HIGH ASPECT RATIO VIRAL NANOPARTICLES FOR CANCER THERAPY By Karin L. Lee Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Dissertation Advisor: Dr. Nicole F. Steinmetz Biomedical Engineering CASE WESTERN RESERVE UNIVERSITY August 2016 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Karin L. Lee candidate for the Doctor of Philosophy degree*. (signed) Horst von Recum (chair of the committee) Nicole Steinmetz Ruth Keri David Schiraldi (date) June 29, 2016 *We also certify that written approval has been obtained for any proprietary material contained therein. TABLE OF CONTENTS Table of Contents List of Tables .................................................................................................................... ix List of Figures and Schemes .............................................................................................x Acknowledgements ........................................................................................................ xiv List of Abbreviations .................................................................................................... xvii Abstract......................................................................................................................... xxiii Chapter 1: Introduction ....................................................................................................1 1.1 Cancer statistics...............................................................................................................1 1.2 Current cancer therapies................................................................................................3 1.2.1 Surgery .................................................................................................................3 1.2.2 Radiation therapy .................................................................................................4 1.2.3 Hormone therapy..................................................................................................5 1.2.4 Photodynamic therapy..........................................................................................6 1.2.5 Immunotherapy ....................................................................................................6 1.2.6 Chemotherapy ....................................................................................................10 1.3 Nanoparticles for cancer therapy ................................................................................11 1.3.1 The importance of nanoparticle shape ...............................................................13 1.4 Viruses as nanoparticles ...............................................................................................17 1.4.1 Vaccines .............................................................................................................18 1.4.2 Immunotherapies................................................................................................20 1.4.3 Gene therapy ......................................................................................................20 1.4.4 Drug delivery......................................................................................................21 1.4.5 Imaging...............................................................................................................22 i TABLE OF CONTENTS 1.5 Plant viral nanoparticles ..............................................................................................23 1.5.1 Tobacco mosaic virus.........................................................................................23 1.5.2 Potato virus X.....................................................................................................24 1.6 Aims of this thesis..........................................................................................................26 1.7 Works cited in this chapter ..........................................................................................27 Chapter 2: Shape matters: comparison of TMV and CPMV diffusion......................42 2.1 Introduction...................................................................................................................42 2.2 Materials and methods .................................................................................................45 2.2.1 CPMV and TMV propagation............................................................................45 2.2.2 Bioconjugate chemistry to modify CPMV and TMV with A555 and O488 .....45 2.2.3 UV/visible spectroscopy ....................................................................................46 2.2.4 Denaturing gel electrophoresis...........................................................................47 2.2.5 Size exclusion chromatography (SEC) ..............................................................47 2.2.6 Transmission electron microscopy (TEM).........................................................47 2.2.7 Dynamic light scattering (DLS) and zeta potential measurements....................47 2.2.8 Preparation of the spheroids...............................................................................48 2.2.9 Confocal imaging ...............................................................................................48 2.2.10 MatLab data analysis........................................................................................49 2.3 Results and discussion ..................................................................................................50 2.3.1 TMV and CPMV properties...............................................................................50 2.3.2 Theory: diffusion rates of TMV and CPMV......................................................54 2.3.3 The spheroid model of the tumor microenvironment.........................................56 2.3.4 Experimental diffusion rates of TMV and CPMV .............................................58 2.4 Conclusions ....................................................................................................................66 ii TABLE OF CONTENTS 2.5 Works cited in this chapter ..........................................................................................67 Chapter 3: Tobacco mosaic virus for delivery of photodynamic therapy..................71 3.1 Introduction...................................................................................................................71 3.2 Materials and methods .................................................................................................74 3.2.1 Zn-EpPor synthesis ............................................................................................74 3.2.2 TMV propagation...............................................................................................75 3.2.3 Zn-EpPor loading into TMV ..............................................................................75 3.2.4 UV/visible spectroscopy ....................................................................................76 3.2.5 Inductively coupled plasma optical emission spectroscopy (ICP-OES)............76 3.2.6 Size exclusion chromatography (SEC) ..............................................................76 3.2.7 Transmission electron microscopy.....................................................................77 3.2.8 Tissue culture .....................................................................................................77 3.2.9 Flow cytometry ..................................................................................................77 3.2.10 Confocal microscopy........................................................................................78 3.2.11 Cell viability.....................................................................................................79 3.3 Results ............................................................................................................................80 3.3.1 Zn-EpPorTMV encapsulation and characterization.................................................80 3.3.2 Cell uptake and intracellular localization of Zn-EpPorTMV in B16F10 melanoma85 3.3.3 Therapeutic efficacy of Zn-EpPorTMV targeting melanoma..................................87 3.4 Discussion.......................................................................................................................90 3.5 Conclusion......................................................................................................................91 3.6 Works cited in this chapter ..........................................................................................92 Chapter 4: Stealth coatings for PVX..............................................................................97 4.1 Introduction...................................................................................................................97 iii TABLE OF CONTENTS 4.2 Materials and methods ...............................................................................................100 4.2.1 PVX propagation..............................................................................................100 4.2.2 Bioconjugation of PVX with fluorophores and PEG.......................................100 4.2.3 UV/visible spectroscopy ..................................................................................101 4.2.4 Denaturing gel electrophoresis.........................................................................102