Polymer and Lipid-Based Nanomedicine of Synergistic Drug Combinations for Improving Chemotherapy of Multidrug Resistant Breast Cancer by Rui Xue Zhang A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Pharmaceutical Sciences University of Toronto © Copyright by Rui Xue Zhang 2016 Polymer and Lipid-Based Nanomedicine of Synergistic Drug Combination for Improving Chemotherapy of Multidrug Resistant Breast Cancer Rui Xue Zhang Doctor of Philosophy Pharmaceutical Sciences University of Toronto 2016 Abstract Chemotherapy is a primary treatment modality for metastatic cancer. The development of multifactorial multidrug resistance (MDR), however, remains a significant obstacle contributing to treatment failure. The goal of this thesis is to investigate an emerging polymer-lipid hybrid nanoparticle (PLN) system for co-delivering synergistic drug combinations to overcome both macro- and microscopic biological barriers associated with MDR that blocks the effectiveness of chemotherapy. Based on our previous research on PLN for drug combination delivery, this thesis is intended to answer three questions: 1) can PLN synchronize the pharmacokinetics and ratio- metrically co-deliver high concentrations of a synergistic drug combination into tumor tissue? 2) Can PLN enhance intracellular drug delivery to the site of drug action e.g., the nucleus? 3) Can PLN overcome the plasma membrane rigidity and target mitochondria that are responsible for apoptosis dysregulation in MDR cancer cells? Results from this thesis show that compared to the drug combination in free solution, PLN synchronized pharmacokinetics and co-delivered two DNA damaging drugs, doxorubicin (DOX) and mitomycin C (MMC), to breast tumors at high concentrations at their synergistic ratios. Compared to clinically used liposomal or free solution of DOX, PLN moderated DOX-induced cardiotoxicity in vivo and enhanced local DOX ii bioavailability as evidenced by the highest intracellular accumulation and nuclear localization in vitro and tumor cell apoptosis in vivo. A new PLN formulation co-encapsulating poly- unsaturated fatty acid docosahexaenoic acid (DHA) and MMC effectively reduced MDR- associated rigidity of cytoplasmic membrane, enhanced drug and nanoparticle uptake, enabled synergistic reaction of MMC and DHA, and subsequently damaged mitochondria through elevated oxidative stress. These rationally designed PLN formulations resulted in superior efficacy against MDR breast cancer cells. The findings in this thesis substantiate PLN as a promising platform for nanoscale co-delivery of synergistic drug combinations and suggest powerful strategies for future design of nanomedicine for overcoming multifactorial MDR in cancer. iii Acknowledgments I would like thank my supervisor, Dr. Xiao Yu (Shirley) Wu, for giving me this mind- challenging but very rewarding opportunity to pursue the Ph.D. projects I loved. With her wise mentor and enormous patience, I have learned and grown to become a better person and more independent scientist. I would also like to thank all my committee members: Dr. Andrew Michael Rauth, Dr. Sandy Pang, Dr. Rob Macgregor, and Dr. Peter O’Brien for their professional critiques and valuable suggestions during my Ph.D. study and annual committee meetings. Without their constant guidance, I would not have pursued the science in the right direction. I want to thank all my colleagues in Dr. Wu’s laboratory. Special appreciations will be given to Dr. Ping Cai for his technical support in all animal work, Dr. Wei Yu (Tom) Weng for guiding me the optimization of drug formulations, Jason Li and Lily Yi Li for their enthusiastic involvement and intelligent contributions in my research projects, Mohammad Ali Amini for obtaining beautiful confocal cell images for my first publication during days of power outage caused by the ice storm. I deeply appreciate all my family members, particularly, my mother and father for the greatest support, strength and hope along the road of pursuing Ph.D. and my life. I acknowledge University of Toronto, Canadian Breast Cancer Foundation (CBCF) – Ontario Region, Natural Sciences and Engineering Research Council (NSERC), Canadian Institutes of Health Research (CIHR), for financial support for conducting these meaningful scientific researches; University of Toronto and the Graduate Department of Pharmaceutical Sciences for the scholarships. iv Table of Contents ACKNOWLEDGMENTS .......................................................................................................... IV TABLE OF CONTENTS ............................................................................................................. V LIST OF TABLES ................................................................................................................... XIII LIST OF FIGURES ................................................................................................................... XV LIST OF EQUATIONS ....................................................................................................... XXVII LIST OF ABBREVIATIONS .............................................................................................. XXIX CHAPTER 1 INTRODUCTION ................................................................................................. 1 MULTIDRUG RESISTANT BREAST CANCER ............................................................ 1 1.1 Breast Cancer Epidemiology ......................................................................................................................... 1 1.2 Chemotherapy and Its Combination ............................................................................................................ 1 1.3 Development of Multidrug Resistance .......................................................................................................... 1 RATIONALE OF PROPOSED STUDIES ......................................................................... 3 2.1 Problems of Free Drug Combination Regimens .......................................................................................... 3 2.2 Advantages of Nanocarriers for Drug Combination Therapy ................................................................... 4 2.3 Polymer Lipid Hybrid Nanoparticles (PLN) as a Drug Combination Carrier System ........................... 5 2.3.1 Polymer Lipid Hybrid Nanoparticles Co-loaded with Doxorubicin and Mitomycin C .............................. 5 2.3.2 Superior Efficacy ......................................................................................................................................... 6 2.3.3 Incomplete Inhibition of MDR in Some Tumor Cells ................................................................................. 7 v GOAL OF THE THESIS ..................................................................................................... 8 3.1 Proposed Approach to Overcoming Barriers in MDR Cancer .................................................................. 8 3.2 Questions ....................................................................................................................................................... 10 3.3 Hypotheses ..................................................................................................................................................... 10 3.4 Objectives ...................................................................................................................................................... 11 SYNOPSIS ........................................................................................................................... 11 CHAPTER 2 LITERATURE REVIEWS ................................................................................. 13 NANOMEDICINE OF SYNERGISTIC DRUG COMBINATIONS FOR CANCER THERAPY – STRATEGIES AND PERSPECTIVES ............................................................ 13 1.1 Abstract ......................................................................................................................................................... 13 1.2 Introduction .................................................................................................................................................. 14 1.3 Rationales and Strategies of Nanocarrier-Based Combination Therapy of Cancer .............................. 16 1.3.1 General Principles and Concept of Combination Therapy ........................................................................ 16 1.3.2 Rational Selection of Drug Combination Candidates and Treatment Schedule ........................................ 19 1.3.3 Common Issues with Free Drug Combinations ......................................................................................... 21 1.3.4 Advantages and Strategies of Nanocarrier-Based Combination Therapy of Cancer ................................ 23 1.4 Examples of Nanocarrier-Based Combination Therapies of Cancer ...................................................... 25 1.4.1 Nanocarrier-Based Combinations of Small Molecule Drugs Only ........................................................... 25 1.4.2 Nanocarrier-Based Combinations Including Large Molecule Drugs ........................................................ 29 1.4.2.1 Co-delivery of Anti-Cancer Genes and Chemotherapeutic Agents ................................................. 30 1.4.2.2 Combining Antibody and Chemotherapeutic Drugs .......................................................................