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
4.2.5 TEM ...... 102
4.2.6 Animals ...... 102
4.2.7 Pharmacokinetics ...... 102
4.2.8 Biodistribution using fluorescence molecular tomography (FMT)...... 103
4.2.9 Immunofluorescence microscopy ...... 104
4.2.10 Immunogenicity ...... 104
4.2.11 Sandwich ELISA...... 105
4.2.12 Tissue culture ...... 106
4.2.13 Flow cytometry ...... 107
4.2.14 Cytokine activation ...... 107
4.2.15 Inflammasome activation ...... 108
4.2.16 Chronic inflammation ...... 109
4.2.17 Statistics ...... 109
4.3 Results ...... 109
4.3.1 Synthesis and characterization of fluorescently labeled and PEGylated PVX filaments...... 109
4.3.2 PEG conformation...... 114
4.3.3 Pharmacokinetics of PEGylated PVX...... 116
4.3.4 Biodistribution and clearance...... 118
iv TABLE OF CONTENTS
4.3.5 Immunogenic properties of PEGylated PVX ...... 122
4.3.6 PVX–cell interactions ...... 125
4.3.7 Cytokine activation in vitro and in vivo ...... 127
4.4 Discussion...... 131
4.5 Conclusions ...... 138
4.6 Works Cited in this chapter ...... 139
Chapter 5: PVX-GE11 for cell-specific targeting ...... 148
5.1 Introduction...... 148
5.2 Materials and methods ...... 152
5.2.1 PVX propagation and purification ...... 152
5.2.2 PVX bioconjugation...... 152
5.2.3 UV/visible spectroscopy ...... 153
5.2.4 Denaturing gel electrophoresis...... 153
5.2.5 Transmission electron microscopy...... 154
5.2.6 Particles stability in serum ...... 154
5.2.7 Tissue culture ...... 154
5.3 Results and discussion ...... 157
5.3.1 Bioconjugation of targeting and imaging moieties ...... 157
5.3.2 Cell binding experiments ...... 161
5.4 Conclusion...... 165
5.5 Works cited in this chapter ...... 167
Chapter 6: PVX-DOX for cancer therapy...... 172
6.1 Introduction...... 172
6.2 Materials and methods ...... 175
v TABLE OF CONTENTS
6.2.1 Potato virus X production ...... 175
6.2.2 Doxorubicin loading onto PVX...... 176
6.2.3 UV/visible spectroscopy ...... 176
6.2.4 Transmission electron microscopy...... 176
6.2.5 Agarose gel electrophoresis ...... 177
6.2.6 Cell culture ...... 177
6.2.7 PVX-DOX efficacy...... 177
6.2.8 Tracking of DOX and PVX-delivered DOX in cells ...... 178
6.2.9 Intracellular tracking of PVX...... 178
6.2.10 Animals ...... 179
6.2.11 Tumor model...... 179
6.2.12 Immunotherapy treatment schedule ...... 180
6.2.13 Chemo-immunotherapy treatment schedule...... 180
6.2.14 Immunohistochemistry of tumors ...... 180
6.3 Results and discussion ...... 181
6.3.1 Synthesis and characterization of PVX-DOX...... 181
6.3.2 PVX-DOX efficacy...... 184
6.3.3 Tracking of DOX and PVX-delivered DOX in cells ...... 185
6.3.4 Intracellular tracking of PVX...... 189
6.3.5 Immunotherapy treatment ...... 191
6.3.6 Chemo-immunotherapy treatment...... 194
6.4 Conclusions and future directions ...... 197
6.5 Works cited in this chapter ...... 199
Chapter 7: Conclusions and future directions ...... 205
vi TABLE OF CONTENTS
7.1 Scope of this work ...... 205
7.2 Shape matters: tobacco mosaic virus as a model high aspect ratio nanoparticle .207
7.3 Potato virus X: a filamentous plant virus for cancer therapy ...... 213
7.4 Future directions ...... 223
7.5 Works cited in this chapter ...... 224
Appendix I: Plasmonic nanodiamonds for cancer therapy ...... 235
A1.1 Introduction ...... 235
A1.2 Materials and methods ...... 237
A1.2.1 Chemicals and solvents ...... 237
A1.2.2 UV-Vis ...... 237
A1.2.3 DLS ...... 238
A1.2.4 Electron microscopy...... 238
A1.2.5 Synthesis of LA-PEG-alkyne ...... 239
A1.2.6 Transferrin-azide (Tf-azide)...... 239
A1.2.7 Nanoshell preparation...... 240
A1.2.8 Stability experiments...... 243
A1.2.9 Cell studies ...... 243
A1.3 Results and discussion...... 247
A1.3.1 Preparation and characterization of particles ...... 247
A1.3.2 Introduction of protective and bioorthogonally reactive PEG coating...... 252
A1.3.3 Modification of particles with Alexa Fluor 647 and transferrin ...... 254
A1.3.4 Targeting of cancer cells ...... 255
A1.3.5 Toxicity study...... 260
A1.3.6 Laser ablation ...... 260
vii TABLE OF CONTENTS
A1.4 Conclusions ...... 261
A1.5 Works cited in this appendix...... 263
Appendix II: Virus-based nanoparticles as vaccines...... 268
A2.1 Introduction ...... 268
A2.2 Virus-based nanoparticles as platform technologies ...... 269
A2.3 Chemical and genetic engineering of virus-based scaffolds ...... 271
A2.3.1 Chemical conjugation strategies...... 272
A2.3.2 Genetic engineering strategies...... 274
A2.4 VNP and VLP vaccines and immunotherapies ...... 275
A2.4.1 Infectious diseases...... 278
A2.4.2 Cancer...... 289
A2.4.3 Addiction (cocaine and nicotine) ...... 298
A2.4.4 Chronic diseases...... 300
A2.5 Conclusions ...... 305
A2.6 Works cited in this appendix...... 305
Bibliography...... 323
viii LIST OF TABLES
List of Tables
Table 2.1 Diffusion coefficients of CPMV and TMV...... 56
Table 4.1. Fluorophore and PEG-loading per PVX for the various A/V-PVX-PEG
formulations used in each experiment ...... 112
Table A2.1. Key VLP/VNP-based vaccines approved or under development...... 276
ix LIST OF FIGURES
List of Figures and Schemes
Figure 1.1. Hallmarks of cancer...... 2
Figure 1.2. The immunosuppressive tumor microenvironment...... 8
Figure 1.3. The importance of nanoparticle shape...... 15
Figure 1.4. Common viral architectures ...... 18
Figure 1.5. Crystal structure of tobacco mosaic virus ...... 23
Figure 1.6. Structure of potato virus X ...... 25
Figure 1.7. Aims of this thesis ...... 27
Figure 2.1. Biochemical characterization of dye-labeled TMV-O488 and CPMV-A555 51
Figure 2.2. Diffusion rates of VNP rod TMV and sphere CPMV into agarose half
spheroids ...... 61
Figure 2.3. Diffusion rates of CPMV sphere versus free O488 dye into agarose half
spheroids ...... 62
Figure 2.4. Diffusion rates of TMV rod versus free Rhodamine red dye into agarose half-
spheroids ...... 63
Figure 3.1. Zn-EpPorTMV conjugation and characterization ...... 82
Figure 3.2. Stability of Zn-EpPorTMV over time ...... 85
Figure 3.3. Zn-EpPorTMV interaction with B16F10 melanoma cells ...... 86
Figure 3.4. B16F10 response to Zn-EpPorTMV and free Zn-EpPor...... 89
x LIST OF FIGURES
Figure 4.1. Characterization of A-PVX-PEG ...... 111
Figure 4.2. Schematic (drawn to scale) showing the various PEG conformations
presented on the PVX filament...... 116
Figure 4.3. Pharmacokinetics of A-PVX-PEG ...... 118
Figure 4.4. Biodistribution of native and PEGylated PVX...... 122
Figure 4.5. Anti-PVX IgG titers and antibody-to-PVX binding...... 124
Figure 4.6. A-PVX-PEG-cell interactions measured by flow cytometry ...... 125
Figure 4.7. Time and concentration-dependent A-PVX-PEG-cell interactions ...... 126
Figure 4.8. Cytokine activation in BMDC induced by PVX-PEG particles...... 128
Figure 4.9. Inflammasone activation as measured by IL-1β production...... 131
Scheme 5.1. Bioconjugation of targeted PVX filaments...... 158
Figure 5.1. Characterization of A647-PVX-GE11 particles...... 160
Figure 5.2. Flow cytometry...... 162
Figure 5.3. Evaluation of targeted filaments in a co-culture of macrophages and cancer
cells...... 164
Figure 6.1. Synthesis and characterization of PVX-DOX...... 183
Figure 6.2. PVX-DOX efficacy in a panel of cell lines...... 185
Figure 6.3. Nuclear accumulation of DOX in A2780...... 188
Figure 6.4. PVX tracking in A2780...... 190
xi LIST OF FIGURES
Figure 6.5. Immunotherapy treatment of B16F10 tumors...... 193
Figure 6.6. Chemo-immunotherapy treatment of B16F10 tumors...... 195
Figure 6.7. Immunohistochemistry of PVX-DOX tumor sections...... 196
Figure A1.1. Preparation of particles...... 249
Figure A1.2. Characterization of particles...... 251
Figure A1.3. Structure of ND@Au-PEG conjugate and its colloidal stability in aqueous
solutions with high ionic strength...... 253
Figure A1.4. SKBR3 cell interactions with particle determined by flow cytometry...... 256
Figure A1.5. Nanodiamond-SKBR3 cell interactions determined by flow cytometry –
competition binding assay...... 257
Figure A1.6. SKBR3 cell interactions with particles observed by confocal microscopy.
...... 259
Figure A1.7. Viability of SKBR3 cells after exposure to ND@Au-Tf...... 260
Figure A1.8. Laser ablation of HeLa cells incubated with ND@Au-Tf nanoparticles. . 261
Figure A2.1. Nanoparticles as vaccination platforms...... 269
Figure A2.2. Categories of viral vaccines...... 270
Figure A2.3. Chemical conjugation strategies (bionconjugation)...... 273
Figure A2.4. Genetic engineering strategies for the display of epitopes on viral coat
proteins...... 275
xii LIST OF FIGURES
Figure A2.5. Structure of HIV-1...... 280
Figure A2.6. Structure of Ebola virus...... 284
Figure A2.7. Structure of influenza A virus...... 286
Figure A2.8. Production facility at Medicago...... 288
Figure A2.9. Human papillomavirus 16 L1 capsid...... 292
Figure A2.10. Human Hepatitis B virus...... 295
Figure A2.11. Chemical structures of nicotine (left) and cocaine (right)...... 299
xiii ACKNOWLEDGEMENTS
Acknowledgements
First, I would like to wholeheartedly thank my advisor Dr. Nicole Steinmetz for her support, encouragement, and guidance throughout my PhD. Without her, none of this would have been possible. She has provided me with wonderful mentorship, training me in research and writing, as well as giving me many opportunities to present my research at conferences around the country.
Next, I would like to thank my committee members for their support and advice over the years: Dr. Ruth Keri, Dr. Horst von Recum, and Dr. David Schiraldi. Their wide range of expertise has been invaluable.
I would also like to thank all the members of the Steinmetz lab throughout my time here. They made my experience in lab mentally stimulating and provided a positive environment, even when research didn’t go as expected. I am especially grateful for Dr.
Amy Wen, who navigated all the grad school experiences with me, as well as being very patient with all my lab and class-related questions. I would also like to thank Dr. Sourabh
Shukla for his guidance and all the hours he spent training and helping me with animal studies. Thank you to the students I had the opportunity to mentor over the years for working so hard and putting up with my (not always so clear) instructions: Paul Chariou,
Nadia Ayat, Kevin Chen, Mengzhi Wu, Sarah Woods, Jacob Schimelman, Sam
Alexander, Stephen Hern, Katherine Krawiec, and Brian So. I would also like to thank all the other members in the Steinmetz lab who gave me a positive and encouraging environment to work in: Dr. Michael Bruckman, Dr. Ibrahim Yildiz, Dr. Jon Whitney, Dr.
Patricia Lam, Dr. Andrzej Pitek, Dr. Brylee Tiu, Dr. Duc Le, Dr. Andy Hu, Anna Czapar,
xiv ACKNOWLEDGEMENTS
Neetu Gulati, Abner Murray, Richard Lin, Frank Veliz, Dan Kernan, Christina Franke, and all our wonderful undergrad trainees.
I am also grateful to Dr. Ruth Keri and her lab for welcoming me into their journal club discussions and teaching me about cancer biology: Jenny Brancato, Darcie
Seachrist, Leslie Cuellar Vite, Dr. Sylvia Gayle, Garrett Dunlap, Bryan Webb, and Dr.
Nicole Restrepo. I would especially like to thank Kristen Weber Bonk for all her help with mouse cancer models.
I am also very thankful for the opportunity to learn from so many collaborators
(listed in no particular order): Dr. Jonathan Pokorski (Macromolecular Science and
Engineering), Jack Edelbrock (Macromolecular Science and Engineering), Dr. George
Dubyak (Physiology and Biophysics), Caroline El Sanadi (Physiology and Biophysics),
Dr. Ulrich Commandeur (RWTH Aachen University, Institute for Molecular
Biotechnology), Dr. Miklos Gratzl (Biomedical Engineering), Logan Hubbard
(Biomedical Engineering), Dr. Phoebe Stewart (Pharmacology), Dr. Petr Cigler (AS CR,
Chemistry), Dr. Ivan Rehor (AS CR, Chemistry), Jan Havlik (AS CR, Chemistry), Dr.
Jana Lokajova (AS CR, Chemistry), Dr. Reza Ghiladi (NCSU, Chemistry), Dr. Bradley
Carpenter (NCSU, Chemistry), Kerstin Uhde-Holzem (RWTH Aachen University,
Institute for Molecular Biotechnology), Dr. Steven Fiering (Dartmouth, Microbiology and Immunology), Dr. Patrick Lizotte (Dartmouth, Microbiology and Immunology), Dr.
Mee Rie Sheen (Dartmouth, Microbiology and Immunology), Dr. Roger French
(Materials Science and Engineering), and Yingfang Ma (Materials Science and
Engineering). I would also like to thank the people who run the Small Animal Imaging
Center for teaching me how to use the instruments there, taking care of my animals, and
xv ACKNOWLEDGEMENTS answering my questions.
I would also like to thank all the administrative assistants in Biomedical
Engineering and the Case Center for Imaging Research: Debra Rudolph, Carol Adrine,
Carol Rice, Barbara Richards, Cena Hilliard, and Kacie Beck.
I also appreciate the members of Center for Nanotechnology and Engineering for letting me visit and for lending me reagents when ours inevitably ran out: Dr. Efstathios
Karathanasis, Dr. Agata Exner, Dr. Randall Toy, Elizabeth Doolittle, Dr. Pubudu Peiris,
Gil Covarrubias, Peter Bielecki, Dr. Prabhani Atukorale, Dr. Vindya Perrera, Chris
Hernandez, Selva Jeganathan, and Dr. Haoyan Zhu.
I would also like to acknowledge the funding sources that have supported my training and research over the years: NIH National Cancer Institute Cancer Pharmacology
Training Grant (R25 CA148052) and NIH Interdisciplinary Biomedical Imaging Training
Grant (T32 EB007509).
I would also like to thank all my friends, although there are far too many of you to name here. Thank you to my friends from high school and college; although it’s been hard to stay in touch they have always been there when I needed to talk. Thank you to my friends in Cleveland who reminded me to have fun and leave lab once in awhile. I would especially like to thank Nick Schmandt for his support and friendship, even when I was stressed and worried about school, writing, and planning for the future.
Lastly, I would like to thank my family for their love and support over the years.
Their encouragement has been invaluable and I want to thank them for pushing me to excel, for encouraging me to follow my passion, and for always believing in me.
xvi LIST OF ABBREVIATIONS
List of Abbreviations
A488 Alexa Fluor 488
A555 Alexa Fluor 555
A647 Alexa Fluor 647
Ad adenovirus
ADCC antibody-dependent cell-mediated cytotoxicity
AIDS acquired immunodeficiency syndrome
AP alkaline phosphatase
APC antigen presencting ceels aPDI antimicrobial photodynamic inactivation
AR aspect ratio
Aβ amyloid-β
BMDC bone marrow derived dendritic cells
BMV brome mosaic virus
BPV bovine papillomavirus
BSA bovine serum albumin
CAR chimeric antigen recetpors
CCMV cowpea chlorotic mottle virus
CMV cucumber mosaic virus
CP coat protein
CPMV Cowpea mosaic virus
CTL cytotoxic T lymphocyte
xvii LIST OF ABBREVIATIONS
CuAAC copper(I)-catalyzed azide-alkyne cycloaddition
CVF cell volume fraction
CytoD cytochalasin
DAPI 4',6-diamidino-2-phenylindole
DC dendritic cell
DLS dynamic light scattering
DMEM Dulbecco's modified Eagle medium
DMSO dimethyl sulfoxide
DOTA 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid
DOX doxorubicin
DPBS Dulbecco's phosphate buffered saline
ECM extracellular matrix
EDC 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
EDTA ethylenediaminetetraacetic acid
EGF epidermal growth factor
EGFR epidermal growth factor receptor
ELISA enzyme-linked immunosorbent assay
EPR enhanced permeability and retention
FACS fluorescence-activated cell sorting
FBS fetal bovine serum
FHV flock house virus
MFI mean fluorescence intensity
FMDV foot and mouth disease virus
xviii LIST OF ABBREVIATIONS
FPLC fast protein liquid chromatography
GAG glycosaminoglycan
GFP green fluorescent protein
GM-CSF granulocyte macrophage colony-stimulating factor
GNS gold nanosphere
GP glycoprotein
GS goat serum
HA hemagglutinin
HBV hepatitis B virus
HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
HER2 human epidermal growth factor receptor 2
HIV human immundeficiency virus
HPV human papillomavirus
HSP heat shock protein
HSV herpes simplex virus
IBDV infectious bursal disease virus
ICP-OES inductively-coupled plasma optical emission spectroscopy
IFN interferon
IL interleukin
KP potassimum phosphate
LPS lipopolysaccharide
MDRAB multidrug-resistant Acinetobacter baumannii
MEM minimum essential media
xix LIST OF ABBREVIATIONS
MPS mononuclear phagocyte system
MPyV murine polyomavirus
MRI magnetic resonance imaging
MRSA methicillin-resistant Staphylococcus aureus
MUC-1 human mucin-1
N. benthamiana Nicotiana benthamiana
NA neuraminidase
ND nanodiamond
NHS N-hydroxysuccinimide
NP nucleoprotein
O488 Oregon Green 488
P10B PEG 10 kDa, branched
P20 PEG 20 kDa, linear
P5B PEG 5000 Da, branched
P5L PEG 5000 Da, linear
PAMP pathogen-associated molecular pattern
PapMV papaya mosaic virus
PBS phosphate buffered saline
PDT photodynamic therapy
PEG polyethylene glycol penstrep penicillin-streptomycin
PET positron emission tomography
PN plasmonic nanostructure
xx LIST OF ABBREVIATIONS
PRINT particle replication in nonwetting templates
PS photosensitizer
PVX Potato virus X
RABV rabeies virus
RES reticuloendothelial system
RF Flory dimension
RH hydrodynamic radius
RNA ribonucleic acid
ROS reactive oxygen species
RPMI Roswell Park Memorial Institute
SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
SEC size exclusion chromatography
SV40 simian virus 40
T-VEC Talimogene Laherparepvec
TACA tumor-asscoiated carbohydrate antigen
TEM transmission electron microscopy
TEOS tetraehtyl orthosilicate
Tf transferrin
TfR transferrin receptor
THPC tetrakis(hydroxymethyl)phosphonium chloride
THPTA tris(3-hydroxypropyltriazolylmethyl)amine
TMV tobacco mosaic virus
TNF tumor necrosis factor
xxi LIST OF ABBREVIATIONS
UV/vis UV/visible
V. unguiculata Vigna unguiculata v/v volume by volume
VLP virus-like particle
VNP viral nanoparticle
VSV vesicuar stomatitis virus w/v weight by volume
WGA wheat-germ aggultinin
Zn-EpPor 5-(4-ethynylphenyl)-10,15,20-tris-(4-methylpyridin-4-ium-1-
yl)porphyrin-zinc(II) triiodide
xxii ABSTRACT
High Aspect Ratio Viral Nanoparticles for Cancer Therapy
Abstract
by
KARIN L. LEE
Each year, one million new cases of cancer are diagnosed in the United States and each case is unique, making it hard disease to prevent and treat. In the past few decades, nanoparticles have emerged as a promising platform for the development of cancer therapies. Unlike small molecule drugs, nanoparticles can be both passively and actively targeted towards tumor sites (both primary and metastatic sites). Additionally, they are able to carry large cargos for delivery of monotherapy or combination therapy, while also decreasing systemic side effects often associated with small molecule cancer therapies.
Nanoparticles in the clinic, as well as in clinical and pre-clinical trials have been predominantly spherical in shape. However, recent data suggest that high aspect ratio nanoparticles may have advantages for cancer treatment, including decreased uptake by phagocytic cells, improved margination, and enhanced tumor homing and penetration.
Despite this, synthesis of these materials remains challenging using chemical approaches.
Therefore, I turned to towards the study of plant virus-based nanoparticles, and my dissertation focused on the study and development of high aspect ratio viral nanoparticles.
Specifically, I focused on the plant viruses tobacco mosaic virus (TMV) and potato virus
X (PVX). My initial studies evaluated TMV as a model high aspect ratio nanoparticle and determined that it had improved diffusion into a 3D spheroid compared to a spherical virus. Additionally, I determined that it could be stably loaded with via non-covalent
xxiii ABSTRACT interactions with a cationic photosensitizer for photodynamic therapy. In the central body of my thesis I developed the filamentous virus PVX for cancer therapy. Stealth coated
PVX filaments exhibited substantially extended circulation time, while also decreasing recognition by PVX-specific antibodies, an important step towards translation of this platform. Addition of targeting ligands on the surface of the PVX filament led to selective targeting of cells in mono- and co-culture. Finally, a chemotherapy-loaded PVX filament was evaluated in in vitro and in vivo models of cancer and showed promise as a drug delivery platform for intravenous administration, as well as an intratumoral chemo- immunotherapy. Overall, my research enhanced the understanding of high aspect ratio viral nanoparticles, and described the development of a filamentous platform technology for cancer therapy. It has laid groundwork towards the development of improved and effective high aspect ratio virus-based cancer therapies.
xxiv CHAPTER 1: INTRODUCTION
Chapter 1: Introduction
1.1 Cancer statistics
Cancer is characterized by an uncontrolled growth and spread of abnormal cells.
However, the disease is defined by more than just the presence of rapidly dividing cells.
A tumor is comprised of multiple cell types that interact with each other as well as their surrounding tumor microenvironment. As cancers grow and develop, they acquire characteristics that enhance survival and have been categorized as the hallmarks of cancer.
The hallmarks of cancer were originally proposed as six biological capabilities within tumor development: sustained proliferative signaling, evading growth suppressors, activating invasion and metastasis, enabling replicative immortality, inducing angiogenesis, and resisting cell death. This list was recently extended to include two additional hallmarks, avoiding immune destruction and deregulating cellular energetics, and two enabling characteristics, genome instability and mutation and tumor-promoting inflammation (Figure 1.1) [1].
1 CHAPTER 1: INTRODUCTION
Figure 1.1. Hallmarks of cancer. (top) The six originally proposed biological capabilities within tumor development. (bottom) Recently added emerging hallmarks and enabling characteristics. Reprinted from Cell, Vol. 144, Douglas Hanahan and Robert A. Weinberg, Hallmarks of Cancer: The Next Generation, 646-674, Copyright 2011, with permission from Elsevier.
From these hallmarks, it is evident that tumors are complex, with a variety of signaling pathways, cell types, and small molecules that all contribute to their survival.
Additionally, cancer is a heterogeneous disease, with no two tumors being exactly alike, leading to wide variability between patients, and even within the same patient who has both primary and metastatic disease. As a result, there is no one-size-fits-all cancer
2 CHAPTER 1: INTRODUCTION therapy. Each individual is unique, and as a result, cancer is hard to prevent, as well as treat.
Each year, over 500,000 people die from cancer in the United States alone.
Additionally, there are one million new cases of cancer are diagnosed [2]. Cancer can be caused by a variety of environmental causes, including chemicals, viruses, and radiation, as well from genetics [3]. As a result, it affects many different organ systems and can present differently depending on where it is diagnosed and in which stage it is diagnosed.
1.2 Current cancer therapies
Depending on the cancer type and time of diagnosis (early versus late stage), there are different prognoses and different treatment options that can be applied as monotherapies or in combination. These include options that specifically target a cancer site such as surgery or radiation therapy, therapies that target a specific subtype of cancer, such as hormone therapy, as well as less specific therapies: photodynamic therapy, immunotherapy, and chemotherapy.
1.2.1 Surgery
Surgical resection is the standard of care for solid cancers, including melanoma, bone cancer, and breast cancer [4]. However, it is limited by the location of the tumor, such as with diffuse glioblastoma multiforme [5]. Surgery is also not applicable to small metastatic sites and cannot be applied to blood cancers, such as leukemia. Nevertheless, for localized, solid tumors that can be completely removed, surgery is highly effective.
3 CHAPTER 1: INTRODUCTION
For example for stage 0 and stage I melanoma as well as prostate cancer that is confined to the prostate gland, surgical resection is the only necessary treatment and is considered curative [6, 7]. However, more commonly surgical resection is administered in combination with radiation or chemotherapy to treat any residual cancer cells, and therefore prevent the patient from relapse due to recurrence of the disease. Surgery may also be used to debulk the tumor, but not entirely remove it, in order to improve responses of other therapies [8]. For example, ovarian tumors, often diagnosed at late stages, are debulked with the goal of removing all tumors larger than 1 cm. This surgery is then followed by chemotherapy to treat the smaller, remaining tumors [9, 10].
1.2.2 Radiation therapy
Radiation therapy is a common cancer treatment, with over 50% of patients requiring it during the course of their disease. It can be used alone as a curative treatment in some early stage cancers, including skin, prostate, cervical, and head and neck cancers, as well as lymphomas [11]. Radiation kills cells by damaging DNA, both directly and through generation of free radicals, leading to cell death [12]. Additionally, it is known to make cancers immune susceptible and is used in combination with immunotherapy or chemotherapy for breast cancer, advanced lymphomas, central nervous system tumors, and pediatric tumors [11, 13]. However, like many cancer therapies, radiation can cause side effects to surrounding healthy tissue. Additionally, there are lifetime limits of radiation depending on the tissue, resulting in a limited number of radiation treatments
[14]. For example, the typical dose of adjuvant radiation given for head and neck cancer
4 CHAPTER 1: INTRODUCTION is between 45 to 60 Gray (Gy). However, this can be delivered as smaller doses over one to two months.
1.2.3 Hormone therapy
Hormone therapy is used to treat breast, prostate, and a subset of endometrial cancers that are hormone receptor-positive. Prostate cancer is designated hormone receptor-positive if it is androgen receptor positive. Similarly, breast and endometrial cancers are designated hormone receptor-positive if it is estrogen or progesterone receptor positive. Hormone therapy can be used as a monotherapy or as an adjuvant therapy to either block hormone receptors or lower hormone levels within the body. For example, in breast cancer, approximately 2/3 of cancers are hormone receptor-positive
[15]. Tamoxifen is a selective estrogen-receptor modulator that binds to the estrogen receptor, and blocks interaction with the native agonist [16]. Another class of therapeutic is aromatase inhibitors, such as Letrozole, which prevent the conversion of androgens to estrogen by blocking aromatase [15]. In part due to the availability of hormone therapies, hormone receptor-positive breast cancers have improved outcomes versus the more aggressive, triple negative breast cancer (90% 5-year survival vs. 77% 5-year survival, respectively) [17]. Nevertheless, hormone therapy is limited to a subset of breast, prostate, and endometrial cancers that are hormone receptor-positive.
5 CHAPTER 1: INTRODUCTION
1.2.4 Photodynamic therapy
Photodynamic therapy (PDT) is used to treat superficial cancers and pre-cancers, including skin cancer, head and neck tumors, and digestive system tumors [18]. It is traditionally used as an adjuvant therapy in combination with surgery; it has also been combined with radiation and chemotherapy [18]. PDT involves three nontoxic components: a photosensitizer (PS), light, and oxygen [19]. When all three are present, cell killing occurs through production of reactive oxygen species, which induce oxidative stress on biomolecules [19]. Additionally, PDT damages the tumor microenvironment by damaging the tumor vasculature and stimulating an anti-tumor immune response [20].
One clinical study targeting basal cell cancer showed that PDT following the systemic administration of Photofrin (a porphyrin-based photosensitizer) resulted in an initial complete response rate of 92% [21]. This same cohort of 1440 patients also had a recurrence rate of less than 10% after 4 years. However, PDT is limited due to the poor bioavailability of the therapeutic and low tumor accumulation of PS. Nanoparticle technologies have been developed to enhance bioavailabity and delivery to the tumor; this will be discussed in Chapter 3.
1.2.5 Immunotherapy
More recently, immunotherapy, specifically immune checkpoint inhibitors and chimeric antigen receptor (CAR) T cells, has emerged as a promising cancer therapy.
Although it is not as commonly used as surgery or radiation therapy, immunotherapy is approved for treatment for a variety of cancers, including breast cancer, leukemia, melanoma, lung cancer, lymphoma, prostate cancer, and lymphoma [22]. Immunotherapy
6 CHAPTER 1: INTRODUCTION regimens can be used as a monotherapy, or in conjunction with surgery, radiation, hormone therapy, and chemotherapy [23].
Immunotherapy is characterized as using components of the immune system to fight cancer. This can be done by via passive strategies, which involve delivery of monoclonal antibodies or donor T cells [22, 24, 25], or by active strategies, which activate and prime the native immune system against cancer [22, 26]. Passive administration of anticancer immune components has had success. These include monoclonal antibodies that target cancer-associated proteins (HER2/neu, VEGF, EGFR,
CD20, CD52, and CD33) to trigger cell death via antibody-dependent cellular cytotoxicity (ADCC), as well as blocking oncogenic signaling pathways [22, 24, 27-32].
Antibodies can be combined with chemotherapies and toxins for dual-pronged approaches, one example of a drug-antibody conjugate is brentuximab vedotin, which has been FDA approved for CD30-positive Hodgkin’s lymphoma [33, 34].
Transplantation of donor lymphocytes is another form of passive immunization and has had success treating a subset of leukemias and lymphomas [25]. In a retrospective study of 53 of 446 patients who received donor lymphocyte infusion, 32% were alive at a median follow up of 30 months [25]. However, passive immunotherapy does not invoke a long-lasting immune response, and requires multiple treatments to remain efficacious. Therefore, recent research has turned towards the development of active immunotherapy to activate the native immune system.
