Novel Amphiphilic Block Copolymers and Their Self-Assembled Injectable Hydrogels for Gene Delivery" (2007)
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Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 2007 Novel amphiphilic block copolymers and their self- assembled injectable hydrogels for gene delivery Ankit Agarwal Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Cell Biology Commons, and the Chemical Engineering Commons Recommended Citation Agarwal, Ankit, "Novel amphiphilic block copolymers and their self-assembled injectable hydrogels for gene delivery" (2007). Retrospective Theses and Dissertations. 15544. https://lib.dr.iastate.edu/rtd/15544 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Novel amphiphilic block copolymers and their self-assembled injectable hydrogels for gene delivery by Ankit Agarwal A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Chemical and Biological Engineering Program of Study Committee: Surya K Mallapragada, Major Professor Balaji Narasimhan Jacqueline V Shanks Marit Nilsen-Hamilton Michael J. Wannemuehler Iowa State University Ames, Iowa 2007 Copyright © Ankit Agarwal, 2007. All rights reserved. UMI Number: 3283092 UMI Microform 3283092 Copyright 2008 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, MI 48106-1346 ii To my parents and grand parents… iii TABLE OF CONTENTS ACKNOWLEDGMENTS viii ABSTRACT ix CHAPTER 1 GENERAL INTRODUCTION 1 Thesis organization 5 References 7 Figures 9 CHAPTER 2 SYNTHETIC SUSTAINED GENE DELIVERY 11 SYSTEMS Abstract 12 Outline 12 1 Introduction 12 2 Design parameters for controlled release systems 15 3 Delivered Factors 17 4 Different modalities of sustained delivery devices 18 4.1 Nano-sphers 19 4.2 Micro-spheres 21 4.3 Hydrogels 27 4.3.1 In-situ forming hydrogels 28 4.3.2 Implantable hydrogels 32 4.3.2.1 Chemically-crosslinked hydrogels 32 4.3.2.2 Photo-crosslinked hydrogels 33 4.4 Implantable scaffolds 35 4.4.1 Biodegradable matrices 36 4.4.2 Synthetic scaffolds 38 4.4.3 Substrate Immobilization 40 5 Applications 41 5.1 Cancer therapy 42 5.2 Vaccination 43 5.3 Tissue engineering 44 6 Conclusions 46 Table 1 47 References 48 Figure 1 59 CHAPTER 3 NOVEL CATIONIC PENTABLOCK COPOLYMERS 61 AS NON-VIRAL VECTORS FOR GENE THERAPY Abstract 61 1 Introduction 62 iv 2 Materials and Methods 63 2.1 Materials 63 2.2 Plasmid DNA 64 2.3 Synthesis of pentablock copolymers 64 2.4 Cell Culture 64 2.5 Polymer-DNA complexes (Polyplexes 65 2.6 Molecular weight analysis and particle size determination 65 2.7 Gel retardation assay 66 2.8 Nuclease resistance assay 66 2.9 Cytotoxicity assay 67 2.10 In-vitro transfection 66 2.10.1 Detection of Green Fluorescent Protein 68 2.10.2 Detection of Luciferase activity 69 3 Results and Discussion 69 3.1 Molecular weight analysis and particle size 69 3.2 DNA complexation 71 3.3 Protection against nuclease degradation 71 3.4 Cytotoxicity 72 3.5 In-vitro transfection 73 3.5.1 Green Fluorescence Protein assay 73 3.5.2 Luciferase assay 74 4 Conclusions 75 Acknowledgements 76 References 76 List of Tables 79 List of Figures 80 CHAPTER 4 INVESTIGATION OF IN VITRO 93 BIOCOMPATIBILITY OF NOVEL PENTABLOCK COPOLYMERS FOR GENE DELIVERY Abstract 93 1 Introduction 94 2 Materials and Methods 96 2.1 Materials 97 2.2 Polymers 97 2.3 Cells 97 2.4 Plasmid DNA 97 2.5 Polyplexes 98 2.6 Lactate dehydrogenase (LDH) assay 98 2.7 MTT assay 99 2.8 Microscopic observations 99 2.9 Agarose diffusion assay 100 2.10 Characterization of polymer induced cell death 101 2.11 Luciferase Transfection 102 2.12 Statistics 103 v 3 Results 103 3.1 Effect of different wt% of PDEAEM on pentablock copolymer 103 cytotoxicity 3.2 Toxicity increases with exposure time 104 3.3 Apoptosis vs necrosis 105 3.4 Luciferase Transfection 106 3.5 Polymer gel cytotoxicity studies 108 4 Discussion 108 5 Conclusions 114 Acknowledgements 115 References 116 List of Tables 120 List of Figures 122 CHAPTER 5 MORPHOLOGY OF THE PLASMID DNA 137 CONDENSATES OF NOVEL CATIONIC AMPHIPHILIC COPOLYMERS AND THEIR INTRA-CELLULAR TRAFFICKING Abstract 138 1 Introduction 139 2 Materials and Methods 140 2.1 Materials 140 2.2 Polymers 141 2.3 Cells 141 2.4 Plasmid DNA 142 2.5 Polyplexes 142 2.6 Cryo-TEM 143 2.7 Labeling 144 2.8 Confocal microscopy 144 3 Results 145 3.1 Morphology 145 3.2 Intra-cellular trafficking 147 3.3 Transfection efficiency 151 4 Conclusions 152 Acknowledgements 152 References 152 List of Figures 156 CHAPTER 6 COLLOIDAL STABILITY AND TRANSFECTION 173 EFFICIENCY OF SELF-ASSEMBLING COPOLYMERIC GENE DELIVERY VECTORS IN THE PRESENCE OF SERUM Abstract 173 1 