Complexation of Cationic Polymers with Nucleic Acids for Gene Delivery A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Engineering and Physical Sciences 2014 Amy Freund School of Physics and Astronomy 3 Abstract The prospect of gene delivery for therapeutic purposes has created much hope for the treatment and prevention of diverse illnesses, both inherited and environmental. One major challenge for the realisation of this potential is the availability of a suitable vector for trafficking foreign DNA into a cell. Many viruses are effective but carry unacceptable risks of dangerous host immune response or infection. Cationic polymers have been identified as promising candidates to neutralise the DNA's negative charge and gain entry to the cell. In this work a structural study was conducted on a range of established and novel cationic polymer vectors in complexation with nucleic acid molecules for gene delivery. The high neutron flux of the recently available SANS2D instrument on the second target station at the ISIS pulsed neutron source was used in conjunction with a stopped-flow apparatus to study the static and kinetic structure of aggregates of complexes formed between different architectures and molecular weights of polyethylenimine, a commonly used, highly cationic polyelectrolyte, and nucleic acids. Findings indicated the stability of high molecular weight branched polyethylenimine was superior to any other architecture studied. Subsequently, the zeta potential of these polymers complexed with biologically relevant siRNA molecules was studied, which suggested was established that aggregation still proceeds, even with the most apparently stable of the complexes. Finally, structural studies of the complexation between a family of synthetic, biocompatible cationic block copolymer vectors which incorporate a stabilising, hydrophilic conjugate block, designed to mitigate aggregation of the extent encountered by PEI complexes upon binding are described. The variation of neutron scattering with charge ratio of polymer to DNA was elucidated for this family of systems. 5 "Wir m¨ussenuns daran erinnern, daß das, was wir beobachten, nicht die Natur selbst ist, sondern Natur, die unserer Art der Fragestellung ausgesetzt ist." W. Heisenberg, "Physik und Philosophie" I gratefully acknowledge significant help and guidance from Prof. Jian Lu, Dr XiuBo Zhao, Dr Fang Pan and Dr Mohammed Yaseen. I also thank Dr Henggui Zhang, Dr Tom Waigh, Dr Salman Rogers, Dr Steven Magennis, David Pearce, Dr Paul Coffey, Faheem Padia, Jamie Fearnley and other members of the Biological Physics Group and others at the University of Manchester for helpful discussions. Additionally, my thanks to Eric Brunner, Dr Stuart Fisher, Dr Liam O'Ryan, Dr Natasha Fox, Elliot Woods and Roger Goldsbrough for their considerable advice, support and assistance. I am grateful to the EPSRC DTA and John and Stephanie Forrest for their financial support. The help of Dr. Sarah Rogers and Dr Richard Heenan for technological work, support, guidance and assistance with preliminary data analysis on SANS data was invaluable, and without whom the SANS work would not have been possible, as well as Dr Ann Terry and Dr Stephen King. Importantly, and not exclusive of those mentioned above, I am extraordinarily grateful to my friends and family for their ongoing support. Table of Contents Table of Contents 1 1 Introduction to Gene Delivery and Study Motivation 9 1.1 Motivation for Complexation . 10 1.1.1 Charge Neutralisation . 11 1.1.2 Cellular Targeting, Extracellular Obstacles and Cellular Entry . 12 1.1.3 Endosomal Escape . 14 1.1.4 Intracellular Trafficking . 15 1.1.5 Protection from Enzymatic Degradation . 15 1.1.6 Dissociation and Transcription . 16 1.1.7 Nuclear Localisation and Entry . 16 1.1.8 Stability of Expression . 17 1.2 Gene Delivery Applications . 17 1.2.1 Plasmid DNA and Underexpression . 