7 CHAPTER 1: INTRODUCTION
Figure 1.2. The immunosuppressive tumor microenvironment. Reprinted by permission from Macmillan Publishers Ltd: Nature Medicine, Vol. 19, Daniela F Quail and Johanna A Joyce, Microenvironmental regulation of tumor progression and metastasis, 1423-1437, Copyright 2013.
The tumor microenvironment is naturally immunosuppressive due to multiple factors. These include, a high number of immunosuppressive factors (i.e. IL-10), impairment of antigen presenting machinery, inhibition of dendritic cell maturation, and upregulation of suppressive immune cells (i.e. regulatory T cells and myeloid-derived suppressor cells) (Figure 1.2) [35-37]. Active immunotherapy aims to stimulate this immunosuppressive environment to initiate immunity. Active immunity can be divided into 4 classes: cancer vaccines, checkpoint inhibitors, adoptive transfer of lymphocytes, and nonspecific immunostimulatory molecules [22, 38, 39].
Multiple therapeutic cancer vaccines are in clinical and preclinical trials [22]. One class of cancer vaccines is peptide and protein vaccines that target cancer antigens. These vaccines are delivered as free protein, recombinant protein, or via a viral vector that
8 CHAPTER 1: INTRODUCTION encodes for the peptide of interest, and showed the most promise when co-administered with an adjuvant [40, 41]. For example, PROSTVAC, which is an initial vaccination with a recombinant vaccinia virus encoding for prostate-specific antigen and a triad of costimulatory molecules, B7-1, lymphocyte function-associated antigen 3, and intracellular adhesion molecule 1, followed by a prime-boost strategy using a fowlpox vector, in combination with granulocyte macrophage colony-stimulating factor (GM-
CSF) improved survival approximately 9 months compared to the control group [42, 43].
Cell-based vaccines are another promising approach towards therapeutic cancer vaccines. This technique involves isolating a patient’s dendritic cells, activating them against tumor associate antigens ex vivo, and then returning them to the patient, along with immunostimulatory molecules [44]. Sipuleucel-T, a cell-based vaccine targeting the antigen prostatic acid phosphatase, was recently approved for treatment of prostate cancer
[45-47].
More recently, checkpoint inhibitors have exhibited promise for active immunotherapy. These monoclonal antibodies target inhibitory pathways that are normally involved in maintaining self-tolerance. However, in cancer, immune checkpoints are utilized by tumors to evade the immune system. An antibody against cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) (ipilimumab) was recently approved for use in metastatic melanoma, for patients with late stage melanoma that has spread or cannot be surgically resected [48-50]. Additionally antibodies disrupting the programmed cell death protein 1 (PD1) – programmed death ligand 1 (PD-L1) are in clinical trials [22, 38].
9 CHAPTER 1: INTRODUCTION
Due to the development of retrovirus vectors that carry chimeric antigen receptors
(CAR), adoptive T-cell therapy has recently expanded to include tumors that express more than just standard targets. CAR-T cells have shown promise in early clinical trials, but safety issues still need to be addressed [22, 51-54].
Other approaches, including the use of immunostimulatory molecules, such as cytokines (interferons and interleukin-2 (IL-2)), have been investigated to boost the immune response within the tumor microenvironment, leading to durable therapeutic responses [39].
Virus-like particles have also been used as immunostimulatory molecules to activate the immune response within the tumor microenvironment. Talimogene
Laherparepvec (T-VEC) is an oncolytic virus engineered to express GM-CSF that was recently approved for the treatment of inoperable melanomas [55]. In addition to oncolytic viruses, viruses used for cancer therapy include those used for gene therapy and in situ vaccination (see Section 1.4).
1.2.6 Chemotherapy
Chemotherapy is one of the most well-known and widely used therapeutic options for cancer. It is approved for use in many cancer types, either as a monotherapy or in combination with other therapies. Chemotherapy is the also the last treatment option for aggressive cancers that have no other available therapeutic regimens [56, 57].
Chemotherapy uses small molecule chemicals to target rapidly dividing cancer cells [58].
There are many classes of chemotherapy drugs, including anthracyclines, taxanes,
10 CHAPTER 1: INTRODUCTION platinum-based agents, alkylating agents, and vinca alkaloids [59, 60]. Each of these classes has a different mechanism of action that takes advantage of the rapid division of cancer cells by causing DNA damage, disrupting DNA transcription and replication, or inhibiting microtubule function [60-62]. Additionally, certain classes of chemotherapy are known to cause immune susceptibility, leaving cancer cells more vulnerable to immune therapies [63-65], making chemoimmunotherapy an attractive option for combination therapy.
However, chemotherapeutics are not targeted specifically towards cancer cells. As a result, when they are administered systemically, they interact with healthy cells that also divide rapidly, such as hair cells, bone marrow, and the epithelial lining of the gastrointestinal (GI) system. These interactions result in the side effects commonly associated with chemotherapy: hair loss, neutropenia, and GI problems [66, 67]. To overcome this, substantial work has been done to limit off-target accumulation in healthy tissues by encapsulating chemotherapies into nanoparticles.
1.3 Nanoparticles for cancer therapy
Due to their size, nanoparticles passively target solid tumors via the enhanced permeability and retention (EPR) effect. The EPR effect takes advantage of the leaky vasculature of rapidly growing tumors; nanoparticles are able to extravasate into tumors and then are retained within the disease site due to the lack of a lymphatic system [68, 69].
However, the EPR effect is controversial due to the heterogeneity of tumors. Barriers to the EPR effect include tumors that do not have leaky vasculature or those with a necrotic
11 CHAPTER 1: INTRODUCTION core [68, 70]. EPR targeting is also not applicable to for non-solid tumors, such as blood cancers, or metastatic sites [71].
Nanoparticles also offer the advantage of multivalent display of targeting ligands for active targeting of tumor cells [72-74]. This allows for improved selectivity for the cancer site, as well as increased uptake by cancer cells. Despite the advantages nanoparticles offer for both passive and active targeting compared to systemic administration of free drug, they still face a major barrier from the organs of the reticuloendothelial system (RES). Accumulation within these organs, including the liver and spleen, results in limited accumulation of therapy within the tumor. Due to this barrier, few nanoparticle formulations have been approved for use in the clinic.
Nanoparticles are able to carry large cargos of cancer therapy (both monotherapy and combination therapy); for example liposomal formulations of doxorubicin carry can carry 10,000 molecules per liposome [75]. For hydrophobic and unstable molecules, nanoparticle encapsulation can improve their biodistribution [76, 77]. At the same time, encapsulation of these small molecules improves their safety profile. Nanoparticles have altered biodistribution compared to free drug, which can decrease off target effects. For example, free doxorubicin administration is limited by cardiotoxicity [78], but when encapsulated into a lipid or polymeric nanoparticles, there was lower cardiac accumulation of the drug [79].
Currently, there are seven FDA-approved nanoparticle formulation delivery systems for cancers including breast cancer, ovarian cancer, HIV-related Kaposi sarcoma, and pancreatic cancer. Of these, six are liposomal formulations: Doxil (doxorubicin),
Myocet (doxorubicin), DaunoXome (daunorubicin), Marquibo (vincristine), MEPACT 12 CHAPTER 1: INTRODUCTION
(mifamurtide), and Onivyde (irinotecan) [80]. Liposomes are closed bilayer phospholipid systems that were first described in 1965 [81]. As a bilayer system, they are able to encapsulate both hydrophobic and hydrophilic molecules. Additionally, since their first description, they have been researched and designed to improve their in vivo properties.
The addition of polyethylene glycol (PEG) decreases clearance by the RES [82, 83], while the addition of targeting ligands enhances cell-specific receptor-mediated endocytosis to increase intracellular delivery of drugs [84, 85]. Liposomes can also be modified so that their contents are released at the target site; this can be achieved through remote triggering (e.g. heat, etc.) [86] or intrinsic triggering (e.g. pH, etc.) [87].
The other clinically approved nanoparticle cancer therapy is an albumin-based nanosphere, Abraxane (paclitaxel), which is approved for use in breast cancer, non-small cell lung carcinoma, and pancreatic cancer [88-90]. Paclitaxel is a hydrophobic molecule and administration of free drug requires the use of a surfactant to aid in solubilization
[91]. The surfactant used, Cremophore, is associated with additional side effects separate from those of paclitaxel itself [92]. However, albumin-bound paclitaxel can be delivered without the surfactant, thereby improving its safety profile [89]. Additionally, albumin has preferential uptake by tumors and inflamed tissues, as well as being biocompatible and biodegradable [93].
1.3.1 The importance of nanoparticle shape
Due to chemical synthesis restraints, particle-based delivery systems of cancer therapies have typically been spherical in shape. In addition to liposomes and albumin nanospheres, other particles currently in clinical and pre-clinical trials for cancer therapy 13 CHAPTER 1: INTRODUCTION include dendrimers, polymeric nanoparticles, and micelles [80], all of which are spherical in nature. In comparison, there are few high aspect ratio (AR) particles under investigated.
High aspect ratio synthetic particles are limited to carbon nanotubes, filomicelles, and particle replication in nonwetting templates (PRINT) nanoparticles. However, carbon nanotubes accumulate in the lungs and cause damage [94], filomicelles cannot yet manufactured on the nanometer scale with low polydispersity [95, 96], and PRINT nanoparticles cannot be manufactured with an axis below 50 nm [97]. Despite these technological challenges, recent data suggest that high aspect ratio nanoparticles hold advantages for cancer treatment (Figure 1.3). Rod-shaped particles have decreased non- specific cell uptake by phagocytic cells, including macrophages [98, 99]. For example, comparison of uptake of gold nanorods and gold nanospheres in murine macrophages found decreased gold content within the macrophages incubated with rod-shaped particles [99]. In a similar study using a plant virus-based nanoparticle, our lab showed that tobacco mosaic virus rods of variable aspect ratios exhibited different uptake into murine macrophages: short rods (AR= 3) had increased uptake by macrophages after 24 h compared to long rods (AR=16.5) [100]. This phenomenon is explained by the fact that uptake rate into macrophages correlates to the contact angle of the nanoparticle with the cell surface. Spherical particles have only one contact angle. However, high aspect ratio nanoparticles are defined by a long and short axis. Parallel alignment of the long axis results in slower uptake rates than parallel alignment of the short axis; uptake rates will vary between these maximum and minimum uptake rates based on contact angle [101].
Lower macrophage uptake results in longer circulation time in the blood, enhancing tumor accumulation [102, 103].
14 CHAPTER 1: INTRODUCTION
Figure 1.3. The importance of nanoparticle shape. Rod-shaped nanoparticles exhibit (top) decreased macrophage uptake, (bottom left) improved extravasation, and (bottom right) improved specific binding due to increased ligand display compared to spherical nanoparticles. Reprinted from Nanomedicine, Vol. 9, Randall Toy, Pubudu M. Peiris, Ketan B. Ghaghada, and Efstathios Karathanasis, 121-134, Copyright 2011, with permission from Future Medicine Ltd.
Nanoparticles are commonly modified with targeting ligands, including small peptides, antibodies/antibody fragments, aptamers, etc., to actively target molecules overexpressed on cancer cells or the associated tumor vasculature. Unlike small molecule targeting conjugates (e.g. antibody-drug conjugates), nanoparticles have multivalent display of ligands, enhancing avidity-based binding [72-74, 103, 104]. High aspect ratio nanoparticles also have improved specific binding due to increased ligand presentation compared to spherical nanoparticles with the same surface area [104, 105]. For example,
15 CHAPTER 1: INTRODUCTION nanorods and nanospheres with the same surface area, displaying antibodies at the same density, exhibited different uptake into targeted cells. Nanorods had significantly increased uptake into ligand-specific cells compared to the spherical nanoparticles [104].
Higher specific binding leads to improved active targeting at the site of disease.
High aspect ratio nanoparticles also have improved margination, or lateral drift towards the endothelial wall, compared to spherical nanoparticles [106-108]. The anisotropic nature of high aspect ratio nanoparticles results in tumbling when traveling through the blood stream, leading to lateral drift towards the blood vessels wall [103, 106,
107]. For example, when gold nanorods and gold nanospheres were evaluated in a microchannel to determine margination rates, the gold nanorods exhibited significantly increased deposition within the channel compared to the gold nanospheres [107].
Improved margination allows nanoparticles to escape the blood flow to interact with cancer cell and vascular targets [109].
After escaping the blood stream and entering the tumor microenvironment, high aspect ratio nanoparticles also have improved tumor penetration compared to spherical nanoparticles [110]. This is due to improved transport through pores within the tumor microenvironment, which is determined by the short dimension of high aspect ratio nanoparticles [110]. Improvement in nanoparticle penetration reduces the likelihood that particles will be washed back out into circulation, as well as increasing the number of cancer cells exposed to the therapy, thereby substantially improving therapeutic efficacy
[110].
In summary, it is clear that high aspect ratio materials have unique properties and may be beneficial for cancer drug delivery. Because the synthesis of such materials still 16 CHAPTER 1: INTRODUCTION remains challenging using purely chemical approaches, I have turned toward the study of plant virus-based nanoparticles, a novel class of biologics that naturally forms high aspect ratio nanotubes and filaments.
1.4 Viruses as nanoparticles
As an alternative to the synthetic nanoparticles discussed above, my dissertation research focused on the study and development of virus-based nanoparticles (VNPs).
VNPs come in a range of shapes and sizes and they are ideal for cargo delivery because they have naturally evolved to carry cargo (i.e. their genome) (Figure 1.4). As biological nanoparticles, viruses are programmed by nucleic acids and as such assemble into monodisperse nanoparticles with a high degree of reproducibility, something not yet achievable using chemical synthesis. They also have highly symmetrical structures that are known to atomic resolution, allowing for site-specific modification. Importantly, for use in vivo, they are biocompatible and biodegradable [111]. Non-mammalian viruses offer the additional advantage of being noninfectious in humans. Nevertheless, as with any novel therapeutic, detailed toxicology studies (including immunotoxicology) need to verify the safety profiles prior to launching novel products.
17 CHAPTER 1: INTRODUCTION
Figure 1.4. Common viral architectures. Reproduced from Chemical Society Reviews, Amy M. Wen and Nicole F. Steinmetz, Design of virus-based nanomaterials for medicine, biotechnology, and energy, doi: 10.1039/c5cs00287g, Copyright 2016 with permission from The Royal Society of Chemistry.
VNPs and nucleic acid-void virus-like particles (VLPs) have been investigated for use as nanoparticles in preclinical and clinical studies for numerous biomedical applications, including vaccines and immunotherapies, gene therapy, drug delivery, and imaging.
1.4.1 Vaccines
One of the earliest uses of viruses for biomedical applications was for the development of vaccines. VNPs and VLPs have many properties that make them ideal candidates for vaccines [112-120]. Early virus-based vaccines, based on inactivated or
18 CHAPTER 1: INTRODUCTION live-attenuated viruses, had success against infectious diseases, including the eradication of polio [121, 122]. While these vaccines continued to be used, more recently developed vaccines have an improved safety profile because they are based on viruses that cannot replicate in humans: either VNPs from non-mammalian viruses or VLPs that are produced in heterologous expression systems, either through fermentation or farming in plants [123]. Additionally, virus-based vaccines have expanded to include VNP/VLPs, which express foreign antigens, either as a conjugate to their surface or via gene delivery
(discussed below) to treat a range of diseases: cancer, infectious diseases, addiction, and chronic diseases [124-127].
Antigens of interest have been attached to VNPs using chemical and genetic engineering. For example, the bacteriophage Qβ displays lysine residues on its exterior surface and these have been modified with molecules, such as nicotine, CCR5, angiotensin II, or a tumor-associated carbohydrate antigen to develop vaccines against nicotine addiction, HIV, hypertension, and cancer, respectively [128-131]. In similar approaches, VNP/VLPs can be genetically modified to produce recombinant coat proteins, expressing an antigen of interest. For example, peptides from amyloid-β, a protein implicated in Alzheimer’s disease progression, were genetically introduced into the coat proteins of human papillomavirus 16 and bovine papillomavirus 1 to produce recombinant VLPs. These VLPs stably assembled and exhibited promising results in preclinical vaccination studies [124, 132].
For additional information on VNP/VLP-based vaccine platforms, refer to
Appendix II, which contains a review article that I authored summarizing the field [133].
19 CHAPTER 1: INTRODUCTION
1.4.2 Immunotherapies
In addition to vaccines, viruses have also been used as immunotherapies for cancer treatment [134-136]. One method is through the use of modified oncolytic viruses.
T-VEC is a herpes simplex virus (HSV)-based oncolytic virus engineered to produce
GM-CSF. The therapeutic has a dual-pronged mechanism: cell killing through the oncolytic activity of the virus and immune-activation through GM-CSF expression. T-
VEC has been approved for in situ treatment of melanoma [55]. Recently, our lab showed the impact of in situ vaccination with a plant based VLP. Treatment with unmodified
VLPs from cowpea mosaic virus (CPMV) resulted in a potent immune-mediated anti- tumor response. For complete efficacy, the response required IL-12, IFNγ, adaptive immunity, and neutrophils. Importantly, this response led to tumor regression and prevented recurrence and metastasis after either intratracheal or intratumoral administration in multiple models of cancer, indicating that a local treatment with CPMV
VLPs leads to overall systemic immunity and most importantly also induces immune memory [134].
1.4.3 Gene therapy
Virus-based gene delivery utilizes the natural infective nature of viruses to introduce foreign nucleic acid sequences into host cells, in order to produce a therapeutic effect [137]. Although there are no clinically available gene therapies approved, there are a number of virus-based gene therapies in clinical trials for cancer, as well as other diseases [137-140]. A variety of viruses have been investigated for gene delivery, including adenovirus, adeno-associated virus, herpes simplex virus, lentivirus, poxvirus,
20 CHAPTER 1: INTRODUCTION and Epstein-Barr virus [141]. These viral genomes incorporate foreign genes, which are then transcribed in target cells. For example, two suicide genes were incorporated into the genome of HSV and successfully delivered to malignant glioma [142].
1.4.4 Drug delivery
As an alternative to delivering genetic material for gene therapy, viruses been utilized to carry cargos for drug delivery [143-149]. Therapeutic drugs can be attached to
VNPs by targeting amino acids on the interior or exterior of the virus. For example, the chemotherapeutic doxorubicin was conjugated to both CPMV and brome mosaic virus
(BMV) utilizing carboxylic acids and thiol groups, respectively [143, 146]. Carboxylic acids on the exterior of CPMV were activated using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to catalyze the reaction with the amine group on doxorubicin [143]. Thiol groups on the exterior of
BMV were targeted using a maleimide bifunctional linker containing a hydrazine group.
The hydrazine group was then coupled to the ketone group of doxorubicin to form a hydrazide linkage [146]. Cell delivery and efficacy was demonstrated in tissue culture.
Alternative loading methods have been developed that utilize non-covalent principles: for example, cucumber mosaic virus (CMV) was incubated with DOX, which then diffused into the viral capsid through pores and was retained after interaction with CMV RNA
[150]. DOX remained active after interacting with CMV RNA; cell delivery and killing was demonstrated in vitro [150]. Making use of electrostatic interactions, TMV was electrostatically loaded with positively charge platinum-based drugs, targeting glutamic
21 CHAPTER 1: INTRODUCTION acid residues on the interior channel. Drug-loaded TMV exhibited improved efficacy versus free drug in an in vivo model of breast cancer [149].
The use of VNP and VLPs for drug delivery is an emerging field, with promising results. However, a majority of previously reported studies have focused on icoasahedral viruses and were only evaluated in vitro. My thesis work will expand this field by focusing on high aspect ratio VNPs, with the central body of my work focusing on in vivo evaluation.
1.4.5 Imaging
Toward the development of theranostic approaches that combine therapy and imaging, VNPs and VLPs have also been modified to carry imaging agents, including fluorescent molecules, gadolinium or iron oxide for magnetic resonance imaging (MRI) contrast, and 64Cu for positron emission tomography (PET) imaging [146, 151-162].
Similar to drug delivery, imaging molecules have been loaded onto VNPs via both infusion/encapsulation, as well as chemical conjugation. For certain imaging moieties, such as green fluorescent protein (GFP), genetic engineering can also be used. For example, our lab constructed a vector containing GFP fused to the N-terminus of potato virus X (PVX) via a foot and mouth disease (FMDV) 2A linker [162]. The FMDV 2A linker induces a ribosomal skip, so that approximately 50% of PVX coat proteins produced are recombinant coat proteins, and the remaining 50% are wild-type [163], allowing for stable assembly of PVX-GFP virions. These fluorescently labeled particles were successfully used for imaging in plants, cells, and mice [162].
22 CHAPTER 1: INTRODUCTION
1.5 Plant viral nanoparticles
In my dissertation research, I specifically consider plant VNPs. They can be produced in large quantities in plants and are noninfectious in mammals. Like all viruses, they come a range of shapes and sizes. I specifically evaluated the high aspect ratio plant
VNPs tobacco mosaic virus (TMV) and potato virus X (PVX).
1.5.1 Tobacco mosaic virus
Tobacco mosaic virus (TMV) is a stiff, rod-shaped virus, whose wild-type form is comprised of 2130 identical coat proteins, which assemble into tubes around a ssRNA template (Figure 1.5). The resulting rod measures 300 x 18 nm and has an interior channel diameter of 4 nm. Each coat protein displays a tyrosine residue on the exterior and two glutamic acid residues on the interior for site-specific chemical modifications
[164, 165]. Additionally, TMV-mutants are available that contain cysteine or lysine residues on the exterior for alternate modification [166, 167].
Figure 1.5. Crystal structure of tobacco mosaic virus. Green = Tyr139; Red = Glu97; Blue = Glu106.
23 CHAPTER 1: INTRODUCTION
TMV is a well-characterized virus and has been used in biomedical applications including imaging, drug delivery, and tissue engineering [149, 153, 168]. Our lab showed that TMV can be modified using bioconjugate chemistry to add targeting ligands for molecular specificity or polyethylene glycol (PEG) chains for stealth coating. At the same time TMV could be functionalized to carry large payloads of Gd(DOTA) enabling molecular MRI of atherosclerosis in a mouse model [153]. Our lab also recently showed that interior glutamic acids can be targeted for infusion and retention of positively charged platinum-based drug candidates, enabling drug delivery and treatment of triple negative breast cancer in a mouse model [149].
1.5.2 Potato virus X
Like TMV, potato virus X (PVX) is a high aspect ratio plant VNP. However, unlike the rigid structure of TMV, PVX is a filamentous virus (Figure 1.6). It is comprised of 1270 identical coat proteins that assemble into a filament measuring 515 x
13 nm. Each coat protein contains a lysine group available for modification [169].
Additionally, it was previously shown that the coat protein can be modified with an N- terminal fusion protein that displays on the exterior of the virus [162].
24 CHAPTER 1: INTRODUCTION
Figure 1.6. Structure of potato virus X. (Left) Transmission electron microscope image of PVX filaments; (Right) CryoEM structure of PVX. CryoEM structure reproduced with permission from Journal of Virology, Vol. 82, Amy Kendall et al., Structure of filamentous plant viruses, 9546-9554, with permission from American Society for Microbiology.
High aspect ratio filamentous nanoparticles, such as PVX, have not been extensively studied due to difficulties in producing them synthetically. Prior to the work discussed here, PVX had been investigated as a vaccine platform for influenza, human papillomavirus, hepatitis C, and HIV [170-175], as well as for enzyme immobilization
[176]. Additionally, towards its development for as a therapeutic delivery vehicle, previous studies showed that PVX could be modified with biotin, fluorophores, and PEG, and investigated how these surface modifications impacted PVX-cell interactions [169].
Our lab also showed that PVX has enhanced tumor homing and tissue penetration versus a spherical virus [177]. While this prior work laid the groundwork for my dissertation research, further investigation into the in vitro and in vivo properties of PVX is necessary if it is to be developed into a cancer therapy platform.
25 CHAPTER 1: INTRODUCTION
1.6 Aims of this thesis
In the past few decades, the use of nanoparticles for cancer therapy has made extensive progress. However, conventional nanoparticles are predominantly spherical in shape, with limited investigation into high aspect ratio particles due to chemical synthesis restraints. Despite this, preliminary results indicate that high aspect ratio particles have advantages for cancer therapy due to improved tumor targeting. To overcome chemical synthesis restraints, I turned towards biological nanoparticles, specifically, the plant viruses TMV and PVX. The purpose of my dissertation research was to develop high aspect ratio viral nanoparticles for applications in cancer therapy (Figure 1.7). In
Chapters 2 and 3 I investigated TMV as a model high aspect ratio nanoparticle to determine the importance of shape, as well as evaluate it as a potential carrier for photosensitizers. In Chapters 4-6 I developed the filamentous plant virus PVX for cancer therapy. I present the first extensive studies focused on the development of the PVX platform towards cancer therapy, investigating the impact of surface modifications on in vitro and in vivo properties (Chapters 4+5) and evaluating it for drug delivery and chemoimmunotherapy (Chapter 6). In Chapter 7, I provide concluding remarks and discuss future directions.
While my dissertation is focused predominantly on high aspect ratio viral nanoparticles for cancer therapy, in Appendix I, I also investigated nanodiamonds as an alternative nanoparticle for targeted cancer therapy. In Appendix II, I review the use of virus-based nanoparticles for vaccine development.
26 CHAPTER 1: INTRODUCTION
Figure 1.7. Aims of this thesis. Towards the development of high aspect ratio viral nanoparticles for cancer therapy, (top) tobacco mosaic virus was used as a model high aspect ratio nanoparticle to determine the importance of shape and in a proof-of-concept study for delivery of cationic photosensitizers. (bottom) The filamentous virus potato virus X was developed for cancer therapy using stealth coatings, targeting ligands, and chemotherapy.
1.7 Works cited in this chapter
1. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011;144:646-74.
2. American Cancer Society. Global Cancer Facts & Figures, 3rd Edition. In: Society AC, editor. 3rd ed. Atlanta: American Cancer Society; 2015.
3. Blackadar CB. Historical review of the causes of cancer. World J Clin Oncol 2016;7:54-86.
4. Sabel MS, Diehl KM, Chang AE. Priniciples of surgical therapy in oncology. Oncology. New York: Springer; 2006. p. 58-72.
27 CHAPTER 1: INTRODUCTION
5. Claes A, Idema AJ, Wesseling P. Diffuse glioma growth: a guerilla war. Acta Neuropathol 2007;114:443-58.
6. Redaniel MT, Martin RM, Gillatt D, Wade J, Jeffreys M. Time from diagnosis to surgery and prostate cancer survival: a retrospective cohort study. BMC Cancer 2013;13:559.
7. Testori A, Rutkowski P, Marsden J, Bastholt L, Chiarion-Sileni V, Hauschild A, et al. Surgery and radiotherapy in the treatment of cutaneous melanoma. Ann Oncol 2009;20 Suppl 6:vi22-9.
8. Silberman AW. Surgical debulking of tumors. Surg Gynecol Obstet 1982;155:577-85.
9. Morice P, Brehier-Ollive D, Rey A, Atallah D, Lhomme C, Pautier P, et al. Results of interval debulking surgery in advanced stage ovarian cancer: an exposed-non-exposed study. Ann Oncol 2003;14:74-7.
10. Morice P, Dubernard G, Rey A, Atallah D, Pautier P, Pomel C, et al. Results of interval debulking surgery compared with primary debulking surgery in advanced stage ovarian cancer. J Am Coll Surg 2003;197:955-63.
11. Baskar R, Lee KA, Yeo R, Yeoh KW. Cancer and radiation therapy: current advances and future directions. Int J Med Sci 2012;9:193-9.
12. Yoshimura M, Itasaka S, Harada H, Hiraoka M. Microenvironment and radiation therapy. Biomed Res Int 2013;2013:685308.
13. Gameiro SR, Jammeh ML, Wattenberg MM, Tsang KY, Ferrone S, Hodge JW. Radiation-induced immunogenic modulation of tumor enhances antigen processing and calreticulin exposure, resulting in enhanced T-cell killing. Oncotarget 2014;5:403-16.
14. A Guide to Radiation Therapy. In: Society AC, editor.2015.
15. Johnston SR, Dowsett M. Aromatase inhibitors for breast cancer: lessons from the laboratory. Nat Rev Cancer 2003;3:821-31.
16. Lam HY. Tamoxifen is a calmodulin antagonist in the activation of cAMP phosphodiesterase. Biochem Biophys Res Commun 1984;118:27-32.
17. Pal SK, Childs BH, Pegram M. Triple negative breast cancer: unmet medical needs. Breast Cancer Res Treat 2011;125:627-36.
18. Agostinis P, Berg K, Cengel KA, Foster TH, Girotti AW, Gollnick SO, et al. Photodynamic therapy of cancer: an update. CA Cancer J Clin 2011;61:250-81.
28 CHAPTER 1: INTRODUCTION
19. Castano AP, Demidova TN, Hamblin MR. Mechanisms in photodynamic therapy: part one-photosensitizers, photochemistry and cellular localization. Photodiagnosis Photodyn Ther 2004;1:279-93.
20. Dougherty TJ, Gomer CJ, Henderson BW, Jori G, Kessel D, Korbelik M, et al. Photodynamic therapy. J Natl Cancer Inst 1998;90:889-905.
21. Zeitouni NC, Shieh S, Oseroff AR. Laser and photodynamic therapy in the management of cutaneous malignancies. Clin Dermatol 2001;19:328-38.
22. Mellman I, Coukos G, Dranoff G. Cancer immunotherapy comes of age. Nature 2011;480:480-9.
23. Drake CG. Combination immunotherapy approaches. Ann Oncol 2012;23 Suppl 8:viii41-6.
24. Nagata Y, Lan KH, Zhou X, Tan M, Esteva FJ, Sahin AA, et al. PTEN activation contributes to tumor inhibition by trastuzumab, and loss of PTEN predicts trastuzumab resistance in patients. Cancer Cell 2004;6:117-27.
25. Bethge WA, Hegenbart U, Stuart MJ, Storer BE, Maris MB, Flowers ME, et al. Adoptive immunotherapy with donor lymphocyte infusions after allogeneic hematopoietic cell transplantation following nonmyeloablative conditioning. Blood 2004;103:790-5.
26. Johansson A, Hamzah J, Payne CJ, Ganss R. Tumor-targeted TNFalpha stabilizes tumor vessels and enhances active immunotherapy. Proc Natl Acad Sci U S A 2012;109:7841-6.
27. Scott AM, Wolchok JD, Old LJ. Antibody therapy of cancer. Nat Rev Cancer 2012;12:278-87.
28. Presta LG, Chen H, O'Connor SJ, Chisholm V, Meng YG, Krummen L, et al. Humanization of an anti-vascular endothelial growth factor monoclonal antibody for the therapy of solid tumors and other disorders. Cancer Res 1997;57:4593-9.
