Viral Mimicking Iron-Oxide Nanoplatforms for Highly Efficient Lymph Node Delivery and Lymphocyte Activation By Ryan Clauson A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Pharmaceutical Sciences) in The University of Michigan 2019 Doctoral Committee: Assistant Professor Beata Chertok, Co-Chair Professor Duxin Sun, Co-Chair Assistant Professor Yu Lei Assistant Professor James Moon Professor David Smith Ryan Clauson [email protected] ORCID iD: 0000-0001-6044-8884 © Ryan Clauson 2019 For my father – Without your sacrifice and guidance, none of this could have been possible. You have always been and will always be my hero. ii Acknowledgements Firstly, I would like to express my sincere gratitude to both of my advisors, Dr. Beata Chertok and Dr. Duxin Sun for their continuous support and guidance during my tenure at the University of Michigan. Dr. Chertok, thank you for developing me as a scientist, writer and problem solver. Dr. Sun, thank you for nurturing my skills as an independent researcher and for challenging me as a presenter. The advancements I have made as a scientist, leader and person are thanks in large part to both of your impacts in my life. I will be forever grateful for your investments in me. Beyond my advisors, I would like to recognize the individual members of my thesis committee including Dr. David Smith, Dr. James Moon and Dr. Yu Leo Lei. Your astute insights and wealth of experience have been invaluable. Additionally, your challenges to my research ideas and methodologies have strengthened my capabilities as an independent critical thinker. Next, I would like to acknowledge my lab mates for their incredible support academically, scientifically and emotionally over the last five years. From the Chertok Lab, I would like to thank Lindsay Scheetz, Dr. Mingsheng Chen and Brendan Berg. From the Sun Lab, I would like to specifically thank the members of the nano group including Dr. Hongwei Chen, Dr. Wei Gao and Hongxiang Hu. Outside of the laboratory, I would be remiss if I did not recognize the friendships built during my time at the University of Michigan that were pivotal in maintaining my happiness and sanity. I will cherish the memories and your friendships for the rest of my life. I would specifically like to acknowledge, Nick Waltz, Phil Rzeczycki and Nate Truchan. Last, but certainly not least, I would like to sincerely thank my family and future bride. To all of my family, your constant support throughout my entire academic career has given me the strength to complete this arduous journey. Without all of you, I would not be the man that I am today. To my loving fiancé Brenna, you are my rock and greatest source of inspiration. Thank you for your unwavering belief in me and the selflessness you exhibited for 800+ miles away as I pursued my dreams. iii Table of Contents Dedication .................................................................................................................................... ii Acknowledgements .................................................................................................................... iii List of Figures ............................................................................................................................ vi List of Tables ............................................................................................................................. ix List of Appendices ..................................................................................................................... x Abstract ....................................................................................................................................... xi Preface .......................................................................................................................................... 1 Part 1 – Iron-Oxide Nanoplatforms for B-cell Activation ........................................................ 2 Chapter 1 - Nanoparticles for Antigen-Specific Antibody Production: The Potential of Viral Mimicry ............................................................................................................................... 4 Chapter 2 – Engineering Inorganic Viruses with Viral Structural and Functional Mimicry for Lymph Node Homing, B-cell Activation and Enhanced Antigen-Specific Antibody Production .................................................................................................................................. 55 Part 2 – Iron-Oxide Nanoplatforms for T-cell Activation ....................................................... 94 Chapter 3 – Size-Controlled Iron Oxide Nanoplatforms with Lipidoid-Stabilized Shells for Efficient MRI-Trackable Lymph Node Targeting and High Capacity Biomolecule Display ..................................................................................................................................................... 95 iv Chapter 4 – The Content of CpG-DNA in Antigen-CpG Conjugate Vaccines Determines Their Cross-Presentation Activity .......................................................................................... 135 Chapter 5 – Conclusions and Future Directions .................................................................. 152 Appendices .............................................................................................................................. 165 v List of Figures Figure 2.1 – Nanoparticle Formulation and Viral-Like Structure Schematic ................................ 77 Figure 2.2 – Inorganic Virus-Like Nanoparticle (IVLN) Formulation and Viral Mimicry ............... 78 Figure 2.3 – Inorganic Virus-Like Nanoparticle (IVLN) enhanced Antigen-Specific Antibody Production in Mice ....................................................................................................................... 80 Figure 2.4 – Prophylactic HER2+ Breast Cancer Anti-Tumor Efficacy ........................................ 81 Figure 2.5 – Lymph Node Distributions and Viral-Like Function Schematic ............................... 82 Figure 2.6 – Viral-Like Functionality Assessment: Lymph Node Homing, Distribution and Immune Cell Uptake .................................................................................................................... 83 Figure 2.7 – In-vivo Germinal Center Formation and Antigen-Specific B-cells ........................... 85 Figure 3.1 – Formulation of Multi-Lipid-Coated Iron-Oxide Nanoparticles (IONP-ML) .............. 118 Figure 3.2 – Characterization of IONP-ML by TeM ................................................................... 119 Figure 3.3 – Size Control of the IONP-ML ................................................................................. 120 Figure 3.4 – Evaluation of IONP-ML’s Magnetic Properties ...................................................... 122 Figure 3.5 – In-Vivo MRI-Tracked Lymph Node Delivery of the IONP-ML Following Intradermal Administration ............................................................................................................................ 123 Figure 3.6 – Conjugation of Model Biomolecules (CpG Oligonucleotides and Peptide) to the Surface of IONP-ML .................................................................................................................. 124 Figure 3.7 – In-Vivo Lymph Node Delivery of Biomolecule-Displaying IONP-ML-CpG Following Intradermal Injection .................................................................................................................. 126 vi Figure 4.1 – Analysis of OVA-CpG Conjugates ......................................................................... 144 Figure 4.2 – In-Vitro Cross-Presentation Assay using B3Z CD8 T-cell Hybridoma ................... 145 Figure 4.3 – Analysis of the Aggregation Propensity in OVA-CpG Conjugates ........................ 146 Figure S1 – Transmission Electron Microscopy of Individual IVLN Components .................... 165 Figure S2 – Peptide Loading of IVLN Standardized on a per Gold Nanoparticle basis ........... 166 Figure S3 – Serum Stability Study of IVLNs ............................................................................. 167 Figure S4 – Antigen-Specific IgG Antibody Production Kinetics .............................................. 170 Figure S5 – Total Non-Specific IgM Antibody Production ........................................................ 171 Figure S6 – IVLN Antigen-Specific IgG Antibody Productions: Au/Fe Ratio and Peptide Density .. ................................................................................................................................................... 172 Figure S7 – Antigen-Specific Antibody D2F2/E2 Cell Specificity ............................................. 173 Figure S8 – IVLN Lymph Node Delivery by Different Administration Routes ........................... 174 Figure S9 – In-Vitro Nanoparticle Cell Uptake – Surface Topography Effects ......................... 175 Figure S10 – Modeling of inter-nanoparticle distance on IVLN surfaces ................................. 180 Figure S11 – IONP Formulation Method Comparison Images ................................................. 184 Figure S12 – Synthesis of Lipidoids ......................................................................................... 186 Figure S13 – Formulation Stability Lipidoid Comparison .......................................................... 187 Figure S14 – TLC analysis of the IONP-ML