Novel amphiphilic block copolymers and their self-assembled injectable hydrogels for gene delivery

264 Pages·2016·10.01 MB·English

by  Ankit Agarwal

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
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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
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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
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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
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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
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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.