29. Matar P, Rojo F, Cassia R, Moreno-Bueno G, Di Cosimo S, Tabernero J, et al. Combined epidermal growth factor receptor targeting with the tyrosine kinase inhibitor gefitinib (ZD1839) and the monoclonal antibody cetuximab (IMC- C225): superiority over single-agent receptor targeting. Clin Cancer Res 2004;10:6487-501.
30. Colombat P, Salles G, Brousse N, Eftekhari P, Soubeyran P, Delwail V, et al. Rituximab (anti-CD20 monoclonal antibody) as single first-line therapy for patients with follicular lymphoma with a low tumor burden: clinical and molecular evaluation. Blood 2001;97:101-6.
29 CHAPTER 1: INTRODUCTION
31. Lundin J, Kimby E, Bjorkholm M, Broliden PA, Celsing F, Hjalmar V, et al. Phase II trial of subcutaneous anti-CD52 monoclonal antibody alemtuzumab (Campath-1H) as first-line treatment for patients with B-cell chronic lymphocytic leukemia (B-CLL). Blood 2002;100:768-73.
32. Caron PC, Schwartz MA, Co MS, Queen C, Finn RD, Graham MC, et al. Murine and humanized constructs of monoclonal antibody M195 (anti-CD33) for the therapy of acute myelogenous leukemia. Cancer 1994;73:1049-56.
33. Hughes B. Antibody-drug conjugates for cancer: poised to deliver? Nat Rev Drug Discov 2010;9:665-7.
34. Younes A, Bartlett NL, Leonard JP, Kennedy DA, Lynch CM, Sievers EL, et al. Brentuximab vedotin (SGN-35) for relapsed CD30-positive lymphomas. N Engl J Med 2010;363:1812-21.
35. Monjazeb AM, Zamora AE, Grossenbacher SK, Mirsoian A, Sckisel GD, Murphy WJ. Immunoediting and antigen loss: overcoming the achilles heel of immunotherapy with antigen non-specific therapies. Front Oncol 2013;3:197.
36. Michielsen AJ, Hogan AE, Marry J, Tosetto M, Cox F, Hyland JM, et al. Tumour tissue microenvironment can inhibit dendritic cell maturation in colorectal cancer. PLoS One 2011;6:e27944.
37. Zou W. Immunosuppressive networks in the tumour environment and their therapeutic relevance. Nat Rev Cancer 2005;5:263-74.
38. Pardoll DM. The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer 2012;12:252-64.
39. Rosenberg SA. IL-2: the first effective immunotherapy for human cancer. J Immunol 2014;192:5451-8.
40. Schwartzentruber DJ, Lawson DH, Richards JM, Conry RM, Miller DM, Treisman J, et al. gp100 peptide vaccine and interleukin-2 in patients with advanced melanoma. N Engl J Med 2011;364:2119-27.
41. Leffers N, Lambeck AJ, Gooden MJ, Hoogeboom BN, Wolf R, Hamming IE, et al. Immunization with a P53 synthetic long peptide vaccine induces P53-specific immune responses in ovarian cancer patients, a phase II trial. Int J Cancer 2009;125:2104-13.
42. Gulley JL, Madan RA, Tsang KY, Jochems C, Marte JL, Farsaci B, et al. Immune impact induced by PROSTVAC (PSA-TRICOM), a therapeutic vaccine for prostate cancer. Cancer Immunol Res 2014;2:133-41.
30 CHAPTER 1: INTRODUCTION
43. Sanda MG, Smith DC, Charles LG, Hwang C, Pienta KJ, Schlom J, et al. Recombinant vaccinia-PSA (PROSTVAC) can induce a prostate-specific immune response in androgen-modulated human prostate cancer. Urology 1999;53:260-6.
44. Mac Keon S, Ruiz MS, Gazzaniga S, Wainstok R. Dendritic cell-based vaccination in cancer: therapeutic implications emerging from murine models. Front Immunol 2015;6:243.
45. Small EJ, Schellhammer PF, Higano CS, Redfern CH, Nemunaitis JJ, Valone FH, et al. Placebo-controlled phase III trial of immunologic therapy with sipuleucel-T (APC8015) in patients with metastatic, asymptomatic hormone refractory prostate cancer. J Clin Oncol 2006;24:3089-94.
46. Cheever MA, Higano CS. PROVENGE (Sipuleucel-T) in prostate cancer: the first FDA-approved therapeutic cancer vaccine. Clin Cancer Res 2011;17:3520-6.
47. Beer TM, Bernstein GT, Corman JM, Glode LM, Hall SJ, Poll WL, et al. Randomized trial of autologous cellular immunotherapy with sipuleucel-T in androgen-dependent prostate cancer. Clin Cancer Res 2011;17:4558-67.
48. Hodi FS, O'Day SJ, McDermott DF, Weber RW, Sosman JA, Haanen JB, et al. Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med 2010;363:711-23.
49. Prieto PA, Yang JC, Sherry RM, Hughes MS, Kammula US, White DE, et al. CTLA-4 blockade with ipilimumab: long-term follow-up of 177 patients with metastatic melanoma. Clin Cancer Res 2012;18:2039-47.
50. Lipson EJ, Drake CG. Ipilimumab: an anti-CTLA-4 antibody for metastatic melanoma. Clin Cancer Res 2011;17:6958-62.
51. Restifo NP, Dudley ME, Rosenberg SA. Adoptive immunotherapy for cancer: harnessing the T cell response. Nat Rev Immunol 2012;12:269-81.
52. Kochenderfer JN, Dudley ME, Feldman SA, Wilson WH, Spaner DE, Maric I, et al. B-cell depletion and remissions of malignancy along with cytokine-associated toxicity in a clinical trial of anti-CD19 chimeric-antigen-receptor-transduced T cells. Blood 2012;119:2709-20.
53. Lee DW, Kochenderfer JN, Stetler-Stevenson M, Cui YK, Delbrook C, Feldman SA, et al. T cells expressing CD19 chimeric antigen receptors for acute lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial. Lancet 2015;385:517-28.
54. Kalos M, Levine BL, Porter DL, Katz S, Grupp SA, Bagg A, et al. T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia. Sci Transl Med 2011;3:95ra73.
31 CHAPTER 1: INTRODUCTION
55. Ott PA, Hodi FS. Talimogene Laherparepvec for the Treatment of Advanced Melanoma. Clin Cancer Res 2016.
56. Kaltsas GA, Mukherjee JJ, Plowman PN, Monson JP, Grossman AB, Besser GM. The role of cytotoxic chemotherapy in the management of aggressive and malignant pituitary tumors. J Clin Endocrinol Metab 1998;83:4233-8.
57. Azzarelli A, Gronchi A, Bertulli R, Tesoro JD, Baratti D, Pennacchioli E, et al. Low-dose chemotherapy with methotrexate and vinblastine for patients with advanced aggressive fibromatosis. Cancer 2001;92:1259-64.
58. Mitchison TJ. The proliferation rate paradox in antimitotic chemotherapy. Mol Biol Cell 2012;23:1-6.
59. Chabner BA, Roberts TG, Jr. Timeline: Chemotherapy and the war on cancer. Nat Rev Cancer 2005;5:65-72.
60. Mihlon Ft, Ray CE, Jr., Messersmith W. Chemotherapy agents: a primer for the interventional radiologist. Semin Intervent Radiol 2010;27:384-90.
61. Cheung-Ong K, Giaever G, Nislow C. DNA-damaging agents in cancer chemotherapy: serendipity and chemical biology. Chem Biol 2013;20:648-59.
62. Perez EA. Microtubule inhibitors: Differentiating tubulin-inhibiting agents based on mechanisms of action, clinical activity, and resistance. Mol Cancer Ther 2009;8:2086-95.
63. Hodge JW, Garnett CT, Farsaci B, Palena C, Tsang KY, Ferrone S, et al. Chemotherapy-induced immunogenic modulation of tumor cells enhances killing by cytotoxic T lymphocytes and is distinct from immunogenic cell death. Int J Cancer 2013;133:624-36.
64. Kaneno R, Shurin GV, Kaneno FM, Naiditch H, Luo J, Shurin MR. Chemotherapeutic agents in low noncytotoxic concentrations increase immunogenicity of human colon cancer cells. Cell Oncol (Dordr) 2011;34:97-106.
65. Menard C, Martin F, Apetoh L, Bouyer F, Ghiringhelli F. Cancer chemotherapy: not only a direct cytotoxic effect, but also an adjuvant for antitumor immunity. Cancer Immunol Immunother 2008;57:1579-87.
66. Morstyn G, Campbell L, Souza LM, Alton NK, Keech J, Green M, et al. Effect of granulocyte colony stimulating factor on neutropenia induced by cytotoxic chemotherapy. Lancet 1988;1:667-72.
67. Carelle N, Piotto E, Bellanger A, Germanaud J, Thuillier A, Khayat D. Changing patient perceptions of the side effects of cancer chemotherapy. Cancer 2002;95:155-63.
32 CHAPTER 1: INTRODUCTION
68. Fang J, Nakamura H, Maeda H. The EPR effect: Unique features of tumor blood vessels for drug delivery, factors involved, and limitations and augmentation of the effect. Adv Drug Deliv Rev 2011;63:136-51.
69. Matsumura Y, Maeda H. A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs. Cancer Res 1986;46:6387-92.
70. Prabhakar U, Maeda H, Jain RK, Sevick-Muraca EM, Zamboni W, Farokhzad OC, et al. Challenges and key considerations of the enhanced permeability and retention effect for nanomedicine drug delivery in oncology. Cancer Res 2013;73:2412-7.
71. Schroeder A, Heller DA, Winslow MM, Dahlman JE, Pratt GW, Langer R, et al. Treating metastatic cancer with nanotechnology. Nat Rev Cancer 2012;12:39-50.
72. Coxon TP, Fallows TW, Gough JE, Webb SJ. A versatile approach towards multivalent saccharide displays on magnetic nanoparticles and phospholipid vesicles. Org Biomol Chem 2015;13:10751-61.
73. Venter PA, Dirksen A, Thomas D, Manchester M, Dawson PE, Schneemann A. Multivalent display of proteins on viral nanoparticles using molecular recognition and chemical ligation strategies. Biomacromolecules 2011;12:2293-301.
74. Montet X, Funovics M, Montet-Abou K, Weissleder R, Josephson L. Multivalent effects of RGD peptides obtained by nanoparticle display. J Med Chem 2006;49:6087-93.
75. Park JW. Liposome-based drug delivery in breast cancer treatment. Breast Cancer Res 2002;4:95-9.
76. Petros RA, DeSimone JM. Strategies in the design of nanoparticles for therapeutic applications. Nat Rev Drug Discov 2010;9:615-27.
77. Chen ZG. Small-molecule delivery by nanoparticles for anticancer therapy. Trends Mol Med 2010;16:594-602.
78. Chatterjee K, Zhang J, Honbo N, Karliner JS. Doxorubicin cardiomyopathy. Cardiology 2010;115:155-62.
79. Couvreur P, Kante B, Grislain L, Roland M, Speiser P. Toxicity of polyalkylcyanoacrylate nanoparticles II: Doxorubicin-loaded nanoparticles. J Pharm Sci 1982;71:790-2.
80. A.C. A, S. M. Nanoparticles in the Clinic. Bioengineering & Translational Medicine 2016.
33 CHAPTER 1: INTRODUCTION
81. Allen TM, Cullis PR. Liposomal drug delivery systems: from concept to clinical applications. Adv Drug Deliv Rev 2013;65:36-48.
82. Klibanov AL, Maruyama K, Torchilin VP, Huang L. Amphipathic polyethyleneglycols effectively prolong the circulation time of liposomes. FEBS Lett 1990;268:235-7.
83. Allen TM, Hansen C, Martin F, Redemann C, Yau-Young A. Liposomes containing synthetic lipid derivatives of poly(ethylene glycol) show prolonged circulation half-lives in vivo. Biochim Biophys Acta 1991;1066:29-36.
84. Lee RJ, Low PS. Delivery of liposomes into cultured KB cells via folate receptor- mediated endocytosis. J Biol Chem 1994;269:3198-204.
85. Allen TM, Brandeis E, Hansen CB, Kao GY, Zalipsky S. A new strategy for attachment of antibodies to sterically stabilized liposomes resulting in efficient targeting to cancer cells. Biochim Biophys Acta 1995;1237:99-108.
86. Weinstein JN, Magin RL, Yatvin MB, Zaharko DS. Liposomes and local hyperthermia: selective delivery of methotrexate to heated tumors. Science 1979;204:188-91.
87. Yatvin MB, Kreutz W, Horwitz BA, Shinitzky M. pH-sensitive liposomes: possible clinical implications. Science 1980;210:1253-5.
88. Miele E, Spinelli GP, Miele E, Tomao F, Tomao S. Albumin-bound formulation of paclitaxel (Abraxane ABI-007) in the treatment of breast cancer. Int J Nanomedicine 2009;4:99-105.
89. Green MR, Manikhas GM, Orlov S, Afanasyev B, Makhson AM, Bhar P, et al. Abraxane, a novel Cremophor-free, albumin-bound particle form of paclitaxel for the treatment of advanced non-small-cell lung cancer. Ann Oncol 2006;17:1263-8.
90. Von Hoff DD, Ramanathan RK, Borad MJ, Laheru DA, Smith LS, Wood TE, et al. Gemcitabine plus nab-paclitaxel is an active regimen in patients with advanced pancreatic cancer: a phase I/II trial. J Clin Oncol 2011;29:4548-54.
91. Gradishar WJ, Tjulandin S, Davidson N, Shaw H, Desai N, Bhar P, et al. Phase III trial of nanoparticle albumin-bound paclitaxel compared with polyethylated castor oil-based paclitaxel in women with breast cancer. J Clin Oncol 2005;23:7794-803.
92. Gelderblom H, Verweij J, Nooter K, Sparreboom A. Cremophor EL: the drawbacks and advantages of vehicle selection for drug formulation. Eur J Cancer 2001;37:1590-8.
93. Zhao D, Zhao X, Zu Y, Li J, Zhang Y, Jiang R, et al. Preparation, characterization, and in vitro targeted delivery of folate-decorated paclitaxel-loaded bovine serum albumin nanoparticles. Int J Nanomedicine 2010;5:669-77.
34 CHAPTER 1: INTRODUCTION
94. Hamilton RF, Jr., Wu Z, Mitra S, Shaw PK, Holian A. Effect of MWCNT size, carboxylation, and purification on in vitro and in vivo toxicity, inflammation and lung pathology. Part Fibre Toxicol 2013;10:57.
95. Oltra NS, Swift J, Mahmud A, Rajagopal K, Loverde SM, Discher DE. Filomicelles in nanomedicine - from flexible, fragmentable, and ligand-targetable drug carrier designs to combination therapy for brain tumors. Journal of Materials Chemistry B 2013;1:5177-85.
96. Simone EA, Dziubla TD, Discher DE, Muzykantov VR. Filamentous polymer nanocarriers of tunable stiffness that encapsulate the therapeutic enzyme catalase. Biomacromolecules 2009;10:1324-30.
97. Xu J, Wong DH, Byrne JD, Chen K, Bowerman C, DeSimone JM. Future of the particle replication in nonwetting templates (PRINT) technology. Angew Chem Int Ed Engl 2013;52:6580-9.
98. Sharma G, Valenta DT, Altman Y, Harvey S, Xie H, Mitragotri S, et al. Polymer particle shape independently influences binding and internalization by macrophages. J Control Release 2010;147:408-12.
99. Arnida, Janat-Amsbury MM, Ray A, Peterson CM, Ghandehari H. Geometry and surface characteristics of gold nanoparticles influence their biodistribution and uptake by macrophages. Eur J Pharm Biopharm 2011;77:417-23.
100. Shukla S, Eber FJ, Nagarajan AS, DiFranco NA, Schmidt N, Wen AM, et al. The Impact of Aspect Ratio on the Biodistribution and Tumor Homing of Rigid Soft- Matter Nanorods. Adv Healthc Mater 2015;4:874-82.
101. Champion JA, Mitragotri S. Role of target geometry in phagocytosis. Proc Natl Acad Sci U S A 2006;103:4930-4.
102. Geng Y, Dalhaimer P, Cai S, Tsai R, Tewari M, Minko T, et al. Shape effects of filaments versus spherical particles in flow and drug delivery. Nat Nanotechnol 2007;2:249-55.
103. Toy R, Peiris PM, Ghaghada KB, Karathanasis E. Shaping cancer nanomedicine: the effect of particle shape on the in vivo journey of nanoparticles. Nanomedicine (Lond) 2014;9:121-34.
104. Barua S, Yoo JW, Kolhar P, Wakankar A, Gokarn YR, Mitragotri S. Particle shape enhances specificity of antibody-displaying nanoparticles. Proc Natl Acad Sci U S A 2013;110:3270-5.
105. Decuzzi P, Ferrari M. The adhesive strength of non-spherical particles mediated by specific interactions. Biomaterials 2006;27:5307-14.
35 CHAPTER 1: INTRODUCTION
106. Gentile F, Chiappini C, Fine D, Bhavane RC, Peluccio MS, Cheng MM, et al. The effect of shape on the margination dynamics of non-neutrally buoyant particles in two-dimensional shear flows. J Biomech 2008;41:2312-8.
107. Toy R, Hayden E, Shoup C, Baskaran H, Karathanasis E. The effects of particle size, density and shape on margination of nanoparticles in microcirculation. Nanotechnology 2011;22:115101.
108. Lee SY, Ferrari M, Decuzzi P. Shaping nano-/micro-particles for enhanced vascular interaction in laminar flows. Nanotechnology 2009;20.
109. Peiris PM, Toy R, Doolittle E, Pansky J, Abramowski A, Tam M, et al. Imaging metastasis using an integrin-targeting chain-shaped nanoparticle. ACS Nano 2012;6:8783-95.
110. Chauhan VP, Popovic Z, Chen O, Cui J, Fukumura D, Bawendi MG, et al. Fluorescent nanorods and nanospheres for real-time in vivo probing of nanoparticle shape-dependent tumor penetration. Angew Chem Int Ed Engl 2011;50:11417-20.
111. Steinmetz NF. Viral nanoparticles as platforms for next-generation therapeutics and imaging devices. Nanomedicine 2010;6:634-41.
112. Bessa J, Jegerlehner A, Hinton HJ, Pumpens P, Saudan P, Schneider P, et al. Alveolar macrophages and lung dendritic cells sense RNA and drive mucosal IgA responses. J Immunol 2009;183:3788-99.
113. Heil F, Hemmi H, Hochrein H, Ampenberger F, Kirschning C, Akira S, et al. Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science 2004;303:1526-9.
114. Hou B, Saudan P, Ott G, Wheeler ML, Ji M, Kuzmich L, et al. Selective utilization of Toll-like receptor and MyD88 signaling in B cells for enhancement of the antiviral germinal center response. Immunity 2011;34:375-84.
115. Jegerlehner A, Maurer P, Bessa J, Hinton HJ, Kopf M, Bachmann MF. TLR9 signaling in B cells determines class switch recombination to IgG2a. J Immunol 2007;178:2415-20.
116. Jennings GT, Bachmann MF. The coming of age of virus-like particle vaccines. Biol Chem 2008;389:521-36.
117. Lua LH, Connors NK, Sainsbury F, Chuan YP, Wibowo N, Middelberg AP. Bioengineering virus-like particles as vaccines. Biotechnol Bioeng 2014;111:425- 40.
36 CHAPTER 1: INTRODUCTION
118. Manolova V, Flace A, Bauer M, Schwarz K, Saudan P, Bachmann MF. Nanoparticles target distinct dendritic cell populations according to their size. Eur J Immunol 2008;38:1404-13.
119. Peacey M, Wilson S, Baird MA, Ward VK. Versatile RHDV virus-like particles: incorporation of antigens by genetic modification and chemical conjugation. Biotechnol Bioeng 2007;98:968-77.
120. Zhang W, Wang L, Liu Y, Chen X, Liu Q, Jia J, et al. Immune responses to vaccines involving a combined antigen-nanoparticle mixture and nanoparticle- encapsulated antigen formulation. Biomaterials 2014;35:6086-97.
121. In: Knobler S, Lederberg J, Pray LA, editors. Considerations for Viral Disease Eradication: Lessons Learned and Future Strategies: Workshop Summary. Washington (DC)2002.
122. Miller M, Barrett S, Henderson DA. Control and Eradication. In: Jamison DT, Breman JG, Measham AR, Alleyne G, Claeson M, Evans DB, et al., editors. Disease Control Priorities in Developing Countries. 2nd ed. Washington (DC)2006.
123. Schneemann A, Young MJ. Viral assembly using heterologous expression systems and cell extracts. Adv Protein Chem 2003;64:1-36.
124. Chackerian B, Rangel M, Hunter Z, Peabody DS. Virus and virus-like particle- based immunogens for Alzheimer's disease induce antibody responses against amyloid-beta without concomitant T cell responses. Vaccine 2006;24:6321-31.
125. De BP, Pagovich OE, Hicks MJ, Rosenberg JB, Moreno AY, Janda KD, et al. Disrupted adenovirus-based vaccines against small addictive molecules circumvent anti-adenovirus immunity. Hum Gene Ther 2013;24:58-66.
126. Pastori C, Tudor D, Diomede L, Drillet AS, Jegerlehner A, Rohn TA, et al. Virus like particle based strategy to elicit HIV-protective antibodies to the alpha-helic regions of gp41. Virology 2012;431:1-11.
127. Tegerstedt K, Lindencrona JA, Curcio C, Andreasson K, Tullus C, Forni G, et al. A single vaccination with polyomavirus VP1/VP2Her2 virus-like particles prevents outgrowth of HER-2/neu-expressing tumors. Cancer Res 2005;65:5953-7.
128. Montoya ID. Biologics (Vaccines, Antibodies, Enzymes) to Treat Drug Addictions. In: Nady el-Guebaly GC, Marc Galanter, editor. Textbook of Addiction Treatment: International Perspectives: Springer; 2015. p. 683-92.
129. Tissot AC, Renhofa R, Schmitz N, Cielens I, Meijerink E, Ose V, et al. Versatile virus-like particle carrier for epitope based vaccines. PLoS One 2010;5:e9809.
37 CHAPTER 1: INTRODUCTION
130. Van Rompay KK, Hunter Z, Jayashankar K, Peabody J, Montefiori D, LaBranche CC, et al. A vaccine against CCR5 protects a subset of macaques upon intravaginal challenge with simian immunodeficiency virus SIVmac251. J Virol 2014;88:2011-24.
131. Yin Z, Comellas-Aragones M, Chowdhury S, Bentley P, Kaczanowska K, Benmohamed L, et al. Boosting immunity to small tumor-associated carbohydrates with bacteriophage qbeta capsids. ACS Chem Biol 2013;8:1253-62.
132. Zamora E, Handisurya A, Shafti-Keramat S, Borchelt D, Rudow G, Conant K, et al. Papillomavirus-like particles are an effective platform for amyloid-beta immunization in rabbits and transgenic mice. J Immunol 2006;177:2662-70.
133. Lee KL, Twyman RM, Fiering S, Steinmetz NF. Virus-based nanoparticles as platform technologies for modern vaccines. Wiley Interdiscip Rev Nanomed Nanobiotechnol 2016.
134. Lizotte PH, Wen AM, Sheen MR, Fields J, Rojanasopondist P, Steinmetz NF, et al. In situ vaccination with cowpea mosaic virus nanoparticles suppresses metastatic cancer. Nat Nanotechnol 2016;11:295-303.
135. Moore AE. The destructive effect of the virus of Russian Far East encephalitis on the transplantable mouse sarcoma 180. Cancer 1949;2:525-34.
136. Andtbacka RH, Kaufman HL, Collichio F, Amatruda T, Senzer N, Chesney J, et al. Talimogene Laherparepvec Improves Durable Response Rate in Patients With Advanced Melanoma. J Clin Oncol 2015;33:2780-8.
137. Naldini L. Gene therapy returns to centre stage. Nature 2015;526:351-60.
138. Biffi A, Montini E, Lorioli L, Cesani M, Fumagalli F, Plati T, et al. Lentiviral hematopoietic stem cell gene therapy benefits metachromatic leukodystrophy. Science 2013;341:1233158.
139. Kochenderfer JN, Dudley ME, Kassim SH, Somerville RP, Carpenter RO, Stetler- Stevenson M, et al. Chemotherapy-refractory diffuse large B-cell lymphoma and indolent B-cell malignancies can be effectively treated with autologous T cells expressing an anti-CD19 chimeric antigen receptor. J Clin Oncol 2015;33:540-9.
140. Bainbridge JW, Mehat MS, Sundaram V, Robbie SJ, Barker SE, Ripamonti C, et al. Long-term effect of gene therapy on Leber's congenital amaurosis. N Engl J Med 2015;372:1887-97.
141. Nayerossadat N, Maedeh T, Ali PA. Viral and nonviral delivery systems for gene delivery. Adv Biomed Res 2012;1:27.
142. Moriuchi S, Wolfe D, Tamura M, Yoshimine T, Miura F, Cohen JB, et al. Double suicide gene therapy using a replication defective herpes simplex virus vector
38 CHAPTER 1: INTRODUCTION
reveals reciprocal interference in a malignant glioma model. Gene Ther 2002;9:584-91.
143. Aljabali AA, Shukla S, Lomonossoff GP, Steinmetz NF, Evans DJ. CPMV-DOX delivers. Mol Pharm 2013;10:3-10.
144. Lockney DM, Guenther RN, Loo L, Overton W, Antonelli R, Clark J, et al. The Red clover necrotic mosaic virus capsid as a multifunctional cell targeting plant viral nanoparticle. Bioconjug Chem 2011;22:67-73.
145. Wu W, Hsiao SC, Carrico ZM, Francis MB. Genome-free viral capsids as multivalent carriers for taxol delivery. Angew Chem Int Ed Engl 2009;48:9493-7.
146. Yildiz I, Tsvetkova I, Wen AM, Shukla S, Masarapu MH, Dragnea B, et al. Engineering of Brome mosaic virus for biomedical applications. Rsc Advances 2012;2:3670-7.
147. Zhao Q, Chen W, Chen Y, Zhang L, Zhang J, Zhang Z. Self-assembled virus-like particles from rotavirus structural protein VP6 for targeted drug delivery. Bioconjug Chem 2011;22:346-52.
148. Pokorski JK, Breitenkamp K, Liepold LO, Qazi S, Finn MG. Functional virus- based polymer-protein nanoparticles by atom transfer radical polymerization. J Am Chem Soc 2011;133:9242-5.
149. Czapar AE, Zheng YR, Riddell IA, Shukla S, Awuah SG, Lippard SJ, et al. Tobacco Mosaic Virus Delivery of Phenanthriplatin for Cancer therapy. ACS Nano 2016;10:4119-26.
150. Zeng Q, Wen H, Wen Q, Chen X, Wang Y, Xuan W, et al. Cucumber mosaic virus as drug delivery vehicle for doxorubicin. Biomaterials 2013;34:4632-42.
151. Aljabali AA, Sainsbury F, Lomonossoff GP, Evans DJ. Cowpea mosaic virus unmodified empty viruslike particles loaded with metal and metal oxide. Small 2010;6:818-21.
152. Bruckman MA, Hern S, Jiang K, Flask CA, Yu X, Steinmetz NF. Tobacco mosaic virus rods and spheres as supramolecular high-relaxivity MRI contrast agents. J Mater Chem B Mater Biol Med 2013;1:1482-90.
153. Bruckman MA, Jiang K, Simpson EJ, Randolph LN, Luyt LG, Yu X, et al. Dual- modal magnetic resonance and fluorescence imaging of atherosclerotic plaques in vivo using VCAM-1 targeted tobacco mosaic virus. Nano Lett 2014;14:1551-8.
154. Farkas ME, Aanei IL, Behrens CR, Tong GJ, Murphy ST, O'Neil JP, et al. PET Imaging and biodistribution of chemically modified bacteriophage MS2. Mol Pharm 2013;10:69-76.
39 CHAPTER 1: INTRODUCTION
155. Huang X, Stein BD, Cheng H, Malyutin A, Tsvetkova IB, Baxter DV, et al. Magnetic virus-like nanoparticles in N. benthamiana plants: a new paradigm for environmental and agronomic biotechnological research. ACS Nano 2011;5:4037- 45.
156. Miyoshi S, Flexman JA, Cross DJ, Maravilla KR, Kim Y, Anzai Y, et al. Transfection of neuroprogenitor cells with iron nanoparticles for magnetic resonance imaging tracking: cell viability, differentiation, and intracellular localization. Mol Imaging Biol 2005;7:286-95.
157. Qazi S, Liepold LO, Abedin MJ, Johnson B, Prevelige P, Frank JA, et al. P22 viral capsids as nanocomposite high-relaxivity MRI contrast agents. Mol Pharm 2013;10:11-7.
158. Shukla S, Steinmetz NF. Virus-based nanomaterials as positron emission tomography and magnetic resonance contrast agents: from technology development to translational medicine. Wiley Interdiscip Rev Nanomed Nanobiotechnol 2015;7:708-21.
159. Steinmetz NF, Ablack AL, Hickey JL, Ablack J, Manocha B, Mymryk JS, et al. Intravital imaging of human prostate cancer using viral nanoparticles targeted to gastrin-releasing Peptide receptors. Small 2011;7:1664-72.
160. Yildiz I, Lee KL, Chen K, Shukla S, Steinmetz NF. Infusion of imaging and therapeutic molecules into the plant virus-based carrier cowpea mosaic virus: cargo-loading and delivery. J Control Release 2013;172:568-78.
161. Washington-Hughes CL, Cheng Y, Duan X, Cai L, Lee LA, Wang Q. In vivo virus-based macrofluorogenic probes target azide-labeled surface glycans in MCF-7 breast cancer cells. Mol Pharm 2013;10:43-50.
162. Shukla S, Dickmeis C, Nagarajan AS, Fischer R, Commandeur U, Steinmetz NF. Molecular farming of fluorescent virus-based nanoparticles for optical imaging in plants, human cells and mouse models. Biomaterials Science 2014;2:784-97.
163. Ryan MD, Drew J. Foot-and-mouth disease virus 2A oligopeptide mediated cleavage of an artificial polyprotein. EMBO J 1994;13:928-33.
164. Bruckman MA, Steinmetz NF. Chemical modification of the inner and outer surfaces of Tobacco Mosaic Virus (TMV). Methods Mol Biol 2014;1108:173-85.
165. Bruckman MA, Kaur G, Lee LA, Xie F, Sepulveda J, Breitenkamp R, et al. Surface modification of tobacco mosaic virus with "click" chemistry. Chembiochem 2008;9:519-23.
166. Demir M, Stowell MHB. A chemoselective biomolecular template for assembling diverse nanotubular materials. Nanotechnology 2002;13:541-4.
40 CHAPTER 1: INTRODUCTION
167. Yi HM, Nisar S, Lee SY, Powers MA, Bentley WE, Payne GF, et al. Patterned assembly of genetically modified viral nanotemplates via nucleic acid hybridization. Nano Letters 2005;5:1931-6.