Introduction 174 vi 2 Materials and Methods 177 2.1 Materials 177 2.2 Polymers 177 2.3 Plasmid DNA 178 2.4 Polyplex formulation 178 2.5 Cell line 179 2.6 DNA condensation, nucleases resistance, serum stabilization 179 2.7 Particle size and zeta(ζ) potential 180 2.8 Cryo-TEM 181 2.9 Transfection and cytotoxicity 181 2.10 Statistics 182 3 Results and discussion 183 3.1 Colloidal stability 183 3.2 DNA integrity and protection 185 3.3 Transfection and cytotoxicity 187 4 General discussion 189 5 Conclusions 193 Acknowledgements 194 References 194 List of Figures 198 CHAPTER 7 SUSTAINED RELEASE OF NOVEL 214 POLYMER/DNA COMPLEXES FROM THEIR INJECTABLE SELF-ASSEMBLED THERMO- REVERSIBLE HYDROGELS Abstract 214 1 Introduction 215 2 Materials and Methods 218 2.1 Materials 218 2.2 Polymer synthesis 219 2.3 Plasmid DNA 219 2.4 Nanoplex formulation 219 2.5 Hydrogel formulation 220 2.6 Dissolution study 221 2.7 Dynamic Mechanical Analysis 221 2.8 Particle size and zeta potential 222 2.9 Agarose gel electrophoresis 223 2.10 Cell line 223 2.11 Transfection efficiency 223 2.12 Statistics 224 3 Results and discussion 224 3.1 Dissolution profile 224 3.2 Mechanical strength 226 3.3 Stability of polyplexes 227 vii 3.4 Transfection efficiency 228 4 General discussion 229 5 Conclusions 232 Acknowledgements 232 References 232 List of Tables 236 List of Figures 238 CHAPTER 8 CONCLUSIONS 244 CHAPTER 9 FUTURE DIRECTIONS 249 CURRICULUM VITA 253 viii ACKNOWLEDGMENTS I must acknowledge that the dissertation presented here is result of not just my efforts, but of many those who are close to me. First, I need to sincerely thank Dr Surya K Mallapragada for the being my mentor, for teaching me to be self-sufficient, and for her intense supervision while also providing all the freedom to carry out the research work. Her enthusiasm was infectious; I could always find her for encouragement and guidance. Thank you. I am extremely thankful to Dr Robert C Unfer at Iowa Cancer Research Institute in whose collaboration this research work was conducted. He taught me a lot of cell culture techniques, devoted countless hours on weekends and evenings to the in vivo studies in mice, and was always there to offer sincere advice. Thanks goes to Dr Yeshayahu Talmon, and Rita Vilensky of Technion- Israel Institute of Technology, Haifa for their help with cryo-TEM imaging. I also want to thank my committee members Balaji Narasimhan, Jacqueline V Shanks, Marit Nilsen-Hamilton, and Michael J. Wannemuehler for their guidance and suggestions that significantly helped me improve the presented research work. Sincere thanks to the members of SKM research group and CBE department for offering their suggestions, encouragement and friendship. I am particularly grateful to Saikat Adhikari, Dinesh Yeragi, Michael Determan, Mathew Kipper, Sikander Hakim, Madhuresh Chaudhary and Maria Torres. My other friends who invested in my life at Iowa State beyond career and academics- Lorena Moeller, Anup Gokarn and Rajneeshwer Nath. Thanks to you for your enduring friendship. Most importantly I want to acknowledge Innan Cervantes- Martinez for her emotional support, encouragement and love that were instrumental in working through every difficulty. Thank you. Last, but not the least, I would like to thank my family for their unconditional love and for teaching me how to dream. Especially, my late grandfather who motivated me to become a good man in life and accomplish big, my father who boosted my morale in all difficult times and infused strength with his support, my brother Vaibhave for providing inspiration to do hard work, and my lovely mother who let me go thousand miles away from her to realize my dreams. Without them, I would not have come to be the person I am today. ix ABSTRACT This work describes the development and investigation of a family of novel “smart” copolymers as non-viral gene delivery vectors. The copolymers have five blocks, and thus named pentablock, with a central block of a hydrophobic polymer, surrounded by two blocks of a hydrophilic polymer, and capped at each terminal end with cationic polymer blocks, arranged in an architecture to provide temperature and pH sensitivity to the copolymers. They are derived from commercially available triblock Pluronic copolymers. The cationic copolymers can efficiently condense negatively charged plasmid DNA in nanostructures with efficient cellular uptake. The amphiphilic nature of copolymers causes them to exist as micelles in aqueous solutions that help them traverse cellular membranes with minimal cell membrane damage.