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High Aspect Ratio Viral Nanoparticles for Cancer Therapy

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High Aspect Ratio Viral Nanoparticles for Cancer Therapy 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
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