168. Wu Y, Feng S, Zan X, Lin Y, Wang Q. Aligned Electroactive TMV Nanofibers as Enabling Scaffold for Neural Tissue Engineering. Biomacromolecules 2015;16:3466-72.
169. Steinmetz NF, Mertens ME, Taurog RE, Johnson JE, Commandeur U, Fischer R, et al. Potato virus X as a novel platform for potential biomedical applications. Nano Lett 2010;10:305-12.
170. Uhde-Holzem K, Schlosser V, Viazov S, Fischer R, Commandeur U. Immunogenic properties of chimeric potato virus X particles displaying the hepatitis C virus hypervariable region I peptide R9. J Virol Methods 2010;166:12- 20.
171. Plchova H, Moravec T, Hoffmeisterova H, Folwarczna J, Cerovska N. Expression of Human papillomavirus 16 E7ggg oncoprotein on N- and C-terminus of Potato virus X coat protein in bacterial and plant cells. Protein Expr Purif 2011;77:146- 52.
172. Morgenfeld M, Segretin ME, Wirth S, Lentz E, Zelada A, Mentaberry A, et al. Potato virus X coat protein fusion to human papillomavirus 16 E7 oncoprotein enhance antigen stability and accumulation in tobacco chloroplast. Mol Biotechnol 2009;43:243-9.
173. Lico C, Mancini C, Italiani P, Betti C, Boraschi D, Benvenuto E, et al. Plant- produced potato virus X chimeric particles displaying an influenza virus-derived peptide activate specific CD8+ T cells in mice. Vaccine 2009;27:5069-76.
174. Marusic C, Rizza P, Lattanzi L, Mancini C, Spada M, Belardelli F, et al. Chimeric plant virus particles as immunogens for inducing murine and human immune responses against human immunodeficiency virus type 1. J Virol 2001;75:8434-9.
175. Cerovska N, Hoffmeisterova H, Moravec T, Plchova H, Folwarczna J, Synkova H, et al. Transient expression of Human papillomavirus type 16 L2 epitope fused to N- and C-terminus of coat protein of Potato virus X in plants. J Biosci 2012;37:125-33.
176. Carette N, Engelkamp H, Akpa E, Pierre SJ, Cameron NR, Christianen PC, et al. A virus-based biocatalyst. Nat Nanotechnol 2007;2:226-9.
177. Shukla S, Ablack AL, Wen AM, Lee KL, Lewis JD, Steinmetz NF. Increased tumor homing and tissue penetration of the filamentous plant viral nanoparticle Potato virus X. Mol Pharm 2013;10:33-42.
41 CHAPTER 2: SHAPE MATTERS
Chapter 2: Shape matters: comparison of TMV and CPMV diffusion
The material in this chapter is adapted with permission from: Lee, K.L.*, Hubbard,
L.C.*, Hern, S., Yildiz, I., Gratzl, M., Steinmetz, N.F. Shape matters: the diffusion rates of TMV rods and CPMV icosahedrons in a spheroid model of extracellular matrix are distinct. Biomater. Sci. 2013, 1: 581-588. Copyright 2013 The Royal Society of
Chemistry
*Both authors contributed equally
As co-first author on this paper, I synthesized the particles, imaged the spheroids, and contributed to paper writing. Logan Hubbard, my co-first author a collaborator in the
Gratzl lab, made the spheroids and performed Matlab analysis.
2.1 Introduction
Shape matters: Nanoparticle-based carrier systems show great promise for applications in tissue-specific imaging and drug delivery, especially for detection and treatment of systemic diseases such as metastatic cancer or cardiovascular disease. The state-of-the-art carrier system is spherical, however, a growing body of data indicates that this may not be optimal. Size and shape (as well as surface chemistry) of nanoparticle carriers determine their cellular and in vivo fate. Distinct advantages of elongated, rod- shaped or filamentous materials over their spherical counterparts have been observed: data indicate that non-spherical materials, such as rods and filaments, have enhanced tumor homing properties [1, 2]. This is explained by the distinct flow properties of rods/filaments versus spheres [3-7]. Based on fluid dynamics elongated, non-spherical
42 CHAPTER 2: SHAPE MATTERS materials marginate better toward the vessel wall, thus have a higher probability to recognize and bind to diseased areas [3, 8, 9]. Further, it is indicated that the elongated materials show enhanced transport across membranes and tissues: nanorods and nanospheres with the same effective hydrodynamic radius and diffusive transport in water show increased penetration rates of rods compared to spheres in gels and tumor tissues [10]. Combined, enhanced margination and transport phenomena contribute to enhanced tumor retention and accumulation of non-spherical materials in tumor tissue.
Still, to date most research is focused on spherical and low-aspect-ratio materials.
Physically and chemically tailoring materials at the nanoscale to create high aspect ratio materials remains technically challenging using synthetic materials. A few examples include polymeric filomicelles (mimicking filamentous viruses) and silica nanorods [4-6,
11, 12]. To overcome this synthetic challenge, we turned toward nature’s materials, specifically our research is focused on the study of viral nanoparticles (VNPs) formed by plant viruses. Some VNPs naturally assemble into high aspect ratio structures, examples include tobacco mosaic virus (TMV) measuring 300x18 nm (aspect ratio: 17), potato virus X (PVX) measuring 515x13 nm (aspect ratio: 40) and grapevine virus A (GVA) measuring 800x12 nm (aspect ratio: 67).
Viral nanoparticles (VNPs): The development and application of VNP-derived materials in the medical field is becoming a growing area of interest and impact. There are many novel types of VNPs in development, with bacteriophages and plant viruses favored because they are considered safer in humans than mammalian viruses [13].
Viruses can be regarded as nature’s carrier systems; they perform the tasks we seek to mimic in nanomedicine, i.e. tissue-specific delivery of cargos. VNPs are genetically
43 CHAPTER 2: SHAPE MATTERS encoded and self-assemble into discrete and monodisperse structures of precise shape and size; their structures are known to atomic resolution. This level of quality control cannot yet be achieved with synthetic nanoparticles.
Although nature provides VNPs in a variety of shapes and sizes, to date most research is focused on the study and development of icosahedral (sphere-like) VNPs.
Based on the emerging data that elongated rods and filaments may provide distinct advantages for drug delivery and imaging, we turned toward a side-by-side evaluation of the tissue penetration properties of VNP rods versus icosahedrons, specifically tobacco mosaic virus (TMV) versus cowpea mosaic virus (CPMV).
In a recent study, we started the evaluation of different shaped VNPs in preclinical tumor models. Data indicate that VNP filaments and icosahedra show differential tumor homing and penetration. Using human tumor xenografts (fibrosarcoma, squamous sarcoma, and colon cancer), it was determined that PEGylated filamentous potato virus X (PVX, 515 by 13 nm) exhibits higher tumor uptake compared to spherical cowpea mosaic virus (CPMV, 30 nm-sized icosahedron), particularly in the core of the tumor. Intravital and ex vivo imaging data were further supported by immunohistochemical analysis of tumor sections, which indicated greater penetration and accumulation of PVX within the tumor tissues [14]. Besides shape-derived advantages of
PVX, surface charge-derived differences also play a role. PVX has a positive, CPMV a negative zeta potential. The collagen-rich extracellular matrix (which is negatively charged) is a major determinant of interstitial transport [15]. Previous studies indicate that charged materials of opposite charges show different tumor homing and penetration properties [16-20]. Therefore it is important to separate charge from geometry to
44 CHAPTER 2: SHAPE MATTERS elucidate how transport phenomena are impacted by shape. To do this, we turned toward two VNP systems of varying shapes by similar surface charge: TMV and CPMV.
2.2 Materials and methods
2.2.1 CPMV and TMV propagation
Previously published procedures were used to propagate CPMV and TMV in
Vigna unguiculata and Nicotiana benthamiana. A combination of chloroform:butanol extraction, PEG precipitation, and ultracentrifugation over sucrose gradients was used to extract CPMV and TMV from infected leaf materials [21, 22]. Virus was resuspended and kept in 0.1 M potassium phosphate buffer pH 7.0; virus concentration was determined by UV/visible spectroscopy using known extinction coefficients at 260 nm
-1 -1 -1 -1 (εCPMV=8.1 mL mg cm and εTMV=3.0 mL mg cm ).
2.2.2 Bioconjugate chemistry to modify CPMV and TMV with A555 and O488
CPMV (in 0.1 M potassium phosphate buffer pH 7.0) was reacted with NHS-
A555 (Invitrogen). Reagents were added in a 10% (v/v) final concentration of DMSO and incubated overnight at room temperature, with agitation. NHS-A555 was added using a molar excess of 2000 per CPMV. CPMV has a molar mass of 5.6x106 g mol-1.
Purification was performed through extensive dialysis. To add alkynes to the interior carboxylic acids, TMV (in 0.1 M potassium phosphate buffer pH 7.0) was first reacted with propargylamine, along with the addition of ethylene carbodiimide (EDC) coupling to activate the carboxylic acids. Propargylamine and EDC were added using an excess of
45 CHAPTER 2: SHAPE MATTERS
25 and 15 equivalents (eq), respectively. To decorate the exterior TMV surface, the phenol ring of tyrosine underwent an electrophilic substitution (pH=9, 30 min) with the diazonium salt generated from 3-ethynylaniline (25 molar eq) to incorporate a terminal alkyne. TMV has a molar mass of 39.4x106 g mol-1. The reaction was incubated overnight at room temperature. TMV-interior alkyne (TMV-iAlk) and TMV-exterior alkyne (TMV- eAlk) were centrifuged at 42,000 rpm (Beckman 50.2 Ti) for 2.5 h at 4 °C. The pellet was resuspended overnight in 1 mL of 0.1 M potassium phosphate buffer (pH 7.0). TMV- iAlk/eAlk was reacted with O488-azide (Invitrogen) using Cu(I)-catalyzed azide-alkyne
1,3-dipolar cycloaddition (CuAAC) reaction. O488-azide was added using a molar excess of 2 eq. The reaction was incubated on ice for 5 min, followed by a 25 min incubation at room temperature. TMV-O488 was purified at 27,000 rpm (Beckman 50.2 Ti) over a 10-
40% sucrose gradient for 2 h. The TMV protein band was collected and subsequently ultracentrifuged at 42,000 rpm (Beckman 50.2 Ti) for 2.5 h, and the resulting pellet was resuspended in 1 mL 0.1 M potassium phosphate buffer (pH 7.0) overnight. We found no differences studying TMV labeled with O488 at the interior versus exterior sites.
2.2.3 UV/visible spectroscopy
UV/visible spectra were recorded using a NanoDrop 2000 spectrophotometer.
46 CHAPTER 2: SHAPE MATTERS
2.2.4 Denaturing gel electrophoresis
10 µg protein samples were analyzed on 4-12% NuPage gels (Life Technologies) in 1x MOPS SDS running buffer. Protein bands were visualized under UV light before staining and under white light after staining the gels with Coomassie blue (0.25% w/v).
2.2.5 Size exclusion chromatography (SEC)
Size exclusion chromatography was performed using a Superose 6 column on the
ÄKTA Explorer chromatography system (GE Healthcare). CPMV-A555 nanoparticles and TMV-O488 nanorods (100 μg in 200 μL) were analyzed at a flow rate of 0.5 mL min-1 in 0.1 M potassium phosphate buffer (pH 7.0).
2.2.6 Transmission electron microscopy (TEM)
CPMV-A555 nanoparticles and TMV-O488 nanorods (20 µL. 0.1 mg mL-1) were negatively stained with 2% (w/v) uranyl acetate for 5 min on a carbon-coated copper grid. Samples were analyzed using a Zeiss Libra 200FE transmission electron microscope operated at 200 kV.
2.2.7 Dynamic light scattering (DLS) and zeta potential measurements
DLS and zeta potential measurements were carried out using a 90 Plus zeta potential analyzer (Brookhaven Instruments Co., USA). Four measurements for CPMV-
A555 nanoparticles and TMV-O488 nanorods (1.5 mL of 0.1 mg mL-1 solutions) were taken, each comprising eight runs. 47 CHAPTER 2: SHAPE MATTERS
2.2.8 Preparation of the spheroids
Spheroids were prepared from 1% (w/v) agarose type VII (2-hydroxyethylagarose type VII, low gelling temperate, Sigma-Aldrich) in PBS at pH 7.4. To dissolve the agarose, the mixture was heated at 50°C for 30 min with magnetic stirring. Upon uniform mixing, the agarose reservoir was then used to generate spheroids in an untreated 24-well dish. In brief, 0.25 – 0.75 μL of liquid agarose was dropped on the bottom of the well where it gelled instantaneously as a half-spheroid. The spheroid hemispheres were kept between 200-800 μm in diameter. After placement of the spheroids, 75 μL of PBS were added to keep them hydrated. Spheroids were prepared fresh for all experiments.
2.2.9 Confocal imaging
An Olympus FV1000 laser scanning confocal microscope was used for imaging.
The spheroid plate was placed on the confocal stage, and the optical scope was used to find a spheroid of appropriate size and shape that was without defects. Imaging was performed using 10x magnification. The imaging plane was kept constant during the entire study. First blank images were taken to correct for autofluorescence. Then, VNPs were added at 0.032 mg mL-1 protein concentration in PBS (total volume was 200 μL).
Image files were saved as .raw files and analyzed using ImageJ software. Data were processed in MatLab.
48 CHAPTER 2: SHAPE MATTERS
2.2.10 MatLab data analysis
RAW (grey scale) images were imported into MatLab, converted into DOUBLE data types, divided by 255, and stored as pixel intensities (ranging from 0-1) in CELL arrays. Within these arrays, all images were normalized according to the same scheme.
Specifically, for each image, the average intensity of the surrounding solution (outside the spheroid) was determined. Each image was then multiplied individually by the inverse of its own solution intensity, resulting in a new normalized solution intensity of
100% for all images. The images were then rescaled to populate the entire plot range. All normalization procedures were performed symmetrically between VNP data, such that
TMV and CPMV diffusion properties could be compared. To generate the figures, four radially oriented pixel sections were selected from each image, spanning outwards from the center of a spheroid to 50 μm past its edge (into the surrounding solution). All pixel sections were equivalent in size (20 pixels wide by 250 pixels long, ~ 25 μm x 325 μm).
Each section was adjusted such that all four sections were aligned in the same direction, and horizontal averaging of sections was performed, resulting in a single average intensity vector with greatly improved signal-to-noise ratio. Using this intensity vector, plots were generated depicting the average intensity versus distance, at multiple time points. Additionally, plots depicting the average intensity versus time, at 100 μm (from the edge of the a spheroid toward its center), were also generated. The rate of intensity change versus time, at 100 μm, was also calculated from the data.
49 CHAPTER 2: SHAPE MATTERS
2.3 Results and discussion
2.3.1 TMV and CPMV properties
TMV and CPMV particles were propagated in Nicotiana benthamiana and Vigna unguiculata plants and purified at yields of 1-2 mg pure VNPs per gram of infected leaf material. TMV particles form a hollow rod-like structure measuring 300 x 18 nm (aspect ratio: 17) with a 4 nm-wide interior channel. CPMV is a 30 nm-sized sphere with icosahedral symmetry. Bioconjugate chemistries on both platforms are well established.
TMV is comprised of 2130 identical copies of a coat protein, and each coat protein displays reactive tyrosine side chains on its exterior surface and carboxylic acids on its interior solvent-exposed surface, each of which can be labeled with chemical modifiers
[23, 24]. CPMV consists of 60 copies of an asymmetric unit that is formed by a small and large coat protein. CPMV displays 300 reactive solvent-exposed lysine side chains that can be labeled using N-hydroxysuccinimide (NHS) active esters [25-28]. Cu(I)-catalyzed azide-alkyne cycloaddition (click chemistry) can also be applied to introduce functionalities to the VNPs [29].
50 CHAPTER 2: SHAPE MATTERS
Figure 2.1. Biochemical characterization of dye-labeled TMV-O488 and CPMV-A555. A) Bioconjugation scheme. B) TEM of UAc negatively-stained TMV-O488 and CPMV-A555, scale bar = 50 nm. C) SDS-PAGE gel under UV light and white light after Coomassie Blue staining, 1 = TMV, 2 = TMV- O488, 3 = CPMV, 4 = CPMV-A555. D) Size exclusion chromatography using FPLC and Superose6 column of TMV-O488 and CPMV-A555, E) UV/visible spectroscopy of CPMV-A555 and TMV-O488.
51 CHAPTER 2: SHAPE MATTERS
TMV was labeled at either external tyrosines or internal carboxylic acids using a two-step reaction: first, an alkyne ligation handle was introduced to the phenol ring of tyrosine through an electrophilic substitution with the diazonium salt generated from 3- ethynylaniline; alternatively, carboxylic acids were labeled with propargylamine via carbodiimide coupling, then Oregon Green 488 was introduced using an azide-activated dye and click chemistry. CPMV was labeled at surface lysine side chains using an NHS- active ester of Alexa Fluor 555. The conjugation sites and chemistries were chosen based on reproducibility in tailoring VNPs with comparable biophysical properties and brightness (see discussion below). Resulting TMV-O488 and CPMV-A555 were purified over sucrose gradients and dialyzed using 10 kDa cut-off spin filters to remove excess reagents.
A combination of techniques was used to confirm structural integrity and quantify the number of dyes covalently attached per VNP (Figure 2.1). The degree of conjugation was quantified based on the UV/visible spectrum using the concentration ratio of O488 or
-1 -1 -1 -1 A555 (Abs at 496, εO488=70,000 cm M , abs at 555, εA555=150,000 cm M ) to TMV
-1 -1 6 and CPMV particles (Abs at 260, εTMV=3.0 mL mg cm , MW of TMV=39.4 x 10
-1 -1 -1 6 -1 gmol , εCPMV = 8.1 mL mg cm , MW of CPMV = 5.6x10 g mol ). TMV was labeled with 530±10% O488 dyes and CPMV was labeled with 80±10% A555 dyes (denoted as
TMV-O488 and CPMV-A555). Transmission electron microscopy (TEM) and dynamic light scattering (DLS) confirmed the presence of monodisperse CPMV nanoparticles and
TMV rods. Zeta potential measurements confirmed negative surface potential for both formulations: CPMV-A555 was measured having an effective diameter of 28 nm and a zeta potential of ζCPMV = -7.5 mV; the effective diameter of TMV was measured to be
52 CHAPTER 2: SHAPE MATTERS
140 nm with a zeta potential of ζTMV = -16.3 mV. (We would like to note that we used agarose, a neutral polymer, as the extracellular matrix model, see details below. We therefore reason that electrostatic interactions of the VNPs with the tissue model can be avoided and that the difference in surface potential of TMV versus CPMV will only minimally impact the results.) Further, size exclusion chromatography using fast protein liquid chromatography and Superose6 column indicated that the particles were intact and showed the VNP-characteristic elution profiles. Co-elution of the fluorophores indicated that the dyes were indeed covalently attached. The latter was further verified using SDS-
PAGE after extensive dialysis; fluorescent appearance of the protein bands under UV light indicated that the labels were covalently attached to the coat proteins (Figure 2.1).
The degree of dye conjugation to TMV and CPMV was found to be comparable: for each formulation approximately 25% of the reactive sites were labeled; 530 dyes were attached to TMV’s 2130 addressable carboxylic acids and 80 dyes were displayed on
CPMV’s 300 available lysine side chains. The dye ratio comparing TMV:CPMV equates the molecular weight ratio: TMV has a 7x higher molecular weight compared to CPMV, and TMV has 7x more dyes attached compared to CPMV; the #dye:MWprotein (MDa) ratio is 13.3 and 13.9 for TMV and CPMV, respectively. Based on the equal chemical labeling with fluorescent dyes, we reasoned that the two formulations would be adequate for the proposed experiments and observed differences could be attributed to differences in shape and size (not the degree of chemical labeling).
53 CHAPTER 2: SHAPE MATTERS
2.3.2 Theory: diffusion rates of TMV and CPMV
Diffusion coefficients of spherical materials can be estimated based on the hydrodynamic radius (RH) of the nanocarrier and the Stokes-Einstein relationship:
D0 = k T / 6 π η RH (1) where k is the Boltzmann constant, T is the temperature, and η is the solvent’s viscosity.
The Stokes-Einstein relationship does not take the shape of the particle into account. For a more accurate estimation of the diffusion coefficient of elongated TMV- based particles, the diffusion coefficient extrapolated to zero concentration of VNPs (D0) was calculated with the Svedberg equation using the particle weight and sedimentation coefficient extrapolated to zero concentration (s0):
D0 = s0 R T / [M (1 - V ρ)] (2) where, R is the gas constant, T is the temperature, M is the molecular weight of the carrier, V is the specific partial volume (V = 0.75 cm3 g-1 for proteins), and ρ is the density of the medium (ρ ≈ 1 g cm-3 for aqueous buffer).
For elongated, approximately cylindrical nanoparticles, such as TMV,
ǁ ┴ translational diffusion coefficients for longitudinal (Dt ) and transverse (Dr ) movement are considered, expressed as
ǁ Dt = (ln (p) + νǁ) k T / 2 π η L (3) and
┴ Dr = (ln (p) + ν┴) k T / 4 π η L (4)
54 CHAPTER 2: SHAPE MATTERS where L and d are the length and diameter of the cylinder, p = L/d, k is the Boltzmann constant, T is temperature, and η is the solvent’s viscosity [30].
Numerical values for of vǁ and v┴ are based on the dimensions of the TMV cylinder and have been determined for TMV as:[31]
2 3 4 νǁ = -0.114 – 0.15 / ln(2p) – 13.5 / (ln 2p) + 37 / (ln 2p) – 22 / (ln 2p) (5) and
2 3 4 ν┴ = 0.866 – 0.15 / ln(2p) – 8.1 / (ln 2p) + 18 / (ln 2p) – 9 / (ln 2p) (6)
Further, the macroscopic (or effective translational) diffusion coefficient (Dt) applies to movements in a random directions and can be expressed as [30]:
Dt = (ln (p) + ν) k T / 3 π η L (7)
where ν = (ν ǁ + ν ┴) / 2.
Data are summarized in Table 2.1. In water/aqueous buffer the smaller CPMV sphere has a predicted 3.9x faster diffusion rate compared to the TMV rod (based on the macroscopic diffusion rate of TMV).
55 CHAPTER 2: SHAPE MATTERS
Table 2.1 Diffusion coefficients of CPMV and TMV
VNP CPMV TMV
Diameter or length x width [nm] 30 300 x 18
14.0 70.0 RH [nm] (measured by DLS)
6 7 Molecular weight [g mol-1] 5.60 x 10 3.94 x 10
-11 -12 Sedimentation coefficient s0 [s] 1.08 x 10 1.94 x 10 (from dbvweb.net database)
2 -1 -7 Stokes-Einstein diffusion coefficient D0 [cm s ] 1.57 x 10 NA
-7 -8 2 -1 1.98 x 10 5.15 x 10 Diffusion coefficient D0 based on s0 [cm s ]
Translational Lengthwise Diffusion coefficient NA 4.86 x 10-8 ǁ 2 -1 Dt [cm s ]
Translational Sideways Diffusion Coefficient NA 3.57 x 10-8 ┴ 2 -1 Dr [cm s ]
Macroscopic Diffusion Coefficient NA 4.00 x 10-8 2 -1 Dt [cm s ]
Phase I: 3.22 ×10-9 Experimental diffusion coefficient Phase I: 5.71 × 10-9 2 -1 -10 -10 DExperimental [cm s ] Phase II: 8.36 ×10 Phase II: 4.76 ×10
2.3.3 The spheroid model of the tumor microenvironment
In healthy tissue the movement of molecules is driven by diffusion (concentration gradients) or convection (pressure gradients), often both. Within tumor tissue, leaky vasculature and lack of functional lymphatics lead to elevated interstitial fluid pressure
56 CHAPTER 2: SHAPE MATTERS
[32], which reduces convective transport from the vessel wall into the interstitial space.
Within the tumor microenvironment, tissue diffusion is the primary mode of nanoparticle and drug transport. Therefore, we chose to mimic the tumor microenvironment using an agarose gel that allows diffusion but no convection. In addition, agarose is a neutral polymer, providing for an uncharged environment that minimizes electrostatic interactions between nanoparticles and matrix [33, 34]. This further allows separation between effects of charge and shape on penetration. The agarose model is cast in the shape of a spheroid: the most commonly used in vitro model of tissue microenvironment.
Spheroids are spherical cultures of thousands of cells, typically a few hundred micrometers to 1 mm in diameter. The spheroid is the classical 3D model of the tumor microenvironment that surrounds a blood capillary [35-37]. Spheroids replicate, to varying degrees, in vivo behavior such as cell-cell interactions, drug penetration and uptake, and interaction with local acidity, oxygenation, and other experimental factors in tumor tissue. We have studied these phenomena in compact spheroids as well as in spheroids made of low-concentration agarose gel with cells suspended in the matrix.
Agarose has pore size distributions similar to the extracellular matrix [38, 39]. By varying cell volume fraction (CVF) from zero up to compact culture, such agarose constructs can be used to obtain information on drug penetration and uptake as they change with increasing CVF.
The aim of this work was to model the penetration of spherical and rod-shaped
VNPs into spheroids made of agarose, with CVF=0. This is to obtain comparable data for
CPMV and TMV based on purely physical interactions and transport within the support matrix and under conditions similar to drug penetration studies conventionally done on
57 CHAPTER 2: SHAPE MATTERS spheroids. It should be noted that while the extracellular matrix of a tumor is primarily composed of a network of collagen fibers and other molecules such as glygosaminoglycans (GAGs) which contribute to a negatively-charged environment, our basic, physical model using agarose spheroids offers an electrostatically neutral environment. In the presented study we chose to use a model made of agarose in order to study the effect of one variable alone between the two viruses, i.e. shape, on penetration.
Of course, one should keep in mind that future models should consider the more complex biological environment, and must take into account local charges as well as the surface chemistries of the nanocarriers. The long-term goal of this work is to compare penetration and uptake of spherical and rod-shaped VNPs and free drug in spheroids of increasing
CVF to compact spheroids so that penetration into tissue can be better understood.
2.3.4 Experimental diffusion rates of TMV and CPMV
To mimic diffusion of the two carrier systems into the tumor environment, we used the well-established spheroid model with one difference: hemispheres were used. It has been problematic to obtain data from different depths of full spherical spheroids during drug penetration. Therefore, in this work, agarose half-spheroids were generated by placing liquid agarose directly onto cover slips. The half-spheroid creates a “window” into the interior of the spheroid from below. This is achieved without altering mass transport, as there would be no net transport perpendicular to the base plane even in the intact spheroid because of radial symmetry of the transport [40]. The use of half- spheroids made it possible in this work to obtain snapshots of the distribution of VNPs inside the spheroid during penetration, as they evolve in time.
58 CHAPTER 2: SHAPE MATTERS
Specifically, 1% (w/v) agarose (type VII) gels were made. Agarose half-spheroids
(200-800 μm in diameter) were seeded on a 24-well plate and kept hydrated in PBS solution. Confocal imaging was performed at room temperature over a 6 h time frame. In brief, samples (free dye and/or VNP sphere and/or VNP rod) were added to the solution and spheroids imaged. First, the penetration of free dye (O488 or rhodamine red) was analyzed, then CPMV-A555 and TMV-O488. Once the imaging parameters were optimized, competition assays comparing CPMV-A555 versus TMV-O488 were conducted. We also compared the diffusion rates of CPMV-A555 versus free O488 dye and TMV-O488 versus free rhodamine red dye (Figures 2.3 and 2.4).
59 CHAPTER 2: SHAPE MATTERS
60 CHAPTER 2: SHAPE MATTERS
Figure 2.2. Diffusion rates of VNP rod TMV and sphere CPMV into agarose half spheroids. A) Snapshots of confocal images showing distinct diffusion of TMV-O488 versus CPMV-A555 (note: the dark spots in the spheroid are air bubbles). The red bars are 300 μm in size and indicate the 4 slices analyzed and averaged in MatLab. B) VNPs were added to the medium and imaging was performed over a 6 h time frame. Imaging data were analyzed using ImageJ and MatLab software. The fluorescence intensity normalized against reservoir fluorescence intensity (saturated with VNPs) is plotted over distance, with 0 μm being the edge of the spheroid structure. Fluorescence intensity versus distance is plotted over time. C) Fluorescence intensity measured at 100 μm distance within the spheroid over time is plotted. D) The integrated fluorescence intensity change over time (d/dt fluorescence intensity %) is plotted against time; data indicate a bi-phasic behavior for TMV and CPMV, with opposite trends of diffusion rates in phase I and II; the diffusion rate in phase I is characterized by Dphase I TMV >> CPMV; in phase II this trend is reversed with Dphase II TMV << CPMV.
Based on the much larger molecular weight and size of the VNP-based carrier
6-7 -1 2 -1 systems compared to free dye (10 g mol versus 10 g mol ), the free dye diffuses much faster and saturates the spheroid within minutes (Figures 2.3 and 2.4).
For the competition diffusion assays, CPMV and TMV were added at comparable concentrations: we performed the imaging keeping the protein concentration (mg/mL
VNP), and most importantly, the amount of cargo (mM dye delivered) constant rather than the number of particles or molar VNP concentration. The overall goal is to define a carrier system that efficiently delivers a high payload of cargo to tumor tissues (not to deliver a high concentration of carrier). Furthermore, this allowed us to keep the fluorescence intensity of each VNP solution comparable: 0.032 mg mL-1 of CPMV-A555 equates to 15 pmolar dye and 220 fmolar of CPMV nanoparticles, and 0.032 mg mL-1 of
61 CHAPTER 2: SHAPE MATTERS
TMV-O488 equates to 15 pmolar of dye, but 30 fmolar concentration of nanorods. Data are plotted in fluorescence intensity versus distance and over time (Figure 2.2).
Figure 2.3. Diffusion rates of CPMV sphere versus free O488 dye into agarose half spheroids. A) Snapshots of confocal images showing distinct diffusion of CPMV-A555 versus O488 dye. Time point 0 sec does not represent 0 sec exactly: this is due to the delay between adding the samples into the reservoir and acquiring the first image. It is apparent from the data that dye diffusion occurs rapidly and the spheroid is saturated within minutes. B) Imaging data were analyzed using ImageJ and MatLab software. The fluorescence intensity normalized against reservoir fluorescence intensity (saturated with VNPs or dyes) is plotted over distance, with 0 μm being the edge of the spheroid structure. Fluorescence intensity versus distance is plotted over time. C) Fluorescence intensity over time is plotted measured at 25 and 200 μm distance within the spheroid.
