FLUOROUS NANOPARTICLE PLATFORM FOR CANCER IMAGING AND TREATMENT By JAQUELINE DIANE WALLAT Dissertation Advisor: Dr. Jonathan K. Pokorski For the degree of Doctor of Philosophy Department of Macromolecular Science & Engineering CASE WESTERN RESERVE UNIVERSITY January, 2018 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Jaqueline Diane Wallat candidate for the degree of Doctor of Philosophy. * Thesis defense date : October 31, 2017 ______________________________ Dr. Jonathan K. Pokorski ______________________________ Dr. David Schiraldi ______________________________ Dr. Gary E. Wnek ______________________________ Dr. Horst A. von Recum *We also certify that written approval has been obtained for any proprietary material contained therein. Signed copy can be viewed in Graduate Studies at Case Western Reserve University. 2 Copyright © 2017 by JAQUELINE DIANE WALLAT All rights reserved 3 Table of Contents List of Figures ................................................................................................................9 List of Tables..................................................................................................................21 Abstract...........................................................................................................................25 Chapter I ........................................................................................................................27 Introduction to Fluorinated Systems for MR Imaging and Drug Delivery: From Perfluorocarbon Emulsions to Multifunctional Fluorinated Polymeric Architectures 1.1. Overview..................................................................................................................28 1.2. A Brief History of Fluorine......................................................................................28 1.3. Fluorine in Biomedical Applications........................................................................29 1.4. Magnetic Resonance Imaging of Fluorinated Nuclei...............................................31 1.5. PFCs in 19F MRI.......................................................................................................33 1.6. Polymeric Materials in 19F Imaging.........................................................................36 1.7. Responsive 19F Contrast Agents...............................................................................39 1.8. Fluorinated Systems for Delivery and Imaging.......................................................41 1.8.1. Multifunctional Fluorine Polymer from CRP Methods................................42 1.9. Conclusions .............................................................................................................48 1.10. Referencecs ...........................................................................................................50 Chapter II........................................................................................................................64 Optical and Magnetic Resonance Imaging by Fluorous Colloidal Nanoparticles 2.1. Background..............................................................................................................65 2.2. Results and Discussion.............................................................................................68 2.2.1. Polymer Synthesis and Characterization........................................................68 4 2.2.2. Investigation of Copolymer Molecular Weight on Size and Imaging Feasibility...............................................................................................................72 2.2.3. Phantom MRI Studies..................................................................................77 2.2.4. Cell Viability and Uptake in Macrophages .................................................79 2.2.5. Tumor Targeting...........................................................................................82 2.3. Conclusions ………………………………………………...…………………….86 2.4. Experimental Methods ……………………………………...……………………87 2.4.1. Reagents ……………………………………………..……………………87 2.4.2. Instrumentation ……………………………………......…………………..87 2.4.3. Synthesis of azide-functionalized ATRP initiator (A)...…………………...88 2.4.4. Synthesis of OEGMEMA/TFEMA Copolymer (1). ..……………………..89 2.4.5. Dynamic Light Scattering …………………….……..………………….....90 2.4.6. Transmission Electron Microscopy …………………………………….....90 2.4.7. Synthesis of Cy5-Labeled Polymer (2) ………………..…………………..90 2.4.8. 19F Magnetic Resonance Imaging Spectroscopy………..……………….... 91 2.4.9. 19F Magnetic Resonance Imaging Phantoms……………..………….......... 91 2.4.10. Cell Culture………………………………………………..…………….. 92 2.4.11. Flow Cytometry ……………………………………………..……………93 2.4.12. Cytotoxicity Assays…………………………………………..…………...93 2.4.13. Confocal Microscopy …………………………………………..…………94 2.4.14. TNBC Targeting………………………………………………...………...94 2.4.15. OCVA Targeting ………………………………………………...………..94 2.5. Supplemental Figures ……………………………………………………...………95 5 2.6. Acknowledgements ………………………………………………………...…….101 2.7. Exploratory Studies………………………………………………………...……..101 2.7.1.Synthetisis Optimization……………………………………………...…….101 2.7.2 Attempted 19F NMR and MRI of Particle Taken Up By Macrophages...…..104 2.8. References ……………………………………………………………………..…109 Chapter III. …………………………………………………………………………....118 Fluorinated Polymer–Photosensitizer Conjugates Enable Improved Generation of ROS for Anticancer Photodynamic Therapy 3.1. Background ………………………………………………………………………119 3.2. Results and Discussion …………………………………………………………...121 3.2.1. Polymer Synthesis and Characterization …………………………………..121 3.2.2. Conjugate Synthesis and Characterization ………………………………...122 3.2.3. Accessing Efficacy of Copolymer-Conjugate 3…………………………....126 3.3. Conclusions …………………………………………………………………….....131 3.4. Experimental Methods…………………………………………………………......132 3.4.1. Reagents…………………………………..………………………………...132 3.4.2. Instrumentation …………………………………..………………………...133 3.4.3. Copolymer (1) Synthesis…………………………………..………………..133 3.4.4. Synthesis of ZnEpPor-Labeled Polymer (3) ....………………………….....134 3.4.5. UV/Visible Spectroscopy ……...…………………………...........................134 3.4.6. Dynamic Light Scattering .............................................................................135 3.4.7. Gel Permeation Chromatography..................................................................135 1 3.4.8. Detection of O2 by ABDA............................................................................135 6 3.4.9. Cell Culture ..................................................................................................136 3.4.10. Confocal Microscopy .................................................................................137 3.4.11. Cytotoxicity Assays ....................................................................................137 3.4.12. LIVE/DEAD Assay.....................................................................................137 3.4.13. MTT Assay .................................................................................................137 3.5. Supplemental Figures ..............................................................................................138 3.6. Exploratory Studies .................................................................................................144 3.7. References ...............................................................................................................146 Chapter IV. .....................................................................................................................152 Fluorous Copolymer Delivers DOX to Cancer Cell 4.1. Background..............................................................................................................153 4.2. Results and Discussion............................................................................................156 4.2.1. Conjugate Synthesis and Characterization....................................................156 4.2.2. In Vitro Cell Studies......................................................................................160 4.3. Conclusions..............................................................................................................165 4.4. Experimental Section ..............................................................................................166 4.4.1. Reagents .......................................................................................................166 4.4.2. Instrumentation ............................................................................................167 4.4.3. Synthesis of tert-butyl-2-(pent-4-ynoyl)hydrazinecarboxylate.....................167 4.4.4. Synthesis of pent-4-ynehydrazine.................................................................168 4.4.5. Synthesis of hydrazone-DOX-alkyne............................................................168 4.4.6. Synthesis of hydrazone-DOX-copolymer.....................................................169 4.4.7. Polymer Characterization..............................................................................169 7 4.4.8. Determination of DOX concentration on the copolymer...............................169 4.4.9. Release Study.................................................................................................170 4.4.10. Cell Culture .................................................................................................170 4.4.11. Tracking DOX in cells.................................................................................170