Synthesis and Characterization of Silicate Ester Prodrugs and Poly(ethylene glycol)-b-poly(lactic-co-glycolic acid) Block Copolymers for Formulation into Prodrug-Loaded Nanoparticles A DISSERTATION SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL OF THE UNIVERSITY OF MINNESOTA BY Adam Richard Wohl IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Doctor of Philosophy Thomas R. Hoye, Advisor September 2012 © Adam Richard Wohl September 2012 Acknowledgements I have been blessed to have the support of my family, friends, and colleagues. Without your help and encouragement, my graduate career would not have been possible. First of all, I would like to thank my advisor Prof. Tom Hoye. Over the past five years, he has been a consistent and patient mentor that leads first and foremost by setting a world-class example. He has taught me so much about analyzing problems, having fun while working hard, and always paying attention to the details. He has constantly taught me during our experimental discussions, results analysis, and late night (or, as I believe Tom would say, “mid-evening”) grant/manuscript writing sessions. I have learned not only many, many chemical concepts and scientific strategies, but also other valuable skills – how to clearly present ideas and make a compelling presentation to the right audience. Thank you, Tom, for all of the time that you have invested in both my scientific career and life – you have my utmost appreciation and respect. I would also like to thank Prof. Chris Macosko for all of his time, help, and expertise that he has provided to this collaborative project. With his guidance, my scientific and personal experiences were broadened tremendously. He has also constantly reminded me to keep the “big picture” in view in both science and life, and for that I am truly grateful. I would also like to thank Macosko group students Dr. Zhengxi Zhu, Jing Han, and Kevin Pustulka for their efforts on this project. In addition, a number of other collaborators have been instrumental to my graduate career. Prof. Jayanth Panyam has provided critical pharmacological expertise to the current project and its future direction during our collaborative biological studies. I owe an especially large debt to his student, Stephen Kalscheuer, for his diligent work on the cell and tumor-bearing animal efficacy studies. Prof. Alon McCormick and Han Seung Lee also provided important theoretical knowledge and experimental skill to the microscopy experiments. I have been lucky to work with many extraordinarily talented colleagues in the Hoye group as well. Dr. Yutaka Miura conducted the initial experiments to test the viability of the silicate ester prodrug strategy, and Dr. Haitao Qian initiated the block copolymer polyesterification project. I learned much from working with both of these group members, especially as I began my graduate research. I also thank Andrew Michel for carrying on this project into the future, and I wish him the best of luck in all of his upcoming experiments. Many other former and current students in the Hoye group have provided valuable technical advice and an amazing working environment (after all, you have to have some fun!). Thank you to Dr. Matthew Jansma, Dr. Dorian Nelson, Dr. Amanda Schmidt, Dr. Susana Emond, Patrick Willoughby, Brian Woods, Susan Brown, Dawen Niu, and Cagri Izgu. My parents, Richard and Laura, have always given me their unwavering support and love. From the time that they helped spark my scientific interest by helping me kill a few marigolds in our basement closet, they have always been unbelievably encouraging. I know that they also made many temporal and financial sacrifices to provide me with the resources to pursue my early scientific interests. I will always cherish your support and all that I learned from both of you during those science projects and the life skills that went with those experiences. Jill, my wife, is my steadfast foundation of support and love throughout the highs and lows that come with graduate school. She celebrated with me in times of success, encouraged me when I was frustrated, and put food in front of me and told me to eat as I studied for my preliminary exams. She has also graciously listened to my incessant “chemistry talk,” patiently allowed me to work late, and always been a much-needed source of balance in my life. Thank you, Jill, for all of the personal sacrifices that you have made in support of my graduate career. Honestly, I couldn’t have done this without your ever-present love and encouragement. I dedicate this thesis to my parents and my wife. Abstract Fine control of the physical and chemical properties of customized materials is a field that is rapidly advancing. This is especially critical in pursuits to develop and optimize novel nanoparticle drug delivery. Specifically, I aim to apply chemistry concepts to test the