62 CHAPTER 2: SHAPE MATTERS
Figure 2.4. Diffusion rates of TMV rod versus free Rhodamine red dye into agarose half-spheroids. A) Snapshots of confocal images showing distinct diffusion of TMV-O488 versus Rhodamine red dye. Time point 0 sec does not represent 0 sec exactly: this is due to the delay between adding the samples into the reservoir and acquiring the first image. It is apparent from the data that dye diffusion occurs rapidly and the spheroid is saturated within minutes. B) Imaging data were analyzed using ImageJ and MatLab software. The fluorescence intensity normalized against reservoir fluorescence intensity (saturated with VNPs or dyes) is plotted over distance, with 0 μm being the edge of the spheroid structure. Fluorescence intensity versus distance is plotted over time. C) Fluorescence intensity over time is plotted measured at 25 and 200 μm distance within the spheroid.
63 CHAPTER 2: SHAPE MATTERS
Overall, we found that TMV rods and CPMV spheres show distinct and bi-phasic diffusion rates, which are characterized by a first, more rapid, loading phase (phase I, ~
10 min) and a second, slower, distribution phase (phase II). Interestingly, the diffusion rate of the TMV rod is faster in phase I compared to the CPMV sphere; in phase II the opposite trend is observed; further phase II is characterized by a slower, constant diffusion rate for both VNPs.
The diffusion rates of CPMV and TMV in agarose were extracted from the experimental data using diffusion theory [22, 41]. At short times after the beginning of
VNP penetration into the spheroid, and relatively close to the edge of the spheroid, the following equation can be used for the evaluation of diffusion coefficients in the initial period, from the data [22]:
CVNP in spheroid (x, t) = CVNP in buffer erfc [x/(√Dt)] (8)
At t = 5 min and x = 100 µm, the equation yields 3.22 ×10-9 cm2 s-1 for CPMV and 5.71 ×
10-9 cm2 s-1 for TMV.
In water/aqueous buffer the smaller CPMV sphere has a 5× faster diffusion rate compared to the TMV rod as predicted, see Table 1. In agarose, this situation is reversed: the spherical CPMV nanoparticle has about 2× the diffusion rate compared to TMV. In other words the diffusion coefficient of CPMV reduces about 60-fold in agarose relative to buffer while the diffusion coefficient of TMV reduces only about 7-fold. However at
60 min, the approximated diffusion coefficient of TMV is 12-fold less compared to its value at 5 min while that for CPMV reduces relatively little (about 4-fold).
64 CHAPTER 2: SHAPE MATTERS
The fast initial loading of TMV rods into the spheroids may be explained by the differences between axial diffusion and diffusion orthogonal to the axis of the high aspect ratio (AR 17) TMV rods (with Daxial >> Dorthogonal). For the rod-shaped TMV molecule its spontaneous movement axially is more facilitated than movement orthogonal to its axis.
This is not apparent for a large assembly of molecules in aqueous buffer because all orientations would be equally probable. However, diffusion within the agarose matrix is more complex; the rods diffuse across channels and pores whose dimensions are not much greater than the particles.
The average pore size of the agarose matrix varies with concentration and type of agarose used. Using a similar experimental setup, others reported that 1% (w/v) agarose
(2-hydroxyethylagarose type VII, low gelling temperate) gels have a pore size distribution of 100-700 nm with a mean of 400 nm [38, 39]. The pores are thus expected to be about an order of magnitude larger than CPMV but are on the same size-scale as
TMV along its axis. We reason that within the channels the rod-like TMV particles are preferentially oriented with the axis parallel to the channels. This would promote favored axial diffusion along channels, i.e., more efficient anisotropic diffusion within the agarose model than isotropic diffusion in the buffer. In the same time the spherical
CPMV particles would have thermal motion in random directions inside the pores; this is slower than axial diffusion of rods of smaller diameter. The dramatic decrease in TMV diffusion rate after about 20 min is probably because once a TMV rod gets trapped in a pore no other TMV particle can pass by. That CPMV diffusion also slows though gradually and to a much lesser extent is because the smaller pores that TMV cannot even
65 CHAPTER 2: SHAPE MATTERS get in can also trap CPMV particles and this reduces the number of available paths for penetration.
2.4 Conclusions
The observed faster initial diffusion rates of rod-shaped versus spherical nanoparticles into the tissue is interesting and in agreement with the recent notion that elongated materials show enhanced tumor homing and penetration properties. The enhanced permeability and retention effect observed in some tumors has been utilized to passively target nanomaterials to solid tumors. If the material, however, does not sufficiently fast extravasate and penetrate from the vessel wall into the tissue, washout effects can reduce the effective carrier, and hence drug load, in the tumor tissue. Rapid initial tissue loading of high aspect ratio materials can provide a distinct advantage, allowing the materials to penetrate into the tissue more rapidly thus increasing access while reducing such washout effects. In addition, we propose that similar effects would be beneficial when actively targeting tumors or metastatic disease, rapid tissue penetration is expected to increase the accumulation of the carrier materials at the target disease sites. The movement and diffusion of extravasated drug carrier materials, as modeled and measured here in a tumor microenvironment model, indicate distinct advantages of elongated rod-shaped carrier systems.
66 CHAPTER 2: SHAPE MATTERS
2.5 Works cited in this chapter
1. Daum N, Tscheka C, Neumeyer A, Schneider M. Novel approaches for drug delivery systems in nanomedicine: effects of particle design and shape. Wiley Interdisciplinary Reviews-Nanomedicine and Nanobiotechnology 2012;4:52-65.
2. Caldorera-Moore M, Guimard N, Shi L, Roy K. Designer nanoparticles: incorporating size, shape and triggered release into nanoscale drug carriers. Expert opinion on drug delivery 2010;7:479-95.
3. Lee S-Y, Ferrari M, Decuzzi P. Shaping nano-/micro-particles for enhanced vascular interaction in laminar flows. Nanotechnology 2009;20:495101.
4. Gentile F, Chiappini C, Fine D, Bhavane RC, Peluccio MS, Cheng MM-C, et al. The effect of shape on the margination dynamics of non-neutrally buoyant particles in two-dimensional shear flows. Journal of biomechanics 2008;41:2312- 8.
5. Geng Y, Dalhaimer P, Cai S, Tsai R, Tewari M, Minko T, et al. Shape effects of filaments versus spherical particles in flow and drug delivery. Nature nanotechnology 2007;2:249-55.
6. Christian DA, Cai S, Garbuzenko OB, Harada T, Zajac AL, Minko T, et al. Flexible filaments for in vivo imaging and delivery: persistent circulation of filomicelles opens the dosage window for sustained tumor shrinkage. Molecular pharmaceutics 2009;6:1343-52.
7. Cai S, Vijayan K, Cheng D, Lima EM, Discher DE. Micelles of Different Morphologies—Advantages of Worm-like Filomicelles of PEO-PCL in Paclitaxel Delivery. Pharmaceutical Research 2007;24:2099-109.
8. Decuzzi P, Godin B, Tanaka T, Lee S-Y, Chiappini C, Liu X, et al. Size and shape effects in the biodistribution of intravascularly injected particles. Journal of Controlled Release 2010;141:320-7.
9. Gentile F, Chiappini C, Fine D, Bhavane RC, Peluccio MS, Cheng MM-C, et al. The effect of shape on the margination dynamics of non-neutrally buoyant particles in two-dimensional shear flows. J Biomech 2008;41:2312-8.
10. Chauhan VP, Popovic Z, Chen O, Cui J, Fukumura D, Bawendi MG, et al. Fluorescent Nanorods and Nanospheres for Real-Time In Vivo Probing of Nanoparticle Shape-Dependent Tumor Penetration. Angewandte Chemie International Edition 2011:n/a-n/a.
11. Decuzzi P, Pasqualini R, Arap W, Ferrari M. Intravascular delivery of particulate systems: does geometry really matter? Pharmaceutical Research 2009;26:235-43.
67 CHAPTER 2: SHAPE MATTERS
12. Decuzzi P, Godin B, Tanaka T, Lee S-Y, Chiappini C, Liu X, et al. Size and shape effects in the biodistribution of intravascularly injected particles. Journal of controlled release : official journal of the Controlled Release Society 2010;141:320-7.
13. Manchester M, Singh P. Virus-based nanoparticles (VNPs): platform technologies for diagnostic imaging. Adv Drug Deliv Rev 2006;58:1505-22.
14. Shukla S, Ablack A, Wen A, Lee K, Lewis J, Steinmetz NF. Increased tumor homing and tissue penetration of the filamentous plant viral nanoparticle Potato virus X. Molecular pharmaceutics 2012.
15. Jain RK, Stylianopoulos T. Delivering nanomedicine to solid tumors. Nat Rev Clin Oncol 2010;7:653-64.
16. Campbell RB, Fukumura D, Brown EB, Mazzola LM, Izumi Y, Jain RK, et al. Cationic charge determines the distribution of liposomes between the vascular and extravascular compartments of tumors. Cancer Research 2002;62:6831-6.
17. Dellian M, Yuan F, Trubetskoy VS, Torchilin VP, Jain RK. Vascular permeability in a human tumour xenograft: molecular charge dependence. British journal of cancer 2000;82:1513-8.
18. Schmitt-Sody M, Strieth S, Krasnici S, Sauer B, Schulze B, Teifel M, et al. Neovascular targeting therapy: paclitaxel encapsulated in cationic liposomes improves antitumoral efficacy. Clinical cancer research : an official journal of the American Association for Cancer Research 2003;9:2335-41.
19. Stylianopoulos T, Diop-Frimpong B, Munn LL, Jain RK. Diffusion anisotropy in collagen gels and tumors: the effect of fiber network orientation. Biophys J 2010;99:3119-28.
20. Stylianopoulos T, Poh MZ, Insin N, Bawendi MG, Fukumura D, Munn LL, et al. Diffusion of particles in the extracellular matrix: the effect of repulsive electrostatic interactions. Biophys J 2010;99:1342-9.
21. Foster GD, Taylor S. Plant Virology Protocols: From Virus Isolation to Transgenic Resistance. Humana Press; 1998.
22. Steinmetz NF, Cho CF, Ablack A, Lewis JD, Manchester M. Cowpea mosaic virus nanoparticles target surface vimentin on cancer cells. Nanomedicine 2011;6:351-64.
23. Schlick TL, Ding Z, Kovacs EW, Francis MB. Dual-surface modification of the tobacco mosaic virus. Journal of the American Chemical Society 2005;127:3718- 23.
68 CHAPTER 2: SHAPE MATTERS
24. Bruckman MA, Kaur G, Lee LA, Xie F, Sepulveda J, Breitenkamp R, et al. Surface modification of tobacco mosaic virus with "click" chemistry. Chembiochem 2008;9:519-23.
25. Steinmetz NF, Manchester M. PEGylated Viral Nanoparticles for Biomedicine: The Impact of PEG Chain Length on VNP Cell Interactions In Vitro and Ex Vivo. Biomacromolecules 2009;10:784-92.
26. Steinmetz NF, Ablack A, Hickey JL, Ablack J, Manocha B, Mymryk JS, et al. Intravital Imaging of Human Prostate Cancer using Viral Nanoparticles Targeted to Gastrin-Releasing Peptide Receptors. Small 2011;in press.
27. Chatterji A, Ochoa WF, Paine M, Ratna BR, Johnson JE, Lin T. New addresses on an addressable virus nanoblock; uniquely reactive Lys residues on cowpea mosaic virus. Chemistry & biology 2004;11:855-63.
28. Basle E, Joubert N, Pucheault M. Protein chemical modification on endogenous amino acids. Chem Biol 2010;17:213-27.
29. Sen Gupta S, Kuzelka J, Singh P, Lewis WG, Manchester M, Finn MG. Accelerated bioorthogonal conjugation: a practical method for the ligation of diverse functional molecules to a polyvalent virus scaffold. Bioconjug Chem 2005;16:1572-9.
30. Tirado MM, Martinez CL, Garcia de la Torre J. J Chem Phys 1984;81:2047-52.
31. Broersma S. J Chem Phys 1981;74:6969-70.
32. Jain RK. Transport of molecules, particles, and cells in solid tumors. Annual review of biomedical engineering 1999;1:241-63.
33. Kelly TA, Ng KW, Wang CC, Ateshian GA, Hung CT. Spatial and temporal development of chondrocyte-seeded agarose constructs in free-swelling and dynamically loaded cultures. J Biomech 2006;39:1489-97.
34. Marinho-Soriano E. Agar polysaccharides from Gracilaria species (Rhodophyta, Gracilariaceae). J Biotechnol 2001;89:81-4.
35. Labarbera DV, Reid BG, Yoo BH. The multicellular tumor spheroid model for high-throughput cancer drug discovery. Expert opinion on drug discovery 2012;7:819-30.
36. Dufau I, Frongia C, Sicard F, Dedieu L, Cordelier P, Ausseil F, et al. Multicellular tumor spheroid model to evaluate spatio-temporal dynamics effect of chemotherapeutics: application to the gemcitabine/CHK1 inhibitor combination in pancreatic cancer. BMC cancer 2012;12:15.
69 CHAPTER 2: SHAPE MATTERS
37. Santini MT, Rainaldi G. Three-dimensional spheroid model in tumor biology. Pathobiology : journal of immunopathology, molecular and cellular biology 1999;67:148-57.
38. Narayanan J, Xiong J-Y, Liu X-Y. Determination of agarose gel pore size: Absorbance measurements vis a vis other techniques. Journal of Physics 2006;28:83-6.
39. Ng KW, Wang CC, Mauck RL, Kelly TA, Chahine NO, Costa KD, et al. A layered agarose approach to fabricate depth-dependent inhomogeneity in chondrocyte-seeded constructs. Journal of orthopaedic research : official publication of the Orthopaedic Research Society 2005;23:134-41.
40. Sheth. Multielectrode platform for measuring oxygenation status in multicellular tumor spheroids. PhD Thesis, CWRU 2011.
41. Crank J. The mathematics of diffusion. Oxford: Oxford University Press; 2004.
70 CHAPTER 3: TMV FOR PDT
Chapter 3: Tobacco mosaic virus for delivery of photodynamic therapy
The material in this chapter is adapted with permission from: Lee, K.L., Carpenter,
B.L., Wen, A.M., Ghiladi, R.A., Steinmetz, N.F. High aspect ratio nanotubes formed by tobacco mosaic virus for delivery of photodynamic agents targeting melanoma. ACS
Biomater. Sci. Eng. 2016, doi: 10.1021/acsbiomaterials.6b00061. Copyright 2016
American Chemical Society.
3.1 Introduction
Melanoma, which arises from melanocytes, is a highly aggressive cancer, and although it accounts for only 4% of skin cancers, it results in approximately 79% of skin cancer related deaths [1]. Even when diagnosed early, approximately 20% of cases cannot be treated using surgical resection [1]. Additionally, it is unresponsive to many known therapies [2, 3]. Recent data suggest that photodynamic therapy (PDT) provides a novel, efficacious regime for treating melanoma that may hold potential as an adjuvant therapy for all stages of melanoma [1, 3-7].
PDT is an emerging technique for cancer therapy, and unlike many current cancer therapies, PDT consists of three nontoxic components: a photosensitizer (PS), light, and oxygen [8]. Only when all three components are present, is cell killing induced, therefore making it a safe alternative to other cancer treatments, such as chemotherapy and radiotherapy [8]. Specifically, when light of a certain wavelength activates the photosensitizer in the presence of oxygen, it causes a photochemical reaction that generates reactive oxygen species (ROS). These ROS lead to oxidative stress of
71 CHAPTER 3: TMV FOR PDT biomolecules to damage organelle function, leading to apoptosis and necrosis [9]. In addition to directly impacting tumor cells, PDT has been shown to cause damage in the tumor microenvironment. ROS cause damage to the tumor vasculature, which can prevent the necessary oxygen and nutrient exchange, therefore enhancing the overall treatment efficacy [10]. Additionally, PDT has been shown to elicit an antitumor immune response at the site of administration, further potentiating the therapy [10].
A few photosensitizers have received FDA approval for clinical use; these include the photosensitizer Photofrin (porfimer sodium) for use in esophageal and non-small cell lung cancers [11]. Aminolevulinic acid (AVA) has been approved for precancerous
Barrett’s esophagus [12]. The development pipeline is rapidly expanding with numerous clinical and preclinical trials investigating novel photosensitizers and their use in PDT targeting various cancers. These include head and neck, skin, cervical, lung, gastric, prostate, and breast cancers [11, 13-19].
Nevertheless, translational challenges exist: photosensitizers are delivered intravenously, but have poor bioavailability and low accumulation in the tumor tissue. To ensure that appropriate amounts reach the site of disease, large amounts have to be delivered, resulting in dispersal of the photosensitizer throughout the body. As a result, patients have to avoid sunlight for four to six weeks following treatment. Additionally, many photosensitizers are hydrophobic, making them insoluble in physiological conditions [18]. Nanoparticle platform technologies hold promise to enhance solubility, bioavailability, and biodistribution allowing drug targeting to the site of disease; the potential of nanoparticle delivery of photosensitizers has been recognized [20].
Nanoparticles can carry large quantities of photosensitizers and enhance cell uptake and
72 CHAPTER 3: TMV FOR PDT therefore cargo delivery. Nanoparticles have a propensity to accumulate in tumors through passive homing based on the enhanced permeability and retention effect or they can be engineered with targeting ligands to impart tissue specificity [21].
Toward the goal to develop a nanoparticle PDT technology, we turned toward a biology-inspired platform, specifically using the nanocarriers formed by plant viruses as the delivery system. Plant virus-based scaffolds can be produced inexpensively at high yields in plants. The protein-based nanoparticles are highly monodisperse, and their structures are known to atomic resolution. Viruses have naturally evolved to deliver cargos, but plant viruses are noninfectious toward mammals. They are biocompatible and biodegradable and therefore offer favorable properties for in vivo medical applications
[22-24].
In this work, we focused on the nucleoprotein components formed by the tobacco mosaic virus (TMV). TMV is a 300 x 18 nm hollow rod, with a 4 nm wide interior channel. Its structure is known to atomic resolution, and the chemistries for modifying the coat protein have been well established [25, 26]. The in vitro and in vivo properties of
TMV have been well characterized: TMV exhibits shape-mediated enhanced tumor homing and penetration compared to spherical viruses [27-30]. Therefore, we reasoned that TMV would be a suitable carrier for delivery of photosensitizers. Specifically, we sought to develop TMV as a carrier for a porphyrin-based photosensitizer: 5-(4- ethynylphenyl)-10,15,20-tris-(4-methylpyridin-4-ium-1-yl)porphyrin-zinc(II) triiodide
(Zn-EpPor) [31].
Zn-EpPor is a cationic porphyrin (Figure 3.1A) previously used in antimicrobial photodynamic inactivation (aPDI) studies. It has been successfully conjugated to 73 CHAPTER 3: TMV FOR PDT cellulose, both as nanocrystals and as fibers, to create photoactivatable materials that were shown to be effective against various strains of drug resistant bacteria, including multidrug-resistant Acinetobacter baumannii (MDRAB), methicillin-resistant
Staphylococcus aureus (MRSA), and vancomycin-resistant Enterococcus faecium, as well as effective against viruses, including dengue-1, influenza A, and human adenovirus-5 [31-33]. Unlike other porphyrin-based PDT molecules, Zn-EpPor has an overall cationic charge and contains a zinc molecule within the porphyrin ring [31].
Recent work indicates that the presence of a cationic charge enhances accumulation within the mitochondria, while the presence of zinc stabilizes the porphyrin ring, both of which improve therapeutic efficacy [14]. Zn-EpPor is unique in that it contains both of these characteristics, making it a suitable candidate for a proof-of-principle study using a nanoparticle strategy targeting cancer. We demonstrate the nanoparticle formulation of
Zn-EpPor and its use in cancer PDT. Specifically, Zn-EpPor was encapsulated into the central TMV channel, allowing for increased therapeutic delivery and efficacy. As a proof-of-concept, melanoma was studied as the test bed.
3.2 Materials and methods
3.2.1 Zn-EpPor synthesis
5-(4-ethynylphenyl)-10,15,20-tris-(4-methylpyridin-4-ium-1-yl)porphyrin-zinc(II) triiodide (Zn-EpPor) was synthesized in a four-step procedure, as described previously
[31].
74 CHAPTER 3: TMV FOR PDT
3.2.2 TMV propagation
TMV was propagated in Nicotiana benthamiana plants. Infection was carried out using 100 ng mL-1 TMV in 0.1 M potassium phosphate (KP) buffer (pH 7.0); to promote the infectious process leaves were dusted with carborundum prior to mechanical inoculation. Leaves were collected 18-20 days postinfection and TMV was isolated using established procedures [34]. Virus concentration was determined by UV/visible
-1 -1 spectroscopy (εTMV = 3.0 mL mg cm ).
3.2.3 Zn-EpPor loading into TMV
Both wild-type TMV and a TMV-Lys mutant (TMVLys; T158K) [35] were investigated for modification with Zn-EpPor. TMVLys was modified with an azide functional handle, followed by click chemistry using previously established methods
[26]. Alternatively, TMV (1 mg mL-1 final concentration, in 0.01 M KP buffer, pH 7.8) was incubated with a 6000 molar excess of Zn-EpPor, with agitation, overnight. TMV-
Zn-EpPor was purified over a 40% (w/v) sucrose cushion using ultracentrifugation at
212,000g for 3 h at 4 °C. Zn-EpPor-loaded TMV (Zn-EpPorTMV) nanoparticles were analyzed using a combination of UV/visible spectroscopy, inductively-coupled plasma optical emission spectroscopy (ICP-OES), transmission electron microscopy (TEM), and size exclusion chromatography (SEC); see below. To confirm that Zn-EpPor was indeed loaded into the central channel of TMV, chemically modified TMV was utilized in which either exterior or interior surface reactive groups were modified with alkynes to shield surface charges; the bioconjugation protocols were as previously described [25].
75 CHAPTER 3: TMV FOR PDT
3.2.4 UV/visible spectroscopy
The number of Zn-EpPor molecules per TMV nanoparticle was determined using both UV/visible spectroscopy and ICP-OES (see below). Using the NanoDrop 2000 spectrophotometer, Zn-EpPor loading was determined using the Beer-Lambert law and
-1 -1 -1 -1 the Zn-EpPor (ε450nm = 195,000 M cm ) and TMV (ε260nm = 3 mL mg cm ) molar absorptivity coefficients.
3.2.5 Inductively coupled plasma optical emission spectroscopy (ICP-OES)
As a complementary method, ICP-OES was used to determine the number of Zn-
EpPor molecules per TMV nanoparticle. This was achieved by quantification of the
Zn:TMV ratio. To release Zn cations from the porphyrin backbone, Zn-EpPorTMV was incubated in 1 M HCl for 2 h at 60 °C. Following incubation, the solution was diluted to
0.1 mg mL-1 TMV and analyzed immediately at λ=202.548. The Zn concentration was determined using a calibration standard curve.
3.2.6 Size exclusion chromatography (SEC)
Zn-EpPorTMV particles were analyzed by SEC using a Superose6 column and
ÄKTA Explorer chromatography system (GE Healthcare). Samples (100 μL, 1 mg mL-1) were analyzed at a flow rate of 0.5 mL min-1 in 0.01 M potassium phosphate buffer, pH
7.
76 CHAPTER 3: TMV FOR PDT
3.2.7 Transmission electron microscopy
Transmission electron microscopy (TEM) was performed before and after light
-1 illumination to assess the stability of the drug delivery system. Zn-EpPorTMV (1 mgmL ) was illuminated in a rectangle (10.5 cm x 11 cm) under white light from a Vivitek
D950HD projector (~10 mW cm-2 at 430 nm) for 30 min (18.1 J cm-2 at 430 nm). Control samples were kept in the dark for 30 min. Samples were then diluted to 0.1 mg mL-1, placed on carbon-coated copper grids, and negatively stained with 2% (w/v) uranyl acetate for 5 min prior to imaging. Samples were analyzed using a Zeiss Libra 200FE transmission electron microscope operated at 200 kV.
3.2.8 Tissue culture
B16F10 melanoma cells were purchased from ATCC, maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) supplemented with 10% (v/v) fetal bovine serum (FBS) (Atlanta Biologicals) and 1% (v/v) penicillin-streptomycin
(Invitrogen) and grown at 37 °C and 5% CO2.
3.2.9 Flow cytometry
Confluent B16F10 cells were removed using Hanks’-based cell dissociation buffer (ThermoFisher) and added to 96-well v-bottom plates (200 μL/well, 2.5 x 106 cells/mL). Native TMV, free Zn-EpPor, or drug-loaded Zn-EpPorTMV was added to wells
5 (1 x 10 particles/cell) in triplicate and incubated for 8 h at 37 °C and 5% CO2. Following incubation, cells were washed 2 times in FACS buffer (1 mM EDTA, 25 mM HEPES,
77 CHAPTER 3: TMV FOR PDT
1% (v/v) FBS in PBS, pH 7.0) and fixed in 2% (v/v) paraformaldehyde in FACS buffer for 10 min at room temperature. Cells were washed 2 times, then resuspended in FACS buffer and analyzed on a BD FACSAria flow cytometer. Live cells were gated, and
10,000 events were recorded; all experiments were carried out in triplicate. Data were analyzed on FlowJo 8.6.3 software.
3.2.10 Confocal microscopy
Confluent B16F10 cells were removed using 0.05% (w/v) trypsin-EDTA and added to 24-well untreated plates with coverslips on the bottom (500 μL/well, 5 x 104
7 cells/mL); cells were grown overnight. Zn-EpPorTMV was added to wells (1 x 10 particles/cell) and incubated for 8 h at 37 °C and 5% CO2. Following incubation, cells were fixed in DPBS containing 5% (v/v) paraformaldehyde and 0.3% (v/v) glutaraldehyde for 10 min at room temperature. Cell membranes were stained with wheat-germ agglutinin (WGA) conjugated to AlexaFluor488 (WGA-A488, Invitrogen) using 1 μg mL-1 WGA-A488 in 5% (v/v) goat serum in DPBS for 45 min at room temperature. Cells were washed 3 times with DPBS in between each step. Coverslips were mounted onto slides using Fluoroshield with DAPI (Sigma) and sealed with nail polish. Slides were imaged using an Olympus FluoView FV1000 confocal laser scanning microscope, and the data were processed in ImageJ 1.47d (http://imagej.nih.gov/ij).
78 CHAPTER 3: TMV FOR PDT
3.2.11 Cell viability
Cell viability was assayed using MTT and LIVE/DEAD assays. Confluent cells were removed using 0.05% (w/v) trypsin-EDTA, added to 96-well plates (100 μL/well, 2
4 x 10 cells/mL), and grown overnight. Native TMV, drug loaded Zn-EpPorTMV, or free Zn-
EpPor were added to cells using 0.025, 0.05, 0.1, 0.25, 0.5, 1.0, and 5.0 μM Zn-EpPor; cells were incubated for 8 h at 37 °C and 5% CO2. Assays were done in triplicate and repeated at least twice. Following incubation, cells were washed twice to remove unbound drug and drug carriers, and then 100 μL of medium was added. Samples were illuminated in a rectangle (10.5 cm x 11 cm) under white light from a Vivitek D950HD projector (~10 mW cm-2 at 430 nm) for 30 min (18.1 J cm-2 at 430 nm). Control samples were kept in the dark for 30 min. After illumination, plates were incubated at 37 °C and
5% CO2 for 48 h. Cell viability was assessed using an MTT cell proliferation assay
(ATCC); the procedure was as per manufacturer’s recommendation. Alternatively, cell viability and cytotoxicity were determined using the LIVE/DEAD Viability/Cytotoxicity
Kit for mammalian cells (ThermoFisher). The staining procedure was as per manufacturer’s recommendation. Plates were imaged on a Zeiss Axio Observer Z1 motorized FL inverted microscope. Images were analyzed for percentage cell viability using ImageJ 1.47d (http://imagej.nih.gov/ij).
79 CHAPTER 3: TMV FOR PDT
3.3 Results
3.3.1 Zn-EpPorTMV encapsulation and characterization
Tobacco mosaic virus (TMV) was produced in and purified from Nicotiana benthamiana plants as previously described [25]. The nucleoprotein components of TMV form a hollow cylinder measuring 300 x 18 nm with a 4 nm-wide interior channel. Each
TMV particle is composed of 2130 identical copies of a coat protein, each containing two solvent-exposed glutamic acids (Glu97, Glu106) on the interior surface and a tyrosine residue (Tyr139) on the exterior (Figure 3.1A). In this study, a lysine-added mutant was also considered (TMVLys, T158K) that also offers an amine ligation handle on the solvent-exposed exterior surface (Figure 3.1A) [35]. Two drug loading strategies were considered: covalent conjugation of Zn-EpPor to the aforementioned amino acid side chains [26] or noncovalent drug loading through electrostatic interactions, as we recently described [36].
The drug candidate, Zn-EpPor, was designed and synthesized with an alkyne handle (Figure 3.1A) for covalent conjugation to an azide-bearing motif using the Cu(I)- catalyzed Huisgen-Meldal-Sharpless 1,3-dipolar cycloaddition reaction. In brief, TMVLys was first modified with an azide functional handle using N-hydroxysuccinimide (NHS) ester-PEG4-azide, followed by the click reaction using Zn-EpPor and reaction conditions as previously described [26]. However, any conjugation attempts resulted in extensive aggregation and loss of the sample, and therefore were not further pursued.
Instead, Zn-EpPor was loaded into TMV making use of the chemically distinct exterior and interior microenvironments. The interior channel of TMV is lined with a high density of negative charges from solvent-exposed glutamic acids Glu97 and Glu106 80 CHAPTER 3: TMV FOR PDT
(Figure 3.1A). We hypothesized that the positively charged drug candidate Zn-EpPor carrying 3 positive charges, one at each of the methylpyridinium side chains, could be loaded into the central channel of TMV based on charge-charge interactions. Zn-EpPor loading was carried out under slightly alkaline conditions (pH 7.8) to promote deprotonation of the carboxylic acids and therefore electrostatic complexation with Zn-
EpPor (Zn-EpPor was used at a 6000 fold molar excess to TMV). The reaction was allowed to proceed overnight, then excess Zn-EpPor was removed using ultracentrifugation (Figure 3.1B). To confirm whether loading occurred and whether free
Zn-EpPor was removed following centrifugation, Zn-EpPorTMV was analyzed using size exclusion chromatography (Figure 3.1D). TMV showed the characteristic elution profiles at 8 mL using the Superose6 column and ÄKTA purifier; broken particles or free coat proteins were not detectable. The coelution of the 260 nm (RNA component), 280 nm (protein component), and 450 nm (Zn-EpPor specific absorbance) peaks indicates successful loading into TMV. No additional peaks were observed at any of the wavelengths measured, indicating that Zn-EpPorTMV was both intact and void of free Zn-
EpPor.
