dr. Raghue Basude, for their help with the in vitro acoustics portion of this research My graduate student colleagues, especially Nikhil Dhoot and Tom Dzuible for their research input and support through rough times My husband Saif Khalil, for his constant support, patience, and loving encouragement My parents, Dr. Mahmoud El-Sherif and Dr. Jeylan El-Mansoury, for encouraging me to go to my limits and to achieve my goals iv Table of Contents List of Tables ..................................................................................................................x List of Figures ................................................................................................................xi Abstract.......................................................................................................................xvii 1. INTRODUCTION..................................................................................................1 1.1. LONG TERM GOALS ........................................................................................1 1.2. THESIS OBJECTIVES .......................................................................................3 2. BACKGROUND AND LITERATURE REVIEW..................................................5 2.1.Diagnostic Imaging.................................................................................................5 2.1.1. Imaging Modalities – Overview ...............................................................5 2.1.2. Ultrasound................................................................................................8 2.1.3. Contrast Agents........................................................................................9 2.2.MICROENCAPSULATION AND DRUG DELIVERY .......................................12 2.2.1. Microencapsulation Techniques .............................................................13 2.2.2. Shell Materials .......................................................................................14 2.2.3. PLGA [poly (lactic-co-glycolic)acid] .....................................................15 2.2.4. PLGA Microcapsule Degradation...........................................................15 2.2.5. Encapsulation and Release of Therapeutics from PLGA Microcapsules ........................................................................................17 2.2.6. Ultrasound Induced Drug Delivery.........................................................18 2.3.CANCER .............................................................................................................19 2.3.1. Anticancer Agents..................................................................................21 2.3.1.1. Doxorubicin (Adriamycin)..........................................................21 v 2.3.1.2. Paclitaxel (Taxol) .......................................................................22 2.3.1.3. Controlled Delivery of Paclitaxel and Doxorubicin .....................23 2.3.2. Nano Applications and Targeting ...........................................................23 2.4.CONTRAST AGENTS AS TARGETED DRUG DELIVERY VEHICLES..........25 2.5.THESIS OVERVIEW...........................................................................................26 3. EXPERIMENTAL METHODS (CONTRAST AGENT FORMULATION AND ANALYSIS) ...............................................................................................32 3.1.POLYMER HANDLING AND STORAGE .........................................................32 3.2.AGENT FABRICATION .....................................................................................33 3.2.1. Native Agent by Double Emulsion .........................................................34 3.2.2. Phase I Agent by Encapsulation of One (Non-Water Soluble) Sublimable Core Material.........................................................40 3.2.3. Phase II Agent by Encapsulation of Two (Water and Non-Water Soluble) Sublimable Core Materials.....................................41 3.2.4. Solvent Extraction – Encapsulating Solid Ammonium Carbonate...........42 3.2.5. Coacervation – Encapsulating Solid Ammonium Carbonate...................43 3.3.ACOUSTIC STUDIES.........................................................................................42 3.3.1. In Vitro Acoustic Testing .......................................................................43 3.3.1.1. In Vitro Dose Response ..............................................................46 3.3.1.2. In Vitro Time Response ..............................................................47 3.3.1.3. In Vitro Attenuation....................................................................48 3.3.2. In Vivo Acoustic Testing........................................................................49 3.3.2.1. In Vivo Dose Response...............................................................49 3.3.2.2. In Vivo Time Duration................................................................50 vi 3.4.CROSS SECTION OF THE POLYMER CONTRAST AGENT...........................50 3.4.1. Embedding in Epoxy and Cross Sectioning ............................................50 3.4.2. Embedding in Epoxy and Polishing........................................................51 3.4.3. Probe Sonication ....................................................................................51 3.5.MICROSCOPY ....................................................................................................51 3.6.CAPSULE DEGRADATION SET-UP.................................................................52 3.7.HPLC ANALYSIS ...............................................................................................53 3.8.Particle Size Distribution ......................................................................................53 4. RESULTS AND DISCUSSION (CONTRAST AGENT FABRICATION AND ANALYSIS) ...............................................................................................55 4.1.A NOVEL METHOD FOR SYNTHESIS OF A POLYMERIC CONTRAST AGENT ...............................................................................................................55 4.1.1. Encapsulation of Solid Ammonium Carbonate .......................................55 4.1.1.1. Challenges Presented by Encapsulating Solid Ammonium Carbonate Particles ......................................................................56 4.1.1.2. Testing to Determine Encapsulation and Sublimation of Ammonium Carbonate .................................................................59 4.1.1.3. Testing to Determine Solvent Effects on Ammonium Carbonate.....................................................................................60 4.1.2. Fabrication of Native Agent by a W/O/W Double Emulsion Process ......60 4.1.2.1. Properties of the Selected Polymers..............................................61 4.1.2.2. Properties of the Selected Surfactant ............................................63 4.1.2.3. Polymer and Surfactant Combinations Used to Fabricate Native Agent ...........................................................................................64 4.1.2.4. Acoustic Testing of Two Possible Native Microcapsules..............67 4.1.3. Fabrication of PLGA Contrast Agents....................................................70 4.1.3.1. Fabrication of Phase I Contrast Agent.........................................70 vii 4.1.3.1.1. Optimal Amount of Camphor for Encapsulation (Comparison to DL25 Native Microcapsules)...............71 4.1.3.1.2. Encapsulation of Camphor (Comparison to DL6 Native Microcapsules)..................................................72 4.1.3.1.3. Acoustic Characterization of Phase I Agents.................73 4.1.3.2. Fabrication of Phase II Contrast Agent........................................74 4.1.3.2.1. Physical Characterization...............................................75 4.1.3.2.2. Effects of PVA Temperature..........................................76 4.1.3.2.3. In Vitro Acoustic Testing of Phase II Contrast Agents ...........................................................................77 4.1.4. Summary of Acoustic Enhancement Properties for Different Polymeric Contrast Agents.....................................................................78 4.2.CROSS-SECTION OF PLGA CONTRAST AGENT ...........................................80 4.2.1. Embedding in