18 1.2.2 Overexpression and Downregulation by ODN or siRNA . 19 1.2.3 In Vivo Gene Delivery - Additional Complications . 21 1.3 Existing Transfection Methods . 22 1.3.1 Physical Techniques . 22 1.3.2 Viral Vectors . 22 1.3.3 Non-viral Vectors . 23 1.4 Aim of Research . 25 1.4.1 Structural Evolution of Cationic Polymer Complexes with DNA . 26 1.4.2 The Impact of Cationic Polymer Structure and Molecular Weight on Zeta Potential . 28 1.4.3 The Structure of Complexes of Cationic Diblock Copolymers with ODNs and Plasmid DNA . 28 2 Relevant Polyelectrolyte Theory and Focus of Study 31 2.1 Fundamental Polymer Theory . 31 2.1.1 Polymers . 31 2.1.2 Polyelectrolyte Binding Models and Complexation Kinetics . 35 2.1.3 Complex Structure and Charge Ratio . 38 1 2 Table of Contents 2.1.4 Solvents, Solubility and Colloidal Stability . 39 2.2 Areas of Interest . 40 3 Relevant Experimental Techniques and Associated Theory 41 3.1 Sample Preparation and Characterisation . 41 3.1.1 Glassware . 41 3.1.2 Nucleic Acids Preparation . 42 3.1.3 Cationic Polymer Preparation . 46 3.1.4 MPC-DEA Copolymers . 46 3.1.5 Polyethylenimine . 46 3.1.6 pH Measurement in H2O and D2O................... 47 3.2 Physical Techniques . 48 3.2.1 Size and Structure . 48 4 Static and Kinetic SANS Study of Polyethylenimine-DNA Complexes 65 4.1 Introduction . 65 4.1.1 Background and Relevance . 65 4.1.2 Choice of Technique . 67 4.1.3 Polyethylenimine as Transfection Agent . 69 4.1.4 Principal Questions . 72 4.1.5 A Notable Study . 77 4.1.6 Selection of the System and Expected model . 78 4.2 Materials and Methods . 79 4.2.1 DNA Preparation . 79 4.2.2 DNA Characterisation . 81 4.2.3 PEI Preparation . 83 4.2.4 pH Calculation in D2O and H2O.................... 85 4.2.5 Solvent Media Selection . 86 4.2.6 Complex Formation . 87 4.2.7 Stopped-flow Configuration . 88 4.2.8 Time Resolution and Complex Age Span . 91 4.2.9 SANS Conditions . 92 4.3 Analysis . 93 4.3.1 Static Analysis . 94 4.3.2 Kinetic Analysis . 108 4.4 Results . 110 4.4.1 Static Data . 111 4.4.2 Kinetic Data . 141 4.4.3 Conclusion - Principal Findings and Their Relevance . 162 4.4.4 Discussion and Citical Evaluation . 165 4.4.5 Further Work . 175 5 Zeta Potential Study of Polyethylenimine Complexes with short in- terfering RNA 179 5.1 Introduction . 179 Table of Contents 3 5.1.1 Relation to SANS and relevant questions . 179 5.1.2 Zeta potential and surface charge . 180 5.2 Theory . 180 5.2.1 SiRNA Mechanism and Applications . 180 5.2.2 Cationic Polymers . 180 5.2.3 Biophysical Similarity to DNA fragments . 181 5.2.4 Importance of Complexation and Optimal Conditions for Successful siRNA Delivery . 182 5.2.5 Zeta Potential . 182 5.2.6 Principle research questions . 184 5.3 Materials and Methods . 185 5.3.1 Polymer Preparation . 185 5.3.2 siRNA preparation . 185 5.3.3 Zeta Potential measurement . 185 5.4 Results and Discussion . 186 5.4.1 Interpretation and relevance . 186 5.5 Future Work . 190 6 SANS Study of DNA Complexation with Cationic Diblock Copolymers193 6.1 Introduction . 193 6.1.1 Nucleic Acids Complexation and Vector Design . 193 6.1.2 Polymer Structure . 194 6.1.3 DNA Types . 194 6.1.4 Preliminary DLS Screening . 194 6.1.5 SANS and structural investigation . 195 6.1.6 Requirements of Complexation for Different Types of DNA . 195 6.1.7 Factors of Importance and Principle Research Questions . 196 6.2 SANS Theory . 197 6.3 Materials and Methods . 198 6.3.1 Sample Preparation . 199 6.3.2 Guinier Analysis . 200 6.3.3 FISH Model Fitting . 200 6.4 Results . 200 6.4.1 DLS . 200 6.4.2 SANS . 203 6.4.3 The Effect of NaCl Concentration on Complex Structure . 208 6.5 Discussion and Further Work . 209 6.6 Potential Future Work . 211 7 Discussion and Future work 213 7.1 Principal Findings . 213 7.1.1 Static and Dynamic Study of the Aggregation of PEI-DNA Complexes213 7.1.2 Time Evolution of Zeta Potential of Complexes . 217 7.1.3 Sterically Stabilised Cationic Diblock Copolymer Complexes.