81 CHAPTER 3: TMV FOR PDT
Figure 3.1. Zn-EpPorTMV conjugation and characterization. (A) (left) TMV-lysine mutant coat protein (T158K) Glu97 = red, Glu 106 = blue, Tyr 139 = green, Lys 158 = purple, RNA = light blue and (right) Zn- EpPor structure; (B) Schematic of Zn-EpPor loading into TMV; (C) Schematic of Zn-EpPor loading efficiencies after interior and exterior modification with alkynes at amino acids Glu97 and Glu106
(interior) and Tyr 139 (exterior); (D) Size exclusion chromatography of Zn-EpPorTMV shows co-elution of intact TMV (260 and 280 nm) and Zn-EpPor specific absorbance (450 nm) at the retention time of 8 mL;
(E) UV/visible spectroscopy of Zn-EpPorTMV, table inset shows quantification of Zn-EpPor loading comparing UV/visible spectroscopy and ICP-OES measurements; (F) Negatively stained TEM images of
Zn-EpPorTMV after light exposure for 30 min (top) and corresponding dark control (bottom).
The loading efficiency was quantified using UV/visible spectroscopy and inductively coupled plasma optical emission spectroscopy (ICP-OES) (Figure 3.1E).
Using the Beer-Lambert law and the TMV- and Zn-EpPor-specific molar absorptivity
-1 -1 -1 -1 coefficients (εTMV = 3.0 mg mL cm at 260 nm; εZn-EpPor = 195,000 M cm at 450 nm), the concentrations of TMV and Zn-EpPor in solution and, hence, the ratio of Zn-
EpPor:TMV were determined. On average, 800 Zn-EpPor were loaded into a TMV
82 CHAPTER 3: TMV FOR PDT particle. The UV/visible spectroscopic method was complemented with ICP-OES measurements to quantify the Zn loading. The latter method may be considered more accurate, because the porphyrin molar absorptivity coefficient is solvatochromic and may be different in the TMV microenvironment than in free solution. For ICP-OES, zinc was released from Zn-EpPorTMV by incubation at 60 °C for 2 h in 1 M HCl. Zinc content was then quantified based on a calibration standard curve. Overall, the data were in good agreement indicating loading of approximately 900±15% Zn-EpPor per TMV, which is in agreement with the spatial constraints of the interior channel of TMV. With a 4 nm interior channel radius, the 300 nm TMV rod has an interior surface area of approximately 3770 nm2. Given 900 Zn-EpPor loaded into TMV, there is approximately
4.2 nm2 (420 Å2) available per Zn-EpPor molecule, greater than either the topological polar surface area of approximately 75 Å2 or the total molecular area of 235 Å2, both calculated from the crystal structure of Zn-EpPor [37], indicating that the high loading efficacy seen is highly feasible. Additionally, similar loading was seen with a 2+ species of platinum drug [36].
To confirm further that Zn-EpPor was indeed loaded into the interior channel, and not nonspecifically adsorbed on the exterior particle surface, TMV was modified on either the interior (TMV-iAlk) or exterior (TMV-eAlk) surface with alkyne groups to mask charged amino acids. In brief, alkyne handles were attached to either the interior or exterior surfaces of TMV using EDC chemistry to target interior glutamic acids or diazonium salt modification to target the exterior tyrosine residue, using previously established protocols [25]. Native TMV, TMV-iAlk, and TMV-eAlk were incubated with
Zn-EpPor as described above. Following purification, Zn-EpPor loading was quantified
83 CHAPTER 3: TMV FOR PDT using ICP-OES (Figure 3.1C). Indeed, interior modification of glutamic acid residues with alkynes indicated a decreased loading efficiency resulting in only 50% loading capacity. A complete reduction in the loading was not observed because it is unlikely that every carboxylic acid was modified with an alkyne, allowing for some electrostatic interactions to remain. On the other hand, exterior modification of TMV showed no difference in Zn-EpPor loading, thus supporting interior loading.
Lastly, we investigated the stability of the Zn-EpPorTMV complex in the dark and after light exposure. Zn-EpPorTMV was kept in the dark or exposed to light - white light from a Vivitek D950HD projector (~10 mW cm-2 at 430 nm) under a rectangle (10.5 cm x 11 cm) - for 30 min and then analyzed using transmission electron microscopy (TEM).
Light-exposed Zn-EpPorTMV showed no apparent differences in their macromolecular structure compared to dark controls (Figure 3.1F), indicating that the treatment with light did not impact the stability of the TMV carrier. Further, we assessed the ability of the
TMV carrier to retain the Zn-EpPor compound during storage: Zn-EpPorTMV was stored at
4 °C for one month in 0.01 M potassium phosphate buffer, pH 7.0, and subsequently analyzed using size exclusion chromatography. The elution profiles were consistent with an intact TMV carrier retaining the Zn-EpPor drug candidate (Figure 3.2).
84 CHAPTER 3: TMV FOR PDT
Figure 3.2. Stability of Zn-EpPorTMV over time. TMV-Zn-EpPor stability was measured using size exclusion chromatography. Zn-EpPorTMV stored at 4 °C for one month retains Zn-EpPor. The characteristic absorbance for TMV RNA (260 nm) and protein (280 nm) coelute at the expected volume with the characteristic absorbance for Zn-EpPor (440 nm).
3.3.2 Cell uptake and intracellular localization of Zn-EpPorTMV in B16F10 melanoma
Photodynamic therapy (PDT) produces reactive oxygen species (ROS) that have very short half-lives [9, 38-40]. Therefore, to ensure that the ROS are able to exert their mechanism of action, it was important to confirm that Zn-EpPorTMV particles are able to bind to and/or be taken up by the cell; particles that remain in the extracellular space will not be effective for PDT.
Cell binding and uptake of the PDT delivery system was measured using both flow cytometry and confocal microscopy. Cell uptake was measured as a function of Zn-
EpPor fluorescence (Figure 3.3A). Free Zn-EpPor had a mean fluorescence uptake (MFI) of 956, while Zn-EpPorTMV exhibited 40% enhanced uptake, with a MFI of 1347
(p<0.05%). Unlabeled TMV did show an increase in MFI compared to cells only control, indicating that the fluorescence observed is from the Zn-EpPor. These studies were
85 CHAPTER 3: TMV FOR PDT complemented with confocal microscopy to determine the intracellular fate of Zn-EpPor delivered by TMV. Following an 8 h incubation of B16F10 cells with Zn-EpPorTMV particles, cells were fixed and stained with wheat germ agglutinin. Confocal fluorescence microscopy confirmed that Zn-EpPor is both taken up by the cells, and also dispersed throughout the cytoplasm (Figure 3.3B), allowing it to exert its mechanism of action.
5 Figure 3.3. Zn-EpPorTMV interaction with B16F10 melanoma cells. (A) Zn-EpPorTMV particles (10 particles/cell) or the corresponding amount of free Zn-EpPor or native TMV were incubated with B16F10 cells for 8 h and analyzed using flow cytometry. (left) Representative histograms; (right) Statistical analysis
(triplicates) and quantitative data show mean fluorescence intensity (MFI) of Zn-EpPorTMV vs. free Zn-EpPor vs. unlabeled TMV vs. cells only control, *p<0.05; (B,C) Confocal microscopy indicates cellular uptake of
Zn-EpPorTMV (green). Nuclei are stained with DAPI (blue) and membranes are labeled with wheat germ agglutinin (red). Scale bar = 10 μm.
86 CHAPTER 3: TMV FOR PDT
3.3.3 Therapeutic efficacy of Zn-EpPorTMV targeting melanoma
To evaluate efficacy in vitro, Zn-EpPorTMV was studied in B16F10 melanoma versus free Zn-EpPor. Untreated cells and drug-free TMV carrier were used as controls.
The TMV concentration was normalized to the drug-loaded concentration and corresponded to the highest amount of TMV used to determine the IC50 curves (Figure
3.4A). Drug candidates and controls were incubated with B16F10 cells for 8 h, washed, and then exposed to white light from a Vivitek D950HD projector under a rectangle (10.5 cm x 11 cm) for 30 min. Following illumination, cells were returned to the incubator for
48 h. Cell viability was assessed using an MTT cell viability assay and analyzed using
GraphPad Prism. The IC50 values were determined as 0.54 μM and 0.24 μM for free Zn-
EpPor and Zn-EpPorTMV, respectively (Figure 3.4A). Dark controls did not show any cell killing (data not shown); neither did any of the controls lead to cytotoxicity.
The MTT assay is based on a yellow tetrazolium dye that is reduced to purple formazan via NAD(P)H-dependent oxidoreductase enzymes. This reduction is highly dependent on cellular metabolism in the mitochondria and is not high in cells with low cellular metabolic activity [41, 42]. Because photosensitizers are known to impact the functionality of the mitochondria [9], the MTT assay may be compromised as a result of impaired mitochondrial activity. Therefore, we also performed LIVE/DEAD cell viability assays to further confirm therapeutic efficacy and cell killing. Cells were incubated with
5.0 μM of free Zn-EpPor or Zn-EpPorTMV, or corresponding controls, for 8 h, washed, and illuminated for 30 min. This drug concentration was based on the MTT assay; it is over four times the IC50 value in B16F10 melanoma and should give maximal cell killing. The
LIVE/DEAD assay was applied the next day, imaged, and images were analyzed using
87 CHAPTER 3: TMV FOR PDT
ImageJ to determine percent cell viability (Figure 3.4B). All samples kept in the dark, as well as cells only and TMV only controls exposed to light, exhibited high cell viability
(98.32 ± 0.53 %). On the contrary, cells exposed to both Zn-EpPor or Zn-EpPorTMV and light had 100% cell killing. It is important to note that although the MTT assay indicated an increased efficacy for Zn-EpPorTMV versus free Zn-EpPor, the LIVE/DEAD assay in
B16F10 cells showed 100% killing for both samples, as expected based on the 5 μM drug concentration.
88 CHAPTER 3: TMV FOR PDT
Figure 3.4. B16F10 response to Zn-EpPorTMV and free Zn-EpPor. (A) Cell viability following 8 h incubation with increasing doses of Zn-EpPor or Zn-EpPorTMV and 30 min illumination with white light (no cell killing was observed when cells were incubated in the dark, not shown). (B) Representative
LIVE/DEAD images of B16F10 cells incubated with 5.0 μM free Zn-EpPor or Zn-EpPorTMV. Cells only, TMV only, and dark controls exhibit no cell killing. Green = live cells, stained with calcein AM; red = dead cells, stained with ethidium homodimer-1; scale bar = 200 μm.
89 CHAPTER 3: TMV FOR PDT
3.4 Discussion
In this study, we loaded a cationic Zn-based porphyrin photosensitizer (Zn-EpPor) into tobacco mosaic virus (Zn-EpPorTMV) for treatment of aggressive melanoma. We compared cell uptake and cell killing efficacy of Zn-EpPorTMV versus free Zn-EpPor. Flow cytometry data indicate that both free Zn-EpPor and Zn-EpPorTMV are indeed taken up by
B16F10 melanoma cells, with loading into the TMV carrier increasing cell uptake of Zn-
EpPor, versus free drug alone. Complementary confocal microscopy of Zn-EpPorTMV confirmed that after an 8 h incubation, Zn-EpPor is delivered to cells and dispersed throughout the cytoplasm. The intracellular distribution of the Zn-EpPor drug was consistent with intracellular release of the cargo. We hypothesize that TMV is taken up and trafficked to the endolysosome, as previously reported [28, 43]. The acidic endolysosomal compartment will result in protonation of the TMV’s interior carboxylic acid resulting in drug release. Furthermore, it is expected that hydrolyases and proteases within the endolysosome will lead to degradation of the proteinaceous nanoparticle, further resulting in release of the Zn-EpPor, which escapes the lysosomes and is dispersed throughout the cytoplasm [43].
In B16F10 cells, loading of Zn-EpPor into TMV improved cell killing efficacy versus free Zn-EpPor alone. The increased efficacy of Zn-EpPorTMV may be attributed to the increased cell uptake of Zn-EpPor due to its delivery by TMV. Additionally, both free
Zn-EpPor and Zn-EpPorTMV performed comparably to previously reported preclinical and clinical photodynamic therapies. In B16F10, phthalocyanine-based drugs had IC50 values between 1.10 and 1.25 μM, while the Japanese drug Talaporfin had an IC50 of 8.50 μM
[13]. In a range of cell lines, including MCF-7, HeLa, and A2780, porphyrin-based
90 CHAPTER 3: TMV FOR PDT
photosensitizers had IC50 values ranging from 0.45 to 5.0 μM [19, 44, 45]. Also, it is of note that the clinically approved Photofrin is ineffective in pigmented melanoma due to inefficient light penetration in melanin-heavy cells, such as the B16F10 cell line [15]. Zn-
EpPorTMV also shows comparable cell killing to other previously reported VNP-based photodynamic therapies. Photosensitizers, including C60 and other porphyrin-based sensitizers, have been attached to viral nanoparticles, including bacteriophages Qβ, MS2, and M13. These VNP-based systems also exhibited cell killing efficacy in the nano- to micromolar range [46-50]. Since the MTT assay can be compromised due to impaired mitochondria, we further confirmed efficacy of both Zn-EpPorTMV and free Zn-EpPor using a LIVE/DEAD assay.
3.5 Conclusion
In this study, a small molecule photosensitizer, Zn-EpPor, previously used in antimicrobial photodynamic inactivation studies [31-33], was applied as a cancer therapeutic, for the first time. Furthermore, to overcome translational challenges of PDT, such as poor solubility and drug targeting, we formulated the Zn-EpPor drug candidate as a nanoparticle therapeutic using the nucleoprotein components of tobacco mosaic virus
(TMV). The drug formulation exhibited a good shelf life, drug release during 1-month storage was not apparent, and the nanoparticles maintained structural integrity. The IC50 was determined as 0.54 μM and 0.24 μM for free Zn-EpPor and Zn-EpPorTMV, respectively. Overall, the IC50 indicates that Zn-EpPor shows comparable efficacy compared to previously reported porphyrin-based PDT therapeutics. The Zn-EpPorTMV particle proved to be stable and efficacious in vitro, improving upon the cell targeting,
91 CHAPTER 3: TMV FOR PDT uptake, and killing versus free Zn-EpPor. Based on the biocompatibility and tumor homing properties of TMV, photosensitizer-TMV platforms such as Zn-EpPorTMV may hold promise for application in PDT or combination therapies targeting melanoma or other cancers.
3.6 Works cited in this chapter
1. Baldea I, Filip AG. Photodynamic therapy in melanoma--an update. J Physiol Pharmacol 2012;63:109-18.
2. Balch CM, Gershenwald JE, Soong SJ, Thompson JF, Atkins MB, Byrd DR, et al. Final version of 2009 AJCC melanoma staging and classification. J Clin Oncol 2009;27:6199-206.
3. Baldea I, Ion RM, Olteanu DE, Nenu I, Tudor D, Filip AG. Photodynamic therapy of melanoma using new, synthetic porphyrins and phthalocyanines as photosensitisers - a comparative study. Clujul Med 2015;88:175-80.
4. Robertson CA, Abrahamse H, Evans D. The in vitro PDT efficacy of a novel metallophthalocyanine (MPc) derivative and established 5-ALA photosensitizing dyes against human metastatic melanoma cells. Lasers Surg Med 2010;42:926-36.
5. Haddad R, Blumenfeld A, Siegal A, Kaplan O, Cohen M, Skornick Y, et al. In vitro and in vivo effects of photodynamic therapy on murine malignant melanoma. Ann Surg Oncol 1998;5:241-7.
6. Teng IT, Chang YJ, Wang LS, Lu HY, Wu LC, Yang CM, et al. Phospholipid- functionalized mesoporous silica nanocarriers for selective photodynamic therapy of cancer. Biomaterials 2013;34:7462-70.
7. Szurko A, Kramer-Marek G, Widel M, Ratuszna A, Habdas J, Kus P. Photodynamic effects of two water soluble porphyrins evaluated on human malignant melanoma cells in vitro. Acta Biochim Pol 2003;50:1165-74.
8. Agostinis P, Berg K, Cengel KA, Foster TH, Girotti AW, Gollnick SO, et al. Photodynamic therapy of cancer: an update. CA Cancer J Clin 2011;61:250-81.
9. Broekgaarden M, Weijer R, van Gulik TM, Hamblin MR, Heger M. Tumor cell survival pathways activated by photodynamic therapy: a molecular basis for pharmacological inhibition strategies. Cancer Metastasis Rev 2015;34:643-90.
92 CHAPTER 3: TMV FOR PDT
10. Dougherty TJ, Gomer CJ, Henderson BW, Jori G, Kessel D, Korbelik M, et al. Photodynamic therapy. J Natl Cancer Inst 1998;90:889-905.
11. Huang Z. A review of progress in clinical photodynamic therapy. Technol Cancer Res Treat 2005;4:283-93.
12. Dolmans DE, Fukumura D, Jain RK. Photodynamic therapy for cancer. Nat Rev Cancer 2003;3:380-7.
13. Obata T, Mori S, Suzuki Y, Kashiwagi T, Tokunaga E, Shibata N, et al. Photodynamic Therapy Using Novel Zinc Phthalocyanine Derivatives and a Diode Laser for Superficial Tumors in Experimental Animals. Journal of Cancer Therapy 2015;6:53-61.
14. Pavani C, Uchoa AF, Oliveira CS, Iamamoto Y, Baptista MS. Effect of zinc insertion and hydrophobicity on the membrane interactions and PDT activity of porphyrin photosensitizers. Photochem Photobiol Sci 2009;8:233-40.
15. Woodburn KW, Fan Q, Kessel D, Luo Y, Young SW. Photodynamic therapy of B16F10 murine melanoma with lutetium texaphyrin. J Invest Dermatol 1998;110:746-51.
16. Nwogu C, Pera P, Bshara W, Attwood K, Pandey R. Photodynamic therapy of human lung cancer xenografts in mice. J Surg Res 2016;200:8-12.
17. Aggarwal N, Santiago AM, Kessel D, Sloane BF. Photodynamic therapy as an effective therapeutic approach in MAME models of inflammatory breast cancer. Breast Cancer Res Treat 2015.
18. Cheng Y, Cheng H, Jiang C, Qiu X, Wang K, Huan W, et al. Perfluorocarbon nanoparticles enhance reactive oxygen levels and tumour growth inhibition in photodynamic therapy. Nat Commun 2015;6:8785.
19. Antoni PM, Naik A, Albert I, Rubbiani R, Gupta S, Ruiz-Sanchez P, et al. (Metallo)porphyrins as potent phototoxic anti-cancer agents after irradiation with red light. Chemistry 2015;21:1179-83.
20. Lucky SS, Soo KC, Zhang Y. Nanoparticles in photodynamic therapy. Chem Rev 2015;115:1990-2042.
21. Danhier F, Feron O, Preat V. To exploit the tumor microenvironment: Passive and active tumor targeting of nanocarriers for anti-cancer drug delivery. J Control Release 2010;148:135-46.
22. Manchester M, Singh P. Virus-based nanoparticles (VNPs): platform technologies for diagnostic imaging. Adv Drug Deliv Rev 2006;58:1505-22.
93 CHAPTER 3: TMV FOR PDT
23. Singh P, Prasuhn D, Yeh RM, Destito G, Rae CS, Osborn K, et al. Bio- distribution, toxicity and pathology of cowpea mosaic virus nanoparticles in vivo. J Control Release 2007;120:41-50.
24. Pokorski JK, Steinmetz NF. The art of engineering viral nanoparticles. Mol Pharm 2011;8:29-43.
25. Bruckman MA, Steinmetz NF. Chemical modification of the inner and outer surfaces of Tobacco Mosaic Virus (TMV). Methods Mol Biol 2014;1108:173-85.
26. Bruckman MA, Kaur G, Lee LA, Xie F, Sepulveda J, Breitenkamp R, et al. Surface modification of tobacco mosaic virus with "click" chemistry. Chembiochem 2008;9:519-23.
27. Lee KL, Hubbard LC, Hern S, Yildiz I, Gratzl M, Steinmetz NF. Shape matters: the diffusion rates of TMV rods and CPMV icosahedrons in a spheroid model of extracellular matrix are distinct. Biomater Sci 2013;1.
28. Shukla S, Eber FJ, Nagarajan AS, DiFranco NA, Schmidt N, Wen AM, et al. The Impact of Aspect Ratio on the Biodistribution and Tumor Homing of Rigid Soft- Matter Nanorods. Adv Healthc Mater 2015;4:874-82.
29. Wu M, Shi J, Fan D, Zhou Q, Wang F, Niu Z, et al. Biobehavior in normal and tumor-bearing mice of tobacco mosaic virus. Biomacromolecules 2013;14:4032-7.
30. Bruckman MA, Randolph LN, VanMeter A, Hern S, Shoffstall AJ, Taurog RE, et al. Biodistribution, pharmacokinetics, and blood compatibility of native and PEGylated tobacco mosaic virus nano-rods and -spheres in mice. Virology 2014;449:163-73.
31. Feese E, Sadeghifar H, Gracz HS, Argyropoulos DS, Ghiladi RA. Photobactericidal porphyrin-cellulose nanocrystals: synthesis, characterization, and antimicrobial properties. Biomacromolecules 2011;12:3528-39.
32. Carpenter BL, Scholle F, Sadeghifar H, Francis AJ, Boltersdorf J, Weare WW, et al. Synthesis, Characterization, and Antimicrobial Efficacy of Photomicrobicidal Cellulose Paper. Biomacromolecules 2015;16:2482-92.
33. Carpenter BL, Feese E, Sadeghifar H, Argyropoulos DS, Ghiladi RA. Porphyrin- cellulose nanocrystals: a photobactericidal material that exhibits broad spectrum antimicrobial activity. Photochem Photobiol 2012;88:527-36.
34. Leberman R. The isolation of plant viruses by means of "simple" coacervates. Virology 1966;30:341-7.
35. Demir M, Stowell MHB. A chemoselective biomolecular template for assembling diverse nanotubular materials. Nanotechnology 2002;13:541-4.
94 CHAPTER 3: TMV FOR PDT
36. Czapar AE, Zheng YR, Riddell IA, Shukla S, Awuah SG, Lippard SJ, et al. Tobacco mosaic virus delivery of phenanthriplatin for cancer therapy. ACS Nano 2016.
37. Feese E. Development of Novel Photosensitizers for Photodynamic Inactivation of Bacteria. [Raleigh, North Carolina]: North Carolina State University; 2012.
38. Niedre M, Patterson MS, Wilson BC. Direct near-infrared luminescence detection of singlet oxygen generated by photodynamic therapy in cells in vitro and tissues in vivo. Photochem Photobiol 2002;75:382-91.
39. Moan J, Berg K. The photodegradation of porphyrins in cells can be used to estimate the lifetime of singlet oxygen. Photochem Photobiol 1991;53:549-53.
40. Castano AP, Demidova TN, Hamblin MR. Mechanisms in photodynamic therapy: part one-photosensitizers, photochemistry and cellular localization. Photodiagnosis Photodyn Ther 2004;1:279-93.
41. Liu Y, Peterson DA, Kimura H, Schubert D. Mechanism of cellular 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction. J Neurochem 1997;69:581-93.
42. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983;65:55-63.
43. Wen AM, Infusino M, De Luca A, Kernan DL, Czapar AE, Strangi G, et al. Interface of physics and biology: engineering virus-based nanoparticles for biophotonics. Bioconjug Chem 2015;26:51-62.
44. Tachikawa S, El-Zaria ME, Inomata R, Sato S, Nakamura H. Synthesis of protoporphyrin-lipids and biological evaluation of micelles and liposomes. Bioorg Med Chem 2014;22:4745-51.
45. Rangasamy S, Ju H, Um S, Oh DC, Song JM. Mitochondria and DNA Targeting of 5,10,15,20-Tetrakis(7-sulfonatobenzo[b]thiophene) Porphyrin-Induced Photodynamic Therapy via Intrinsic and Extrinsic Apoptotic Cell Death. J Med Chem 2015;58:6864-74.
46. Stephanopoulos N, Tong GJ, Hsiao SC, Francis MB. Dual-surface modified virus capsids for targeted delivery of photodynamic agents to cancer cells. ACS Nano 2010;4:6014-20.
47. Rhee JK, Baksh M, Nycholat C, Paulson JC, Kitagishi H, Finn MG. Glycan- targeted virus-like nanoparticles for photodynamic therapy. Biomacromolecules 2012;13:2333-8.
95 CHAPTER 3: TMV FOR PDT
48. Gandra N, Abbineni G, Qu X, Huai Y, Wang L, Mao C. Bacteriophage bionanowire as a carrier for both cancer-targeting peptides and photosensitizers and its use in selective cancer cell killing by photodynamic therapy. Small 2013;9:215-21.
49. Cohen BA, Bergkvist M. Targeted in vitro photodynamic therapy via aptamer- labeled, porphyrin-loaded virus capsids. J Photochem Photobiol B 2013;121:67- 74.
50. Wen AM, Ryan MJ, Yang AC, Breitenkamp K, Pokorski JK, Steinmetz NF. Photodynamic activity of viral nanoparticles conjugated with C60. Chem Commun (Camb) 2012;48:9044-6.
96 CHAPTER 4: STEALTH FILAMENTS
Chapter 4: Stealth coatings for PVX
The material in this chapter is adapted with permission from: Lee, K.L., Shukla,
S., Wu, M., Ayat, N.R., El Sanadi, C.E., Wen, A.W., Edelbrock, J.F., Pokorski, J.K.,
Commandeur, U., Dubyak, G.R., Steinmetz, N.F. Stealth filaments: Polymer chain length and conformation affect the in vivo fate of PEGylated potato virus X. Acta Biomater.
2015, 19: 166-179. doi:10.1016/j.actbio.2015.03.001 Copyright 2015 Elsevier.
4.1 Introduction
The development of nanoparticles for drug delivery and imaging is a rapidly growing area with many novel platform technologies in the pipeline. For example, nanoparticles engineered with appropriate surface chemistries and equipped with therapeutics allow targeting of toxic payloads to cancerous tissues through passive or active targeting mechanisms. This partitioning toward the diseased tissues increases payload delivery and reduces systemic side effects often associated with potent chemotherapies or toxins. Commonly used nanoparticles include lipid-based micelles, polymeric capsules, iron oxide nanoparticles, gold nanoparticles, carbon nanotubes, and protein-based particles and nanocages; each system has its advantages and disadvantages in terms of nanomanufacturing, engineerability, and in vivo properties. Surface chemistries, such as modification with stealth coatings or targeting ligands, and nanoparticle shape are handles to tailor tissue specificity and bioavailability.
Mounting evidence suggests advantageous behaviors of elongated, filamentous nanomaterials: (i) non-spherical materials show increased margination toward the vessel
97 CHAPTER 4: STEALTH FILAMENTS wall and increased tumor homing [1-7]; (ii) elongated materials present ligands more effectively to the larger and flat vessel wall or target cells compared to their spherical counterparts [8-10]; and (iii) elongated materials have increased immune evasion and reduced macrophage uptake, therefore further contributing to synergistic target enhancement [11, 12]. Most platform technologies currently under development are spherical or elongated low aspect ratio materials (AR < 5). Exemptions are carbon nanotubes and filomicelles; however carbon-based materials have low biocompatibility
[13] and filomicelles are in the micron-size regime [14]. Synthetic approaches to high aspect ratio materials remain challenging because of polydispersity. Synthetic chemistry and nanotechnology seek to mimic what nature has achieved, i.e. self-assembly and programmability at the atomic level. Therefore, we turned toward a bio-inspired approach and are studying and developing filamentous plant viruses for nanomedical applications.
Mammalian virus-based nanoparticles for gene therapy and oncolytic virotherapy are in clinical investigations [15-17], so the potential of virus-based materials for medical applications has clearly been recognized. There are many novel viruses in the development pipeline including bacteriophages and plant viruses; these non-mammalian pathogens may be advantageous because they are non-infectious toward humans.
Specifically, we turned toward the filamentous plant virus potato virus X (PVX), which measures 515 nm in length and 13 nm in width. The filaments can be obtained in gram scales through farming using Nicotiana benthamiana plants as the production species. The proteinaceous scaffold is amenable to chemical modification and genetic engineering. For example, we recently demonstrated expression of green fluorescent protein (GFP) and other fluorescent proteins as genetic coat protein fusions [18].
98 CHAPTER 4: STEALTH FILAMENTS
Furthermore, solvent-exposed lysine side chains offer a convenient means of modification with non-peptide-based ligands (e.g. therapeutics or contrast agents) via chemical bioconjugation [19].
We have shown that plant virus-based materials accumulate in tumors; targeting is achieved based on passive accumulation via the enhanced permeability and retention
(EPR) effect [20] or active receptor targeting of cancer signatures [21-25]. Data indicate that filaments show more efficient passive tumor partitioning compared to spherical nanoparticles; this shape-mediated enhanced tumor homing and penetration is reproducible in a variety of models, including human tumor xenografts of fibrosarcoma, squamous cell sarcoma, colon cancer, and breast cancer [20, 26]. Together, these data provide strong support for the further development and investigation of filamentous plant viruses for biomedical applications.
Like other nanomaterials, the proteinaceous carriers are cleared by the mononuclear phagocyte system (MPS) [26]. Conjugation of stealth polymers to coat the nanocarriers allows to reduce interactions with the MPS, therefore enhancing the pharmacokinetics of the carrier. The most extensively studied stealth polymer is polyethylene glycol (PEG) [27-31]. PEG is a non-charged, hydrophilic polymer with low toxicity and immunogenicity; a wide-variety of functionalized PEG monomers and chains are available for nanoparticle modification. The hydrophilic shield provided by the PEG coating of nanoparticles decreases serum protein adsorption, resulting in the “stealth” properties commonly reported for PEGylated nanoparticles (e.g. increased circulation time, decreased accumulation in liver and spleen) [27-31].
In the present studies, we set out to develop and study stealth filaments using 99 CHAPTER 4: STEALTH FILAMENTS
PVX-PEG hybrids. While we previously reported the in vivo properties of PEGylated
PVX modified with linear PEG chains of 5000 Da [20], this study set out to determine whether the pharmacokinetic profiles could be further optimized to generate long- circulating stealth filaments with favorable properties for drug delivery. To do this, we considered PEG chains of various molecular weights and conformations using linear and branched PEGs with molecular weights of 5000 Da and 20,000 Da. The biological fate of stealth filaments as a function of surface coating was analyzed in vitro and in vivo to study pharmacokinetics, biodistribution, immunogenicity, and immune cell interactions.
4.2 Materials and methods
4.2.1 PVX propagation
PVX was propagated in N. benthamiana and purified using previously described procedures [19]. Purified PVX was stored in 0.1 M potassium phosphate buffer pH 7.0 at
4 °C. PVX concentration was determined by UV/visible spectroscopy (εPVX= 2.97 mL mg-1 cm-1 at 260 nm).
4.2.2 Bioconjugation of PVX with fluorophores and PEG
PVX was modified with Alexa Fluor 647 (A647) or VivoTag-S 750 and/or polyethylene glycol (PEG) using N-hydroxysuccinimide (NHS)-activated esters. First,
PVX (at 1-2 mg mL-1 in 0.1 M potassium phosphate buffer, pH 7.0) was reacted with a
2000 molar excess of NHS-A647 (Life Technologies) or VivoTag-S 750 (Perkin-Elmer) at a 10% (v/v) final concentration of DMSO. PVX has a molar mass of 35x106 g mol-1.
