UNIVERSITY OF CALIFORNIA, SAN DIEGO Cell Membrane-Cloaked Nanoparticles for Targeted Therapeutics A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Bioengineering by Brian Tsengchi Luk Committee in charge: Professor Liangfang Zhang, Chair Professor Shu Chien, Co-Chair Professor Michael J. Heller Professor Andrew C. Kummel Professor Dong-Er Zhang 2016 © Brian Luk, 2016 All rights reserved The Dissertation of Brian Tsengchi Luk is approved, and it is acceptable in quality and form for publication on microfilm and electronically: Co-Chair Chair University of California, San Diego 2016 iii DEDICATION This dissertation is dedicated to all those who have helped me reach this point in my life—from teachers to coaches to friends and colleagues; all have been a mentor to me in some shape or form. Without them, reaching this point would have been an impossibility, and I am eternally grateful for the support and guidance each person has provided. A special thanks goes to my loving family, who have supported me in all my endeavors and never cease to push me to be the person they know I can be. iv EPIGRAPH Nature composes some of her loveliest poems for the microscope and the telescope. Theodore Roszak v TABLE OF CONTENTS Signature Page ............................................................................................................... iii Dedication ...................................................................................................................... iv Epigraph ......................................................................................................................... v Table of Contents .......................................................................................................... vi List of Figures ................................................................................................................ xi List of Tables ............................................................................................................... xiv Acknowledgements ...................................................................................................... xv Vita .............................................................................................................................. xix Abstract of the Dissertation ........................................................................................ xxii Chapter 1 Introduction .................................................................................................... 1 1.1 Clinical Development of Polymeric Nanotherapeutics ................................ 3 1.2 Development of Stealth Functionalization Strategies .................................. 7 1.2.1 Synthetic Polymers for Stealth Functionalization ......................... 9 1.2.2 Biopolymers for Stealth Functionalization .................................. 12 1.2.3 Biologically Inspired Stealth Strategies ...................................... 14 1.2.3.1 Polymeric NPs Tagged with Self Markers ................... 16 1.2.3.2 Polymeric NPs Camouflaged in Cellular Membranes .. 18 1.3 Conclusions ................................................................................................ 21 1.4 References .................................................................................................. 22 Chapter 2 Understanding the Fundamentals of Membrane Coating on Polymeric Nanoparticles ............................................................................................... 41 2.1 Interfacial Interactions between the Membrane and Core .......................... 42 2.1.1 Introduction ................................................................................. 42 2.1.2 Experimental Methods ................................................................. 43 2.1.2.1 Preparation and Characterization of RBC-NPs ............ 43 2.1.2.2 Membrane Coverage Assay .......................................... 45 2.1.2.3 Membrane Sidedness Assay ......................................... 46 2.1.2.4 Preparation of Positively Charged Cores ..................... 47 2.1.2.5 Preparation of RBC-NPs with Differently Sized Polymeric Cores ........................................................... 48 2.1.3 Results and Discussion ................................................................ 49 2.1.4 Conclusions ................................................................................. 61 2.1.5 References ................................................................................... 62 2.2 “Marker-of-Self” Functionalization ........................................................... 66 vi 2.2.1 Introduction ................................................................................. 66 2.2.2 Experimental Methods ................................................................. 68 2.2.2.1 Preparation of RBC-NPs .............................................. 68 2.2.2.2 Identification of Membrane Proteins and CD47 .......... 69 2.2.2.3 Quantification of CD47 on RBC-NPs .......................... 70 2.2.2.4 Transmission Electron Microscopy .............................. 70 2.2.2.5 Macrophage Uptake Study ........................................... 71 2.2.3 Results and Discussion ................................................................ 72 2.2.4 Conclusions ................................................................................. 80 2.2.5 References ................................................................................... 81 Chapter 3 Cell Membrane-Coated Nanoparticles for Targeted Biomimetic Nanodelivery ................................................................................................ 85 3.1 Red Blood Cell Membrane-Cloaked Nanoparticles for Drug Delivery ..... 86 3.1.1 Introduction ................................................................................. 86 3.1.2 Experimental Methods ................................................................. 87 3.1.2.1 Preparation of RBC-NP and RBC-NP(DOX) .............. 87 3.1.2.2 In Vitro Cytotoxicity and Uptake ................................. 89 3.1.2.3 In Vivo Antitumor Efficacy .......................................... 89 3.1.2.4 In Vivo Safety Studies .................................................. 90 3.1.2.5 In Vivo Immunogenicity Studies ................................... 90 3.1.3 Results and Discussion ................................................................ 91 3.1.4 Conclusions ............................................................................... 100 3.1.5 References ................................................................................. 101 3.2 Functionalization of Targeting Ligands to RBC-NPs .............................. 106 3.2.1 Introduction ............................................................................... 106 3.2.2 Experimental Methods ............................................................... 108 3.2.2.1 Synthesis of Ligand-Linker-Lipid Conjugates ........... 108 3.2.2.2 Preparation of Ligand-Inserted RBC Ghosts .............. 109 3.2.2.3 Characterization of Ligand-Inserted RBC Ghosts ...... 109 3.2.2.4 Synthesis of Ligand-Functionalized RBC-NPs .......... 110 3.2.2.5 Colocalization Studies of Functionalized RBC-NPs .. 111 3.3.2.6 Cellular Uptake Studies .............................................. 112 3.2.2.7 Cytotoxicity Study ...................................................... 114 3.2.3 Results and Discussion .............................................................. 114 3.2.4 Conclusions ............................................................................... 122 2.3.5 References ................................................................................. 122 3.3 Platelet Membrane-Cloaked Nanoparticles for Targeted Drug Delivery . 127 3.3.1 Introduction ............................................................................... 127 3.3.2 Experimental Methods ............................................................... 128 3.3.2.1 Human Platelet Isolation and Platelet Membrane Derivation ................................................................... 128 3.3.2.2 Platelet Membrane-Cloaked Nanoparticle (PNP) Preparation and Characterization ............................... 130 vii 3.3.2.3 Examination of Platelet Membrane Proteins .............. 132 3.3.2.4 Examination of Protein Sidedness of PNPs ................ 133 3.3.2.5 Platelet Aggregation Assay ........................................ 134 3.3.2.6 Collagen Binding Study ............................................. 135 3.3.2.7 Differential Adhesion to Endothelial and Collagen Surfaces ...................................................................... 135 3.3.2.8 Cellular Uptake Study with Macrophage-like Cells ... 136 3.3.2.9 Complement Activation Study ................................... 137 3.3.2.10 PNP Adherence to Human Carotid Artery ............... 137 3.3.2.11 Pharmacokinetics, Biodistribution and Safety of PNPs in a Rat Model of Angioplasty-Induced Arterial Denudation ................................................................. 139 3.3.2.12 Treatment of Experimental Coronary Restenosis ..... 141 3.3.2.13 Staphylococcus aureus (MRSA252) Bacteria Adherence Study ........................................................ 142 3.3.2.14 Antimicrobial Efficacy Study ................................... 142 3.3.3 Results and Discussion .............................................................. 144 3.3.4 Conclusions ..............................................................................