hypothesis “Silicate ester prodrugs of paclitaxel, customized to have the proper hydrophobicity and hydrolytic lability, can be formulated with well-defined, biocompatible, amphiphilic block copolymers into nanoparticles that are effective drugs.” Chapter 1 briefly describes the context and motivation of the scientific pursuits described in this thesis. In Chapter 2, a family of model silicate esters is synthesized, the hydrolysis rate of each compound is benchmarked, and trends are established based upon the steric bulk and leaving group ability of the silicate substituents. These trends are then applied to the synthesis of labile silicate ester prodrugs in Chapter 3. The bulk of this chapter focuses on the synthesis, hydrolysis, and cytotoxicity of prodrugs based on paclitaxel, a widely used chemotherapeutic agent. In Chapter 4, a new methodology for the synthesis of narrowly dispersed, “random” poly(lactic-co-glycolic acid) polymers by a constant infusion of the glycolide monomer is detailed. Using poly(ethylene glycol) as a macroinitiator, amphiphilic block copolymers were synthesized. Co-formulating a paclitaxel silicate and an amphiphilic block copolymer via flash nanoprecipitation led to highly prodrug- loaded, kinetically trapped nanoparticles. Studies to determine the structure, morphology, behavior, and efficacy of these nanoparticles are described in Chapter 5. Efforts to develop a general strategy for the selective end- functionalization of the polyether block of these amphiphilic block copolymers are discussed in Chapter 6. Examples of this strategy include functionalization of the polyether with an azide or a maleimide. Finally, Chapter 7 provides an outlook for future development of the strategies described in this thesis and summarizes the results and conclusions of the experimental results that led to the development of the therapeutic, paclitaxel silicate-loaded, polymeric nanoparticles. Table of Contents Acknowledgements i Dedication iii Abstract iv List of Tables xiii List of Figures xv List of Schemes xxi List of Abbreviations xxvi Chapter 1. General Overview 1 1.1. General Introduction 1 1.1.1. A Philosophical Viewpoint 1 1.1.2. Flash Nanoprecipitation 1 1.1.3. Previous Efforts to Encapsulate Paclitaxel via FNP 4 1.2. General Project Goals 5 1.2.1. The Over-Arching Hypothesis 5 1.2.2. Simultaneous Control of Physical and Chemical Properties of Small Molecules 5 1.2.3. Synthesis of Well-Defined Block Copolymers FNP 6 Chapter 2. Model Silicate Ester Prodrugs: Synthesis and Determination of Hydrolysis Rate Trends 7 2.1. Introduction 7 2.1.1. Silicate Nomenclature 7 2.1.2. Previous Work Utilizing Silicate Esters and Silyl Ethers in Biomaterials and Prodrugs 8 2.1.3. Established Orthosilicate Chemistry 11 2.2. Methodology Development 14 2.2.1. Considerations and Early Attempts 14 2.2.2. In situ 1H NMR Spectroscopic Measurements 15 2.2.3. Establishment of “Standard” Hydrolysis Conditions and Reproducibility of the 1H NMR Measurements 17 2.3. Hydrolysis Rates of Symmetrical Tetraalkoxy Silicates 19 2.3.1. Controlling the Hydrophobicity 19 2.3.2. Slowing the Hydrolysis Rate: Branched Tetraalkoxy Silicates 21 2.3.3. Rate Determining Step of the Hydrolytic Cleavage 24 2.3.4. Symmetrical Tetraaryl Orthosilicates 26 2.4. Observed Hydrolysis Rates of Unsymmetrical, Tetraalkoxy Silicate Esters 28 2.4.1. Mixed Menthoxy Trialkoxy Silicate Esters 28 2.4.2. A Mixed Triethyl Cresol Orthosilicate 30 2.4.3. Steroid-Containing Silicate Esters 32 2.4.4. Controlling the Hydrolysis Rate: Acyloxy Silicate Esters 34 2.5. Aminotrialkoxysilanes 43 2.5.1. Introduction 43 2.5.2. Attempts to Synthesize Mixed Cyclic Aminoalkoxy Silicate Esters 44 2.5.3. Control Experiments 46 2.5.4. Trialkoxy Silicate Esters as Labile Amine Protecting Groups 49 2.6. Conclusions 51 2.7. Experimental Section 52 Chapter 3. Synthesis and Development of Silicate Ester Prodrugs of Paclitaxel 82 3.1. Introduction 82 3.2. The Silicate Ester Prodrug Strategy 83 3.2.1. Silicate Esters as Prodrugs 83 3.2.2. Toxicological Profile of Orthosilicic Acid 84 3.3. Paclitaxel 86 3.3.1. A Brief Overview of Clinical Formulations of Paclitaxel 86 3.3.2. Selected, Relevant Pre-Clinical Formulations of Paclitaxel 89 3.3.3. Docetaxel and its Formulation as Taxotere® 90 3.3.4. Hydrophilic Derivatives of PTX 91 3.3.5. Hydrophobic Derivatives of PTX 92 3.3.6. PTX Silicate Prodrug Design and Synthesis of Tetraalkoxy PTX Silicate Prodrugs 94 3.3.7. PTX Tetraalkoxy Silicate Prodrug Hydrolysis Studies 98 3.3.8. Acyloxy-Containing PTX Silicate Prodrug Design and Synthesis 102 3.3.9. PTX Acyloxy Silicate Prodrug Hydrolysis Studies 104 3.3.10. Mechanistic Studies of the Hydrolysis of 3.16 107 3.3.11. PTX Silicate Prodrug in Vitro Cytotoxicity Studies 110 3.3.12. Summary and Future Work 111 3.4. Curcumin 112 3.4.1. Introduction 112 3.4.2. Efforts toward a Curcumin Silicate Ester 113 3.4.3.