100 CHAPTER 4: STEALTH FILAMENTS
The reaction was allowed to proceed for 2 hours at room temperature, with agitation, and purified using 10-kDa cut-off centrifugal filters (Millipore). Second, A647-modified
PVX (A-PVX) or VivoTag-S 750-modified PVX (V-PVX) was reacted with linear or branched PEG-NHS with molecular weights of 5000 Da (P5L and P5B) or linear PEG-
NHS with a molecular weight of 20,000 Da (P20) (Nanocs). PEG was added at a molar excess of 10,000 in a 10% (v/v) final concentration of DMSO and incubated overnight at room temperature, with agitation. PVX-A647-PEG (A-PVX-PEG) and PVX-VivoTag-
PEG (V-PVX-PEG) were purified using 10- and 100-kDa-cut off centrifugal filter units
(Millipore). A-PVX-PEG and V-PVX-PEG filaments were stored in 0.1 M potassium phosphate buffer, pH 7.0 at 4 °C and characterized using a combination of UV/visible spectroscopy, denaturing gel electrophoresis, and transmission electron microscopy
(TEM).
4.2.3 UV/visible spectroscopy
The number of A647 or VivoTag molecules per PVX filament was determined by
UV/visible spectroscopy using a NanoDrop 2000 spectrophotometer (Thermo Fisher).
Dye number was determined by the ratio of fluorophore-to-PVX concentration, using the
Beer-Lambert law and the fluorophore and PVX-specific extinction coefficients: εPVX =
-1 -1 -1 -1 2.97 mL mg cm at 260 nm, εA647 = 270,000 M cm at 650 nm, and εVivoTag = 240,000
M-1 cm-1 at 775 nm.
101 CHAPTER 4: STEALTH FILAMENTS
4.2.4 Denaturing gel electrophoresis
SDS-PAGE was used to determine the number of PEG molecules per PVX filament. 15 μg of denatured protein samples were loaded and run on 4-12% NuPage gels
(Life Technologies) in 1x MOPS SDS running buffer (Life Technologies). Protein bands were visualized under white light before and after staining with Coomassie blue (0.25% w/v).
4.2.5 TEM
A-PVX-PEG filaments (20 μL, 0.1 mg mL-1) were negatively stained with 2%
(w/v) uranyl acetate for 5 min on a carbon-coated copper grid (Ted Pella). Samples were analyzed using a Zeiss Libra 200FE transmission electron microscope operated at 200 kV.
4.2.6 Animals
All experiments were carried out in accordance with Case Western Reserve
University’s Institutional Animal Care and Use Committee. Balb/C mice (Charles River) and C57/BL6 mice (Taconic) were used.
4.2.7 Pharmacokinetics
Pharmacokinetics and biodistribution (see below) were evaluated in healthy
Balb/C mice (Charles River) (n=3). Animals were maintained on an alfalfa-free diet
(Teklad) for two weeks prior to the administration of fluorescently labeled PVX filaments 102 CHAPTER 4: STEALTH FILAMENTS to reduce tissue autofluorescence. A-PVX, A-PVX-P5B, or A-PVX-P20 formulations were injected via the tail vein (at 100 μg in 100 μL sterile PBS) and blood was collected into heparin-coated tubes (Fisher) using retro-orbital bleeding; time course studies were conducted and samples were collected up to 36 hours post-administration. Serum was isolated from samples by centrifuging at 10,000g for 10 min. Fluorescence was read (λEx
= 600 nm, λEm = 660 nm) using a Tecan microplate reader, and the fluorescence reading was correlated to a standard curve normalized for each particle formulation to determine the amount of particle (in μg) at each time point. Percent injected dose was determined based on the fluorescence reading and amounts of PVX per 50 μL of serum from each time point and assuming a total blood volume of 1.5 mL.
4.2.8 Biodistribution using fluorescence molecular tomography (FMT)
Biodistribution and clearance were evaluated using healthy Balb/C mice with
FMT imaging as well as immunofluorescence microscopy (see below). Animals were maintained on an alfalfa-free diet (Teklad) for two weeks prior to the study to reduce tissue autofluorescence. For FMT imaging, mice were injected with V-PVX (n=2, one animal died prior to the start of the experiment), V-PVX-P5B (n=3), or V-PVX-P20
(n=3) via the tail vein (at 100 μg in 100 μL sterile PBS) and imaged using a FMT 2500 quantitative tomography in vivo imaging system (Perkin-Elmer) at 0 min (pre-scan), 10 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 24 h, 32 h, and 52 h post-administration. For each image, regions of interest (ROIs) were chosen for the whole body, liver and spleen, and bladder. Fluorescence intensity was calculated using the normalized total fluorescence within each ROI (based on the number of dyes per particle). 103 CHAPTER 4: STEALTH FILAMENTS
4.2.9 Immunofluorescence microscopy
Biodistribution and clearance were evaluated using immunofluorescence microscopy. Mice were sacrificed at 0 h, 6 h, 24 h, 52 h, 72 h, 96 h, and 7 days post- administration of A-PVX-P20 (via the tail vein at 100 μg in 100 μL sterile PVX), and livers and spleens were harvested. Ten μm thick tissue sections were prepared using a
Leica CM1850 cryostat. Slides were fixed in 95% (v/v) ethanol for 20 min on ice and permeabilized using 0.2% (v/v) Triton X-100 (EMD Chemicals) in PBS for 2 min. Slides were blocked using 10% (v/v) goat serum (GS) (Life Technologies) in PBS. PVX staining was carried out using a rabbit anti-PVX antibody (Pacific Immunology, at 1:250 in 1% (v/v) GS in PBS), followed by Alexa Fluor 488-labeled goat anti-rabbit secondary antibody (Life Technologies, 1:500 in 1% (v/v) GS in PBS); each step was carried out for
1 h at room temperature and slides were rinsed three times in PBS in between each step.
Slides were mounted with Fluoroshield with DAPI (Sigma Aldrich) and stored at -20°C until imaged. Analysis of sections was performed on an Olympus Fluoview FV1000 confocal microscope.
4.2.10 Immunogenicity
Antibody titers were evaluated in healthy male Balb/C mice after repeated administration of PVX, PVX-P5L, PVX-P5B, or PVX-P20. PVX-based formulations were administered intravenously via tail vein injections at 100 μg protein in 100 μL sterile PBS at days 0, 5, 10, and 66. Blood was collected on days 0 (pre-bleed), 5, 10, 18,
24, 66, 74, 85, and 97 using heparin-coated tubes (Fisher Scientific) and retro-orbital bleeding. On days on which both blood collection and particle injection were carried out,
104 CHAPTER 4: STEALTH FILAMENTS blood was collected first. Serum was isolated by centrifuging samples at 10,000g for 10 min and analyzed using enzyme-linked immunosorbent assay (ELISA) as follows: 96- well Nunc Polysorp Immuno plates (Thermo Scientific) were coated with 10 μg/well of
PVX in coating buffer (0.05 M Na2CO3, 0.05 M NaHCO3, 0.015 M NaN3 in dH2O, pH
9.6) and incubated overnight at 4 °C. After coating, wells were blocked using 200
μL/well blocking buffer (2.5% (w/v) milk, 25% (v/v) FBS in 1x PBS, pH 7.4) at 37 °C for 1 h. After blocking, 100 μL sera (at various dilutions in blocking buffer) were added to the wells and incubated at 37 °C for 2 h. After serum incubation, 100 μL of alkaline phosphatase-labeled goat anti-mouse IgG (Life Technologies, at 1:3000 in blocking buffer) was added and incubated at 37 °C for 1 h. In between each step, plates were washed four times with washing buffer (0.1% (v/v) Tween 20 in PBS, 200 μL/well).
Wells were developed by adding 100 μL of 1-step PNPP substrate (Fisher) for 10 min at
4 °C. The reaction was stopped using 100 μL of 2M NaOH. Absorbance was read at 405 nm using a Tecan microplate reader.
4.2.11 Sandwich ELISA
Sandwich ELISAs were performed using sera collected from mice treated with various PVX formulations on day 85 (see above). 96-well Nunc Polysorp Immuno plates were coated overnight at 4 °C with a rabbit anti-PVX antibody (Pacific Immunology,
1:500 in 150 μL coating buffer (15 mM Na2CO3, 35 mM NaHCO3 in dH2O, pH 5.6)).
After coating, wells were blocked using 150 μL/well blocking buffer (1.5% (w/v) BSA in coating buffer) for 1 h at 37 °C. After blocking, 5 μg of PVX, PVX-P5B, or PVX-P20 in
105 CHAPTER 4: STEALTH FILAMENTS
150 μL incubation buffer (0.5% (w/v) BSA and 0.5% (v/v) Tween 20 in PBS, pH 7.4) was added and incubated for 1 h at 37 °C. Next, 150 μL of PVX serum were added at a dilution of 1:25,000 in incubation buffer and incubated for 1 h at 37 °C. Then, 150 μL of alkaline phosphatase-labeled goat anti-mouse IgG (Life Technologies, 1:3,000 in incubation buffer) was added and incubated for 1 h at 37 °C. After each incubation step, plates were washed four times with washing buffer (0.5% (v/v) Tween 20 in PBS, pH
7.4, 200 μL/well). Wells were developed by adding 100 μL of 1-step PNPP substrate
(Fisher) for 10 min at 4 °C. The reaction was stopped using 100 μL of 2 M NaOH.
Absorbance was read at 405 nm using a Tecan microplate reader.
4.2.12 Tissue culture
RAW264.7 macrophages (ATCC) were cultured in Dulbecco’s Minimum
Essential Media (DMEM, Life Technologies), supplemented with 10% (v/v) FBS, 1%
(v/v) penicillin-streptomycin (PenStrep), and 1% (v/v) L-glutamine and cultured at 37 °C and 5% CO2. Bone marrow derived dendritic cells (BMDC) were isolated from wild-type
C57/BL6 mice (Taconic) as described previously [32]. Briefly, mice were euthanized by
CO2 inhalation and femurs and tibiae were collected and sterilized in 70% (v/v) ethanol.
Marrow cavity plugs were washed out using PBS. BMDC were resuspended in DMEM
(Sigma Aldrich) and supplemented with 10% (w/v) bovine calf serum, 100 unit/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine, and 15 ng/mL granulocyte macrophage colony-stimulating factor (GM-CSF), plated onto 150-mm dishes and cultured at 37 °C and 10% CO2. Three days post-isolation, 80% of the non-adherent cells were removed and centrifuged at 300g for 5 min at room temperature and fresh medium 106 CHAPTER 4: STEALTH FILAMENTS was added. Five days post-isolation, loosely adherent BMDC were collected and resuspended at 1x106 cells/mL. Cells were used between 7 and 10 days post-isolation.
4.2.13 Flow cytometry
RAW264.7 were grown to confluency, washed three times with PBS, and collected using enzyme-free Hank’s based cell dissociation buffer (Fisher). Cells were added to 96-well v-bottom plates (1x106 cells/200 μL/well) and incubated with 10,000 or
100,000 PVX particles/cell, in triplicate, using A-PVX, A-PVX-P5L, A-PVX-P5B, or A-
PVX-P20 for either 20 min or 2 h at 37 °C and 5% CO2. BMDC were removed from plates using Lidocaine-EDTA (40 mg/mL Lidocaine, 10 mM EDTA in PBS, pH 7.4), added to 96-well v-bottom plates (1x106 cells/200 μL/well) and incubated for 2 h with
10,000 particles/cell of A-PVX, A-PVX-P5L, A-PVX-P5B, or A-PVX-P20 at 37 °C and
5% CO2. Following incubation, cells were washed twice in FACS buffer (1 mM EDTA,
25 mM HEPES, 1% (v/v) FBS in PBS, pH 7.0) and fixed in 2% (v/v) paraformaldehyde
(Electron Microscopy Sciences) in FACS buffer for 10 min at room temperature. Cells were washed twice after fixation, resuspended in 400 μL FACS buffer, and stored at 4 °C until analysis. Cells were analyzed using a BD LSRII Flow Cytometer and 10,000 gated events were recorded. Data were analyzed using FlowJo 8.6.3 software.
4.2.14 Cytokine activation
BMDC were seeded onto treated plates at a concentration of 1x106 cells/mL and allowed to grow for 7 days. Before the start of the assay, plates were centrifuged at 400 g
107 CHAPTER 4: STEALTH FILAMENTS for 5 min. Culture medium was aspirated and fresh complete high glucose DMEM (HG-
DMEM) was added. PVX, PVX-P5B, or PVX-P20 was added to the cells at a concentration of 100,000 particles/cell for 6 or 24 h. Media was collected by centrifugation and analyzed for TNFα, IL-6, or IL-12 using ELISA kits (Biolegend mouse ELISA MAX Standard (TNFα and IL-6); R&D Systems mouse IL-12/IL-23 p40
Non Allele-specific DuoSet (IL-12)). IL-6 activation was analyzed following 6-hour treatment with cytochalasin D (CytoD, Sigma Aldrich). CytoD (10 μg/mL final concentration) was added 10 min prior to addition of PVX formulations.
Lipopolysaccharide (LPS) (30 ng/mL final concentration), Pam3Cys (200 ng/mL final concentration), and PolyI:C (25 μg/mL final concentration) were used as positive controls. Untreated media was used as a negative control. All methods were performed as described by the manufacturer’s instructions. Concentration of cytokine for each sample was determined using a standard curve of TNFα, IL-6, or IL-12 of known concentration supplied with each kit.
4.2.15 Inflammasome activation
BMDC were prepared as described above. Following 8 days of growth, cells were divided into two groups. All groups were treated with PVX, PVX-P5B, or PVX-P20
(100,000 particles/cell) for 6 h. Group 1 was untreated, except for PVX stimuli. Group 2 was primed with LPS (1 μg/mL final concentration) for 4 h prior to addition of stimuli.
Alum (480 μg/mL final concentration), LPS (1 μg/mL final concentration), Nigericin (10
μM final concentration), and untreated media were used as controls. Media was collected as described above and analyzed for IL-1β using mouse IL-1β/ILF2 DuoSet ELISA kit 108 CHAPTER 4: STEALTH FILAMENTS
(R&D Systems). All methods were performed as described by the manufacturer’s instructions. Concentration of IL-1β for each sample was determined using a standard curve of IL-1β of known concentration supplied with the kit.
4.2.16 Chronic inflammation
Sera were collected from mice on days 66 and 97 post treatment with PVX formulations (see immunogenicity study) and subsequently analyzed for IL-6 content using mouse IL-6 ELISA MAX standard (Biolegend), as described above. Pooled sera were analyzed and analysis was performed in triplicate.
4.2.17 Statistics
Statistical analysis was calculated in Microsoft Excel using a two-tailed Student’s t-Test, assuming unequal variances between the two data sets. Each data set contained a minimum of n=3. Specific n are indicated for each experiment in the methods section.
4.3 Results
4.3.1 Synthesis and characterization of fluorescently labeled and PEGylated PVX filaments
PVX was produced through farming in N. benthamiana plants using previously established protocols and extracted at yields of 20 mg of pure PVX from 100 g of infected leaf material [19]. PVX filaments were chemically modified with fluorescent
109 CHAPTER 4: STEALTH FILAMENTS dyes and PEG at solvent-exposed lysine side chains [33]. Chemical conjugation was achieved using N-hydroxysuccinimide (NHS)-activated esters of Alexa Fluor 647 (A647),
VivoTag-S 750, and/or PEG (conjugated particles denoted A-PVX or V-PVX depending on dye). Specifically, we set out to determine the impact of PEG chain length and conformation on the biological fate of PVX; therefore we considered coating PVX using
PEG with a molecular weight of 5000 Da in linear or branched conformation (P5L and
P5B) or linear PEG with a molecular weight of 20,000 Da (P20). Three particle configurations were prepared: PVX-P5L, PVX-P5B, and PVX-P20; for some assays fluorophores were added to allow direct detection of the nanoparticles in cells, blood, or tissues. PEG was added using 10,000 molar excess per PVX and incubated overnight to force maximum coverage. The reaction mixtures were purified by dialysis and the final products were characterized by UV/visible spectroscopy, SDS-PAGE, and TEM (Figure
4.1).
UV/visible spectroscopy was used to determine the degree of fluorescent labeling.
Beer-Lambert law and the fluorophore- and PVX-specific extinction coefficients were used determine the concentration of fluorophores and PVX in solution. (The PVX-
-1 -1 specific extinction coefficient is εPVX = 2.97 mL mg cm at 260 nm with PVX molecular weight MW = 35 Mda; and the fluorophore-specific extinction coefficients are
-1 -1 -1 -1 εA647 = 270,000 M cm at 650 nm, and εVivoTag = 240,000 M cm at 775 nm). The ratio of fluorophore:PVX concentration was used to determine the number of dyes per particle formulation (Figure 4.1A). On average 20-50% of the surface lysine side chains were modified with fluorophores; the dye concentration was kept constant or was normalized between particle formulations for various assays (see Table 4.1). For dual-labeled PVX,
110 CHAPTER 4: STEALTH FILAMENTS i.e. PVX filaments with dyes and PEG, dyes were conjugated prior to addition of PEG.
We tested the conjugation of PEG with and without fluorophores and found that the presence of dyes did not reduce the PEG conjugation efficiency (see below).
Figure 4.1. Characterization of A-PVX-PEG. A) UV/visible spectroscopy of A-PVX-PEG particles. B) SDS-PAGE after staining with Coomassie blue. Ladder = SeeBlue Plus2 protein standard, numbers represent MW in kDa. C) Negatively stained TEM images of A-PVX-PEG particles. Scale bar = 100 nm.
111 CHAPTER 4: STEALTH FILAMENTS
Table 4.1. Fluorophore and PEG-loading per PVX for the various A/V-PVX-PEG formulations used in each experiment
aResults were normalized for dye loading using a separate standard curve for each particle formulation. bSamples were made separately for three studies; data were normalized for each experiment. cVivoTag was used for FMT and A647 was used for A-P20 tissue sections and biodistribution.
SDS-PAGE and lane analysis tool using ImageJ software was used to determine the number of PEG chains per particle (Figure 4.1B). Higher molecular weight bands indicated successful PEGylation; in addition to the PVX coat protein band at ~25 kDa, bands corresponding to coat proteins modified with PEG of molecular weights of 5 or 20 kDa were detected (i.e., ~30 kDa and ~45 kDa). Dimers and oligomers of PEGylated
PVX coat proteins were also apparent. The degree of oligomerization for each sample
112 CHAPTER 4: STEALTH FILAMENTS was calculated using band density analysis to determine the percentage of PVX coat proteins in dimers and/or oligomers vs. total coat protein monomers. Unmodified coat proteins (MW ~25 kDa) and coat proteins modified with a single PEG (MW ~30 kDa or
~45 kDa) were considered coat protein monomers. Any additional higher molecular weight bands were considered dimers or oligomers of PVX-PEG. We determined that 6%,
17%, and 35% of the coat proteins were present as dimers or higher order oligomers for the A-PVX-P5L, A-PVX-P20, and A-PVX-P5B, respectively. Formation of oligomers may be explained by the entangling of the PEGylated coat proteins during the process of denaturing and electrophoresis. A significantly increased number of oligomers were observed for the A-PVX-P5B formulation; in this case, it is possible that multiple coat proteins are entangled or covalently linked together since the branched PEG presents four
NHS groups, one at each distal end, therefore possibly interlinking adjacent coat proteins
(see Figure 4.2). Interparticle crosslinking or aggregation was not observed for any of the formulations (Figure 4.1C).
To estimate the degree of PEGylation (number of PEG chains per PVX particle), band density analysis was performed and the number of modified PEG coat proteins was compared to total number of coat proteins. Data indicate that on average 25-50% of the coat proteins (i.e., up to ~600 coat proteins per PVX filament) were modified with PEG, independent of molecular weight or conformation (see Table 4.1 for a detailed analysis of PEG-per-particle ratios). The batch-to-batch variation may be explained with the inherent instability of the NHS-functional group, which is prone to hydrolysis in aqueous buffer conditions; fresh aliquots were prepared and data were normalized to keep the formulations as consistent as possible.
113 CHAPTER 4: STEALTH FILAMENTS
TEM imaging confirmed that all particle formulation were structurally sound post-chemical modification. Elongated filaments of approximately 515x13 nm were observed in TEM imaging (Figure 4.1C). While the addition of PEG would increase the hydrodynamic radius of PVX, this was not detectable in TEM imaging using negatively- stained samples.
4.3.2 PEG conformation
Immobilized PEG molecules exist in different conformations based on their grafting density and chain length; one differentiates the brush vs. mushroom conformation (as well as various intermediates) [34]. We estimated the conformation of the PEG chains grafted to PVX based on the grafting density (PEG chains per PVX particle), the resulting distance between conjugated PEG chains (D), and the Flory dimension (RF) of the various PEG chains. If RF < D, PEG is assumed to exist in a mushroom conformation, and if RF > D, PEG is assumed to exist in a brush conformation
[34].
RF for each PEG was calculated using the following equations:
3/5 For linear polymers: RF = an (1),
-2/5 3/5 and for branched polymers: RF = af N (2), where a = length of one monomer (0.35 nm for PEG), N = fn (total number of monomers), n = number of monomers (for linear polymers) or number of monomers per branch (for branched polymers), and f = number of branches. For P5L and P20, n is 110 and 440,
114 CHAPTER 4: STEALTH FILAMENTS
respectively. For P5B, f is 4 and N is 110. Based on these equations, we calculated RF for
P5L, P5B, and P20 to be 5.87 nm, 3.37 nm, and 13.49 nm.
Distance (D) was calculated considering the available surface area of PVX and numbers of PEGs per PVX; we assumed a statistically random distribution and homogenous distribution of PEGs on the PVX surface and considered a ratio of CP:CP-
PEG of 3:1, or one PEG for every 3 coat protein residues (Table 4.1). The 515x13 nm
2 PVX filament has a surface area of APVX = 21,033 nm with each of the 1270 identical
2 coat proteins (CP) providing a surface area of ACP =16.6 nm of space. If the PVX CP is assumed to be a square, we determine the distance between two lysines to be 4.1 nm.
Since one PEG is found per every three lysine residues, each PEG is 12.2 nm apart
(therefore D=12.2 nm).
From these calculations, it appears that for the PVX formulations with short PEG chains, i.e. PVX-P5B and PVX-P5L, the PEG chains are likely to exist in a mushroom conformation (D > RF) and a brush conformation is achieved for the PVX formulations with higher molecular weight PEG, PVX-P20 (Figure 4.2), however it is also possible that the PEG chains exist in a more complex conformation. For the branched PEG formulation PVX-P5B, as discussed above, it is possible some PEG chains are attached to multiple adjacent coat proteins due to the presence of an NHS group on each arm. In a fully extended conformation, the distance between NHS groups is 19 nm, while considering a more relaxed configuration, the estimated distance would be in the order of
6 nm. The distance between two adjacent lysine residues is estimated to be 4.1 nm.
Therefore, in either configuration, it would be feasible for the branched P5B to interlink
115 CHAPTER 4: STEALTH FILAMENTS
PVX coat proteins within a single particle. If multiple coat proteins are interlinked, the
PEG chains may be presented closer to the PVX surface (Figure 4.2).
Figure 4.2. Schematic (drawn to scale) showing the various PEG conformations presented on the PVX filament. The distance between PEG molecules was estimated based on the grafting density and the dimensions of PVX. The conformation and inter-PEG spacing (D) was determined based on the Flory dimension (RF) of the various PEGs under consideration.
4.3.3 Pharmacokinetics of PEGylated PVX
The conjugation of PEG to nanoparticle carriers is known to reduce non-specific interactions with blood components, cells, and tissues, therefore increasing nanoparticle bioavailability. We thus set out to investigate how the different PEG configurations affect
116 CHAPTER 4: STEALTH FILAMENTS the PVX pharmacokinetic properties. We previously reported the pharmacokinetics of A-
PVX-P5L [35]; for comparison these data are shown as a gray line in Figure 4.3. In this study, we determined the plasma half-life of A-PVX, A-PVX-P5B, and A-PVX-P20.
PVX-based formulations were administered intravenously in the tail vein of
Balb/C mice, and blood was collected prior to injection and up to 36 h post- administration. Sera were collected and the percent injected dose (%ID) was determined based on fluorescence obtained in each sample (normalized against a standard curve based on A-PVX samples of known concentration). Data were fitted for one-phase decay or two-phase decay using GraphPad Prism to determine the circulation time and half-life
(Figure 4.3). Data indicate that non-PEGylated PVX exhibited a one-phase decay, while
PEGylated PVX formulations follow a two-phase decay. Data indicate that PVX has a half-life of t1/2 = 19 min. A-PVX-P5L, A-PVX-P5B, and A-PVX-P20 all showed two- phase decay, which is characterized by an initial fast clearance rate, up to 100 min, and a
I slower clearance for the remaining time: A-PVX-P5B had a fast initial half-life of t 1/2 =
II 14 min and a slower second half-life of t 1/2 = 1142 min, while A-PVX-P20 had a fast
I II half-life of t 1/2 = 27 min, and a slower second half-life of t 1/2 = 231 min. Previously
I II reported [35], A-PVX-P5L had a t 1/2 = 11 min and a t 1/2 = 409 min.
Overall, the half-life of PVX is significantly improved upon PEGylation. After a fast initial clearance rate (~10-30 min), particles remained in circulation for several hours
II with t 1/2 of reaching up to 19 h for the A-PVX-P5B formulation.
117 CHAPTER 4: STEALTH FILAMENTS
Figure 4.3. Pharmacokinetics of A-PVX-PEG. A-PVX-PEG particles were administered intravenously into Balb/C mice (100 μg/mouse); plasma was collected and analyzed at various time points over 2160 minutes (inset). Percent injected dose (%ID) was determined based on a standard curve of known PVX concentration. GraphPad Prism software was used to analyze and fit the data; plasma half-lives are shown in the Table. PVX = light blue line, data points as circles, A-PVX-P5L = gray line, previously reported in the literature [35], A-PVX-P5B = red line, data points as squares, A-PVX-P20 = purple line, data points as triangles, A-PVX-P5 = red line, data points as squares.
4.3.4 Biodistribution and clearance
Fluorescence molecular tomography (FMT) was used to observe biodistribution of V-PVX, V-PVX-P5B, and V-PVX-P20 up to 52 h post intravenous administration into the tail vein of Balb/C animals. (We previously reported the biodistribution of PVX-P5L
[26] and therefore evaluated only the new formulations. The PVX-P5L biodistribution and clearance is discussed in the context of this new data; see Discussion section). For all
PVX formulations, fluorescence signals were no longer detectable at 52 h post-
118 CHAPTER 4: STEALTH FILAMENTS administration using FMT imaging, indicating clearance from the body (Figure 4.4A).
Images were analyzed and the following regions of interest (ROIs) were considered: whole body, liver and spleen, and bladder. Data were normalized to number of dyes per particle for each formulation (Figure 4.4B).
V-PVX (shown in blue) showed maximum fluorescence intensity at one hour post-injection; by 24 h the fluorescence intensity had dropped below 10% of that maximum. V-PVX showed relatively consistent levels of accumulation in the liver and spleen until 8 h, at which point the fluorescent signals dropped significantly (which is in agreement with the whole body ROI). At early time points, up to 2 h post-administration,
V-PVX showed increased fluorescence intensity in the bladder compared to the liver and spleen. These data are in good agreement with the pharmacokinetics study, with the one- phase decay for PVX indicative of renal clearance.
It was apparent that V-PVX-P5B and V-PVX-P20 showed much lower tissue accumulation (as indicated by lower fluorescence intensities) overall, with whole body fluorescence intensity levels being about 3-fold lower compared to signals observed for the non-PEGylated PVX formulation. This reduced tissue accumulation is a desired outcome consistent with conjugation of PEG. PEGylation of nanocarriers reduces interaction with blood components, cells, and tissues, and therefore results in a longer circulation time for PEGylated vs. native PVX. It should be noted that the threshold of the FMT instrument was set to measure particles deposited in tissues; signals from particles diluted in circulation are not detected. Overall, V-PVX-P5B and V-PVX-P20 showed similar trends. Both formulations accumulated in the liver and spleen until approximately 6-8 h post-administration, and neither showed significant accumulation in
119 CHAPTER 4: STEALTH FILAMENTS the bladder. These data also are in good agreement with pharmacokinetics studies, which indicated a two-phase decay for the PEGylated formulations. The increased diameter of the PEGylated filaments (see Figure 4.2) may reduce renal filtration, leading to the two- phase pharmacokinetics inherent with combined renal and mononuclear phagocyte system (MPS) clearance.
Immunofluorescence imaging was used to confirm that fluorescence signals detected in FMT correlated with the presence of PVX particles. The possibility exists that the fluorophores would be cleaved in vivo, resulting in imaging of free dye and not PVX particles. To evaluate co-distribution of fluorescent cargo and PVX carriers, confocal microscopy studies were carried out using the PVX-P20 formulation. FMT imaging indicated maximum accumulation of PVX-P20 in liver and spleen sections at 6 h post- administration and clearance at 52 h post-administration. Imaging of tissue sections using fluorescence microscopy indicated colocalization of PVX-specific antibodies with the conjugated A647 dye (Figure 4.4C), which suggests that the fluorescent cargo remains attached to PVX in vivo.
We have previously shown that PVX accumulated in liver macrophages and in the white pulp of the spleen, where it colocalizes with the B cell marker B220 [26]; this general pattern is consistent with the present study. While PVX-derived signals were no longer detectable in liver sections 24 h post-administration, PVX remained detectable in the spleen for up to 96 h, indicating that imaging of tissue sections is more sensitive than
FMT imaging.
120 CHAPTER 4: STEALTH FILAMENTS
121 CHAPTER 4: STEALTH FILAMENTS
Figure 4.4. Biodistribution of native and PEGylated PVX. A) FMT imaging of Balb/C mice following intravenous tail vein injection of V-PVX, V-PVX-P5B, or V-PVX-P20. B) Quantification of FMT signal for the regions of interest (ROIs): whole body (top), liver and spleen (middle), or bladder (bottom); V-PVX (n=2, one animal died prior to start of this study, in blue), V-PVX-P5B (n=3, in red), or V-PVX-P20 (n=3, in purple). C) Fluorescence microscopy tissue sections of liver (left) and spleen (right) from mice post A- PVX-P20 administration (intravenous in the tail vein). Sections were stained with a rabbit anti-PVX antibody and secondary Alexa Fluor 488-labeled antibody (green); the antibody staining colocalized with A-PVX-P20 (red, signals derived form conjugated A647 dye). Nuclei were stained with DAPI (blue). Arrows indicate A-PVX-P20 in liver. Scale bar is 100 μm.
4.3.5 Immunogenic properties of PEGylated PVX
Like other viruses, protein-based formulations, or other nanoparticles, plant viruses interact with immune cells and are cleared from the body via the MPS (see
Figure 4.4). In addition, a recent study suggested prevalence of anti-tobacco mosaic virus
(TMV) antibodies in humans [36]. Therefore, we set out to determine the IgG titers after repeat administration of various PVX-based formulations and to test whether produced antibodies would recognize the stealth PVX-PEG filaments.
Balb/C mice were administered PVX-based carriers on days 0, 5, 10, and 66. The initial 5-day interval was chosen because this would follow a typical chemotherapy schedule (the long-term goal is to develop PVX as a drug delivery vehicle), while the late time point (66 days) was chosen as a secondary booster. Blood and sera were collected over a 3-month time frame (see immunization schedule, Figure 4.5A). The sera were analyzed for IgG content specific for PVX using ELISA in which sera were probed against PVX-coated plates. Data indicate that for each formulation analyzed, PVX- specific IgG antibodies were raised. It was interesting to note that the overall IgG titers
122 CHAPTER 4: STEALTH FILAMENTS were lower for animals treated with PVX-P20 compared to all other PVX-based formulations (Figure 4.5A).
It was found that PVX-specific antibodies were raised in mice regardless of shielding. Therefore we sought to address whether these antibodies would recognize the stealth filaments. A sandwich ELISA was performed, in which plates were coated with an anti-PVX antibody produced in rabbits immunized with native PVX, and native and
PEGylated PVX particles were subsequently captured. The captured PVX samples were then probed with the sera from immunized mice and detection was carried out using an anti-mouse secondary antibody. Data indicate a significant decrease in particle recognition for both PEGylated samples studied, PVX-P5B and PVX-P20, compared to non-PEGylated PVX (Figure 4.5B), with PVX-P20 being more efficiently shielded compared to PVX-P5B. These data indicate that when covering a sufficiently large surface area with stealth coatings, effective shielding from antibodies is indeed achieved.
To further test this, we synthesized a PVX modified with 4-arm branched PEG but with a molecular weight of 10,000 Da to increase surface coverage and therefore minimize antibody recognition. PVX-P10B was synthesized with 609 PEG chains; based on the RF = 5.11 nm for P10B, and the number of PEG chains attached per PVX, the PEG chains are likely displayed in a mushroom conformation (D = 8.14 nm, and therefore D >
RF) covering the entire surface area of the filamentous particle (Table 4.1). Using a sandwich ELISA, we determined that absorbance at 405 nm was 0.32 and 0.17 for PVX and PVX-P10B, respectively, confirming that PVX-P10B was effectively shielded from antibody recognition (Figure 4.5B).
123 CHAPTER 4: STEALTH FILAMENTS
Figure 4.5. Anti-PVX IgG titers and antibody-to-PVX binding. A) Timeline of treatment schedule; time points of PVX administration and blood collection are indicated. Anti-PVX IgG titers of sera from mice treated with PVX, PVX-P5L, PVX-P5B, and PVX-P20. B) Sandwich ELISA testing the reactivity of anti- PVX antibodies against PVX, PVX-P5B, PVX-10B, and PVX-P20. All data were analyzed using Excel software and Student’s t-test * p < 0.001; Ab = antibody; AP = alkaline phosphatase.
124 CHAPTER 4: STEALTH FILAMENTS
4.3.6 PVX–cell interactions
To correlate the in vivo properties, PVX uptake by immune cells was studied.
RAW264.7 macrophages (ATCC) and primary bone marrow derived dendritic cells
(BMDC) were used. Concentration- (10,000 vs. 100,000 particles per cell) and time- dependent studies (20 min vs. 2 h incubation) were performed and cell interactions were quantified using flow cytometry (Figure 4.6 and Figure 4.7).
Figure 4.6. A-PVX-PEG-cell interactions measured by flow cytometry. A-PVX-PEG formulations were incubated with RAW264.7 and BMDC at a concentration of 10,000 particles/cell for 2 hours. A) Histograms of A647 signal versus count for A-PVX-PEG particles in RAW264.7 (top) and BMDC (bottom). Gray = cells only, light blue = A-PVX, orange = A-PVX-P5L, red = A-PVX-P5B, purple = A- PVX-P20. Any counts within the indicated gate represent positive cells. At least 10,000 events were considered; all studies were done in triplicate and data were analyzed using FlowJo software. B) Statistical analysis and quantitative data showing percent cell uptake of A-PVX-PEG particles in RAW264.7 or BMDC. * p<0.05.
125 CHAPTER 4: STEALTH FILAMENTS
Figure 4.7. Time and concentration-dependent A-PVX-PEG-cell interactions using RAW264.7 cells and flow cytometry. * p<0.05 vs. A-PVX.
Data indicate that immune cell uptake of PVX is concentration- and time- dependent. Overall PEGylated PVX particles are less prone to macrophage; i.e. uptake of
PEGylated PVX formulations are delayed and reduced as a result of PEG coating and shielding (Figure 4.6). While 29.0% of macrophages and 19.4% of BMDC stained positive for A-PVX, only 8.1%, 6.0%, and 6.3% of macrophages and 11.7%, 6.9%, and
6.0% of BMDC stained positive for A-PVX-P5L, A-PVX-P5B, and A-PVX-P20, respsectively. No significant differences in cell uptake were observed between any particles (native and PEGylated) when incubated for 20 min at a concentration of 10,000 particles/cell; at early time points and low particle concentrations, PVX–cell interactions are negligible (Figure 4.7). Similarly, no significant differences were observed when
126 CHAPTER 4: STEALTH FILAMENTS particles were incubated with cells for 2 h at a concentration of 100,000 particles/cell; at longer time points and high concentration all PVX formulations are taken up by macrophages (Figure 4.7). Differences between PEGylated and native PVX became apparent when particles were incubated for 20 min at a concentration of 100,000 particles/cell and when particles were incubated for 2 h at a concentration of 10,000 particles/cell. Under these conditions, statistically significant decreases in percent cell uptake for PEGylated particles were detected compared to native PVX. However, there were no differences in percent cell uptake comparing the different PEGylated formulations, A-PVX-P5L, A-PVX-P5B, and A-PVX-P20. PVX–cell interactions in macrophages and BMDC showed similar trends (Figure 4.6). (The two populations observed using BMDC maybe explained by a mixture of non-activated and activated cells; cells were not sorted for the uptake studies.) Overall, these data indicate that
PEGylation results in a decreased rate of cell uptake in immune cells.
4.3.7 Cytokine activation in vitro and in vivo
Knowing that PVX interacts with macrophages and BMDC, we studied whether
PVX–cell interactions would trigger inflammatory cytokine signaling. To do this, we incubated PVX, PVX-P5B, or PVX-P20 with BMDC for 6 or 24 h and analyzed the media for TNFα, IL-6, or IL-12 using ELISA kits.
127 CHAPTER 4: STEALTH FILAMENTS
Figure 4.8. Cytokine activation in BMDC induced by PVX-PEG particles. Induction of A) TNFα or B) IL-12 after 6 (solid) or 24 hour (striped) incubation with PVX-PEG formulations. C) IL-6 production after 6 hours in the absence (solid) or presence (striped) of cytochalasin D. D) Analysis of mouse sera for IL-6 after repeat intravenous administration of the PVX-PEG particles. * p<0.05 compared to PVX. 128 CHAPTER 4: STEALTH FILAMENTS
Treatment with non-PEGylated PVX resulted in the largest amount of TNFα activation, with detectable levels of 2834 pg/mL at 6 and 24 h post-treatment. PEGylated
PVX-P5B and PVX-P20 showed significantly reduced amounts of TNFα activation compared to PVX after both 6 and 24 h of incubation (2057 and 1330 pg/mL, respectively at 6 h and 2146 and 2269 pg/mL, respectively at 24 h). There were no differences comparing the two PEGylated formulations (Figure 4.8A). Reduction of cytokine activation is in agreement with reduced cell interactions of PEGylated PVX.
Treatment of BMDC with PVX, PVX-P5B, and PVX-P20 induced IL-12 production. However, modest levels of 939 pg/mL and 756 pg/mL of IL-12 were reached when treated with PVX and PVX-P20, respectively (Figure 4.8B). Treatment of PVX-
P5B resulted in elevated levels of IL-12 production, reaching levels of 2734 pg/mL at 24 h post-treatment, which is a three-fold increase compared to IL-12 levels from cells treated with PVX or PVX-P20. These data indicate that the branched PEG may be processed differently, which may be a result of interparticle crosslinking (see Figures 4.1 and 4.2).
PVX, PVX-P5B, and PVX-P20 triggered production of IL-6, reaching levels of up to 4000 pg/mL (Figure 4.8C). To determine whether cytokine activation is correlated with cell uptake, we studied different cell uptake mechanisms. BMDC were treated with cytochalasin D (CytoD) to block actin polymerization, therefore preventing phagocytosis
[37]. In all cases, IL-6 production was reduced upon CytoD treatment, with statistically significant differences observed for the PEGylated PVX formulations: IL-6 production was reduced by 23% and 45% when cells were treated with CytoD prior to treatment with
PVX-P5B and PVX-P20, respectively (Figure 4.8C).
129 CHAPTER 4: STEALTH FILAMENTS
Although both PVX-P5B and PVX-P20 showed statistically significant decreases in IL-6 production after treatment with CytoD, a complete knockdown of IL-6 production was not achieved. This indicates that multiple cell entry mechanisms may play a role and that IL-6 production is not solely linked to phagocytosis. PVX-induced IL-6 activation may be a result of either particle interaction with cell surface receptors or non- phagocytosis cellular uptake.
In addition to studying inflammatory cytokine signaling, we also set out to determine whether PVX-treatment would result in inflammasome-mediated cytokine expression, since activation of the inflammasome has been linked to nanoparticles [38-
40]; specifically we studied IL-1β activation (Figure 4.9). Prior to incubation with PVX formulations, samples were primed with lipopolysaccharide (LPS) for 4 hours to increase the amount of pro-IL-1β available; alum treatment was used as a positive control for inflammasome activation. Data indicate that IL-1β is not produced in BMDC after PVX treatment. No significant differences were observed comparing non-PEGylated and
PEGylated PVX formulations, and there was no difference comparing non-primed and
LPS-primed BMDCs. Overall, these results indicate that PVX, PVX-P5B, and PVX-P20 do not activate inflammasome-mediated cytokine, IL-1β.
Data from in vitro studies suggest that PVX-based formulations are taken up by macrophages and dendritic cells, with PEGylated formulations showing reduced cell uptake rates. Further data suggest that PVX–BMDC interactions trigger expression of pro-inflammatory cytokines when incubated with BMDCs in vitro (Figure 4.8A-C).
Therefore, we studied whether repeated treatment of mice resulted in chronic inflammation; this was assessed by analyzing IL-6 levels from sera collected from
130 CHAPTER 4: STEALTH FILAMENTS immunized mice during the immunogenicity studies on days 66 and 97 (Figure 4.8D).
By day 66, the mice had received three treatments of PVX (on days 0, 5, and 10), and by day 95, the mice were treated with the initial three injections followed by an additional booster on day 66 (see Figure 4.5A). Significantly elevated levels of IL-6 were not detectable for any of the sera analyzed (Figure 4.8D), therefore indicating that chronic inflammation is not induced.
Figure 4.9. Inflammasone activation as measured by IL-1β production. A) Untreated and B) LPS- primed BMDC cells after exposure to PVX-PEG formulations.
4.4 Discussion
In summary, we synthesized stealth filaments using the PVX platform modified with PEG. Pharmacokinetics, biodistribution, as well as potential immune and inflammatory responses were evaluated in response to stealth filaments coated with PEG chains of varying length and conformation. PEGylation effectively reduces (but does not prevent) interaction with immune cells in vitro and in vivo. While stealth coatings do not prevent production of antibodies against the carrier, immune recognition and antibody 131 CHAPTER 4: STEALTH FILAMENTS neutralization can be overcome upon polymer coating. Stealth filaments show rapid tissue clearance through combined renal and MPS clearance mechanisms. Further, tissue compatibility was indicated, with no apparent inflammatory signaling in vivo.
Furthermore, stark differences were noted in the pharmacokinetic properties; tailoring
PEG chains length and conformation (brush vs. mushroom) allows tuning of the pharmacokinetics, yielding long-circulating stealth filaments for applications in nanomedicine.
While the development pipeline of nanoparticle-based nanomedicines is moving rapidly, a fundamental understanding of the in vivo biology of various systems is lacking.
Recent research has shown cell target specificity results from a combined contribution by particle morphology and surface chemistry [11, 12]. Stealth coating is an accepted technology to increase the bioavailability of inorganic and organic nanoparticle formulations. Increased plasma circulation is a result of PEG conjugation to nanoparticles and has been observed for various synthetic platforms [41, 42], and this is in agreement with the data presented in this study (Figure 4.3). Previous studies have shown that protein adsorption is impacted by PEG density and molecular weight on both 2D surfaces and 3D nanoparticle surfaces and increased pharmacokinetics is explained by the fact that the hydrophilic PEG shell reduces the rate of formation of the so-called ‘protein corona’, which consists of plasma proteins (opsonins, complement, albumin, fibrinogen, immunoglobulins, etc.[43-46]) surrounding the nanocarrier, which in turn results in reduced uptake by immune cells and diminished deposition in non-target organs [42, 47-
49]. Indeed, we show that PEGylation of PVX results in decreased liver and spleen deposition in vivo (Figure 4.4) and reduced uptake in cells of the MPS in vitro (Figure
132 CHAPTER 4: STEALTH FILAMENTS
4.6). These findings are in good agreement with reports on other PEGylated plant viruses, such as cowpea mosaic virus (CPMV) [50], mammalian viruses, including adenoviruses under development for gene therapy applications [51], as well as synthetic nanoparticles
[52-55].
Pharmacokinetic data indicate that the addition of PEG induced a switch from one-phase to two-phase plasma clearance kinetics: native PVX exhibited a one-phase clearance, which is indicative of clearance by primarily renal filtration. Upon PEGylation,
PVX exhibited a two-phase clearance pattern, which is due to combined renal filtration and MPS clearance (Figure 4.3); these findings are in agreement with other reports [56].
Biodistribution profiles of PVX-PEG stealth filaments reflect the pharmacokinetic studies.
As predicted by pharmacokinetics, injection of native PVX is followed by rapid renal clearance. The size cut-off for glomerular filtration is sub-20 nm [57]; therefore, PVX aligned in the flow is theoretically thin enough to pass through the renal filtration system.
Indeed, others have reported that carbon nanotubes are cleared through renal filtration [58,
59], supporting the hypothesis that elongated, rod-shaped nanomaterials with diameters less than 20 nm can be cleared by renal filtration. PEGylation of PVX increases its hydrodynamic radius (Figure 4.2), thereby reducing but not completely eliminating renal filtration. Reduction in renal clearance rates leads to MPS clearance; however, it should be noted that liver and spleen deposition of stealth PVX-PEG particles is generally reduced compared to naked PVX (Figure 4.4).
Whole mouse imaging using FMT indicates rapid tissue clearance of stealth filaments (within 3 days, Figure 4.4). Sectioning and immunofluorescence also confirm rapid tissue clearance of the filamentous nanoparticles from the liver, while small
133 CHAPTER 4: STEALTH FILAMENTS quantities of particles were still detectable in the spleen at 96 h post-administration, most likely as a result of less metabolic activity in the spleen. Within the spleen, PVX-PEG particles travel through the red pulp (~6 h post-administration) and then accumulate within the white pulp of the spleen (at ~24 h post-administration), where the B cells reside. This pattern is in agreement with our previous observation of PVX [26] and TMV
[60], and it is also consistent with the deposition of gold nanoparticles within the spleen
[61].
Overall, biodistribution of native and PEGylated PVX is consistent with trends seen for a wide range of other nanoparticles, including various viral nanoparticle systems
[60, 62-65]. For example, 30 nm-sized icosahedrons CPMV and phage Qβ predominantly accumulate in the liver, while 300x18 nm rod TMV, 30 nm-sized phage MS2, and 900x6 nm filamentous phage M13 were found in both the liver and spleen [60, 62-65]. Of note, a different biodistribution pattern was observed for the 30 nm-sized plant virus cowpea chlorotic mottle virus (CCMV) with much of the administered dose accumulating in the thyroid [66].
While the overall biodistribution of PVX-PEG stealth filaments is consistent with synthetic nanocarriers, it is of importance to note that compared to synthetic nanoparticles, several of which persist within the body for weeks and longer, virus-based materials are biodegradable and thus are removed from the body within days (Figure 4.4).
For some nanoparticles, such as gold, silica, and carbon nanostructures, clearance by the
MPS, and ultimately from the body, can be slow. Synthetic materials, such as gold [67,
68], polymeric materials [69], iron oxide nanoparticles [70], and carbon nanotubes [71], may persist in the liver for months after injection, which may lead to long-term adverse
134 CHAPTER 4: STEALTH FILAMENTS effects. Fast breakdown and clearance of the protein-based stealth filaments may be explained by the fact that the cellular machinery such as proteases, hydrolyases, and other enzymes are naturally trained to break down proteinaceous materials. In addition, some synthetic nanoparticles have previously been reported to accumulate in lungs, heart, and skin [68, 72-74]. PVX-based stealth filaments were not detected in any of these tissues. It is especially interesting to note that lung deposition did not occur, because other elongated, synthetic systems commonly accumulate in the lungs [72, 74]. A main difference is the flexible nature of the soft, PVX-based nanomaterials vs. rigid hard materials such as carbon nanotubes or silica-based nanorods.
Our data show that PEGylation successfully reduces the immunogenic properties of filamentous PVX, including reduced uptake in immune cells and overcoming antibody recognition (Figures 4.5 and 4.6). Since most foreign nanomaterials, including synthetic nanoparticles, are immunogenic, leading to production of carrier-specific antibodies [66,
75-79], it is most important to understand whether stealth particles are recognized and potentially neutralized upon repeated administration. As expected, we found that while
PEGylation did not prevent the production of carrier-specific antibodies, PEGylation indeed significantly reduced immune recognition of PEGylated stealth filaments (Figure
4.5). This is consistent with reports on adeno-associated viruses and adenoviruses, both of which are protected from neutralizing antibodies in vitro and in vivo upon PEGylation
[80-83]. The stealth filaments therefore provide an attractive platform for drug delivery applications, overcoming immune recognition opens the door for repeat administration, a requirement for treatment of oncological diseases.
135 CHAPTER 4: STEALTH FILAMENTS
To gain insight into the potential inflammatory response, we investigated in vitro uptake by immune cells and cytokine activation, both in vitro and in vivo. As with other stealth nanomaterials [50-55], PEGylated PVX showed a decreased rate of uptake into immune cells, and this correlated to reduced production of inflammatory cytokines, such as TNFα, IL-12, and IL-6, which also is in agreement with previous reports investigating the in vivo biology of other virus-based platforms [51, 84]. Most interesting, neither native nor PEGylated PVX induced activation of the inflammasome-mediated cytokine,
IL-1β; this is in contrast to many synthetic nanoparticles.[72, 74, 76, 85-87] It is also important to note that while in vitro induction of inflammatory signaling was observed, no chronic inflammation was observed in vivo after repeat administration of naked or stealth filaments, as monitored by IL-6 levels (Figure 4.8); this is in contrast to mammalian virus-based gene delivery vectors, where elevated IL-6 levels have been reported [51]. The plant pathogen is less likely to trigger adverse side effects because it is less likely to interact with the mammalian cell machinery or receptors triggering signal transduction events.
A common challenge for nanoparticle-based delivery systems is to determine the correct balance between circulation time, targeting, and clearance. Tailoring PVX with various PEG coatings, we were able to tune the pharmacokinetic properties of stealth
PVX. For the linear PEG formulations we found that higher molecular weight PEG increases circulation time more significantly [48, 88] than lower molecular weight PEG.
Biochemical data (Figure 4.1) in combination with modeling (Figure 4.2) indicate that
PVX-P20 presents the PEGs in a brush-like conformation, while PVX-P5L presents PEG chains in a mushroom conformation. More efficient surface coverage in combination with
136 CHAPTER 4: STEALTH FILAMENTS the brush conformation of the higher molecular weight PEGs results in a more efficient stealth effect. This is consistent with other reports that demonstrate that a brush-like conformation of PEG is most effective to decrease clearance by immune cells [89].
Furthermore, our data indicate that branched PEG (PVX-P5B) proved more effective than linear PEG (PVX-P5L) in increasing circulation time. Based on Flory
Dimension and grafting distance along PVX, both P5L and P5B present in a mushroom conformation. However, P5B is more densely packed and closer to the surface of PVX due to simultaneous binding of multiple coat proteins, therefore potentially shielding the particle more efficiently. While modeling indicates that the PEG arms of the P5B formulations are folded into a mushroom-based conformation, it is also possible that the non-conjugated PEG arms are presented as a brush-like structure, extending off the surface and leading to the highly increased pharmacokinetic profiles observed, with a phase II half-life of almost 20 hours. In future studies, it would be interesting to use
CryoEM in combination with tomography to gain further structural insights.
The ability to tailor circulation times based on surface chemistry is interesting and allows customization of PVX-PEG filaments for specific desired applications. For example, longer circulation times may be desirable for therapeutic applications and passive tumor targeting [90], while shorter circulation times are advantageous for receptor-targeted contrast agents or to reduce adverse systemic effects when highly toxic payloads are delivered [91, 92].
Understanding the in vivo properties of nanoparticles is important for translation, and it is equally important to understand efficacy and sensitivity, as well as clearance profiles. In addition to safety and biocompatibility considerations, another important 137 CHAPTER 4: STEALTH FILAMENTS point to consider for a nanoparticle formulation’s foray into the clinical world is its reproducibility and the completeness of the material’s characterization. In particular, it is important that each compound/nanoparticle is the same. The nano-manufacturability and engineerability of plant virus-based materials is a strength of the material, as size and monodispersity impact in vivo distribution. Therefore, the goal must be to make protocols for the synthesis of uniform nanoparticle formulations. Since they are genetically controlled, to a first approximation, every particle is identical. Furthermore, genetic engineering provides a means of controlling the formulation with atomic precision. Most chemical modifications are stochastic, but it is relatively straightforward to determine concentrations of the protein-based carrier along with how many sites are conjugated per nanoparticle through the use of colorimetric assays, gel electrophoresis, UV/visible spectroscopy, and/or other methods. The same level of quantification is difficult to achieve with liposomes or any other nanoparticle, as it is challenging to determine the number of nanoparticles in solution.
4.5 Conclusions
Filamentous plant viruses, such as PVX, present a novel class of nanomaterials with potential applications in drug delivery and tissue-specific imaging. We previously showed that filamentous plant virus-based nanoparticles home to tumor tissues, extravasate, and penetrate into the cancerous tissue; data indicate that this passive tumor accumulation is shape-mediated [35]. Recent data indicating advantageous behaviors of elongated nanomaterials for in vivo medical applications have instigated the search and development for novel chemistries and synthetic routes to yield high aspect ratio
138 CHAPTER 4: STEALTH FILAMENTS nanomaterials. Produced by nature, the highly symmetrical and proteinaceous filaments offer an ideal scaffold for the manufacturing of such materials. In this paper, we discuss the development of stealth filaments by grafting PEG chains to the 515x13 nm PVX- based protein scaffold. We demonstrate increased bioavailability with pharmacokinetics tunable through PEG chain length and conformation. Polymer coating using PEG effectively reduced interaction with immune cells and limited immune recognition by antibodies. The stealth filaments were found to be compatible with tissues and did not induce extended inflammatory responses upon repeat administration in animals. Further improvements may be achieved through incorporation of alternative polymers, such as zwitterionic species. Furthermore, higher polymer densities and favorable brush conformations may be achieved using higher-order branched PEG molecules (e.g. 8-arm
PEGs vs. 4-arm PEGs; as an alternative one might consider grafting polymers from the protein scaffold rather than grafting-to) [93]. Further surface chemistry optimization and a more comprehensive understanding of the biocompatibility of this filamentous platform technology will pave the way for potential translational research in the future.
4.6 Works Cited in this chapter
1. Cai S, Vijayan K, Cheng D, Lima EM, Discher DE. Micelles of different morphologies--advantages of worm-like filomicelles of PEO-PCL in paclitaxel delivery. Pharm Res 2007;24:2099-109.
2. Chauhan VP, Popovic Z, Chen O, Cui J, Fukumura D, Bawendi MG, et al. Fluorescent nanorods and nanospheres for real-time in vivo probing of nanoparticle shape-dependent tumor penetration. Angewandte Chemie (International ed 2011;50:11417-20.
3. Christian DA, Cai S, Garbuzenko OB, Harada T, Zajac AL, Minko T, et al. Flexible filaments for in vivo imaging and delivery: persistent circulation of
139 CHAPTER 4: STEALTH FILAMENTS
filomicelles opens the dosage window for sustained tumor shrinkage. Molecular pharmaceutics 2009;6:1343-52.
4. Decuzzi P, Godin B, Tanaka T, Lee SY, Chiappini C, Liu X, et al. Size and shape effects in the biodistribution of intravascularly injected particles. Journal of controlled release : official journal of the Controlled Release Society 2010;141:320-7.
5. Geng Y, Dalhaimer P, Cai S, Tsai R, Tewari M, Minko T, et al. Shape effects of filaments versus spherical particles in flow and drug delivery. Nat Nanotechnol 2007;2:249-55.
6. Gentile F, Chiappini C, Fine D, Bhavane RC, Peluccio MS, Cheng MM, et al. The effect of shape on the margination dynamics of non-neutrally buoyant particles in two-dimensional shear flows. J Biomech 2008;41:2312-8.
7. Lee SY, Ferrari M, Decuzzi P. Shaping nano-/micro-particles for enhanced vascular interaction in laminar flows. Nanotechnology 2009;20:495101.
8. Doshi N, Prabhakarpandian B, Rea-Ramsey A, Pant K, Sundaram S, Mitragotri S. Flow and adhesion of drug carriers in blood vessels depend on their shape: a study using model synthetic microvascular networks. Journal of controlled release : official journal of the Controlled Release Society 2010;146:196-200.
9. Lee S-Y, Ferrari M, Decuzzi P. Shaping nano-/micro-particles for enhanced vascular interaction in laminar flows. Nanotechnology 2009;20:495101.
10. Tan J, Shah S, Thomas A, Ou-Yang HD, Liu Y. The influence of size, shape and vessel geometry on nanoparticle distribution. Microfluid Nanofluidics 2013;14:77-87.
11. Vacha R, Martinez-Veracoechea FJ, Frenkel D. Receptor-mediated endocytosis of nanoparticles of various shapes. Nano Lett 2011;11:5391-5.
12. Arnida, Janát-Amsbury MM, Ray A, Peterson CM, Ghandehari H. Geometry and surface characteristics of gold nanoparticles influence their biodistribution and uptake by macrophages. European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft für Pharmazeutische Verfahrenstechnik eV 2011;77:417-23.
13. Firme CP, 3rd, Bandaru PR. Toxicity issues in the application of carbon nanotubes to biological systems. Nanomedicine : nanotechnology, biology, and medicine 2010;6:245-56.
14. Geng Y, Dalhaimer P, Cai S, Tsai R, Tewari M, Minko T, et al. Shape effects of filaments versus spherical particles in flow and drug delivery. Nat Nanotechnol 2007;2:249-55.
140 CHAPTER 4: STEALTH FILAMENTS
15. Chitale R. Merck hopes to extend gardasil vaccine to men. J Natl Cancer Inst 2009;101:222-3.
16. Liu TC, Galanis E, Kirn D. Clinical trial results with oncolytic virotherapy: a century of promise, a decade of progress. Nat Clin Pract Oncol 2007;4:101-17.
17. Shirakawa T. Clinical trial design for adenoviral gene therapy products. Drug News Perspect 2009;22:140-5.
18. Shukla S, Dickmeis C, Nagarajan AS, Fischer R, Commandeur U, Steinmetz NF. Molecular farming of fluorescent virus-based nanoparticles for optical imaging in plants, human cells and mouse models. Biomaterials Science 2014;2:784 - 97.
19. Lee KL, Uhde-Holzem K, Fischer R, Commandeur U, Steinmetz NF. Genetic engineering and chemical conjugation of potato virus X. Methods Mol Biol 2014;1108:3-21.
20. Shukla S, Ablack AL, Wen AM, Lee KL, Lewis JD, Steinmetz NF. Increased tumor homing and tissue penetration of the filamentous plant viral nanoparticle Potato virus X. Molecular pharmaceutics 2013;10:33-42.
21. Steinmetz NF, Cho CF, Ablack A, Lewis JD, Manchester M. Cowpea mosaic virus nanoparticles target surface vimentin on cancer cells. Nanomedicine (Lond) 2011;6:351-64.
22. Steinmetz NF, Ablack A, Hickey JL, Ablack J, Manocha B, Mymryk JS, et al. Intravital Imaging of Human Prostate Cancer using Viral Nanoparticles Targeted to Gastrin-Releasing Peptide Receptors. Small 2011;in press.
23. Huang RK, Steinmetz NF, Fu CY, Manchester M, Johnson JE. Transferrin- mediated targeting of bacteriophage HK97 nanoparticles into tumor cells. Nanomedicine (Lond) 2011;6:55-68.
24. Hovlid ML, Steinmetz NF, Laufer B, Lau JL, Kuzelka J, Wang Q, et al. Guiding plant virus particles to integrin-displaying cells. Nanoscale 2012;4:3698-705.
25. Pokorski JK, Hovlid ML, Finn MG. Cell targeting with hybrid Qbeta virus-like particles displaying epidermal growth factor. Chembiochem : a European journal of chemical biology 2011;12:2441-7.
26. Shukla S, Wen AM, Ayat NR, Commandeur U, Gopalkrishnan R, Broome AM, et al. Biodistribution and clearance of a filamentous plant virus in healthy and tumor-bearing mice. Nanomedicine (Lond) 2014;9:221-35.
27. Harris JM, Chess RB. Effect of pegylation on pharmaceuticals. Nat Rev Drug Discov 2003;2:214-21.
141 CHAPTER 4: STEALTH FILAMENTS
28. Jokerst JV, Lobovkina T, Zare RN, Gambhir SS. Nanoparticle PEGylation for imaging and therapy. Nanomedicine (Lond) 2011;6:715-28.
29. Roberts MJ, Bentley MD, Harris JM. Chemistry for peptide and protein PEGylation. Adv Drug Deliv Rev 2002;54:459-76.
30. Veronese FM, Pasut G. PEGylation, successful approach to drug delivery. Drug discovery today 2005;10:1451-8.
31. Wattendorf U, Merkle HP. PEGylation as a tool for the biomedical engineering of surface modified microparticles. J Pharm Sci 2008.
32. Stoll S, Delon J, Brotz TM, Germain RN. Dynamic imaging of T cell-dendritic cell interactions in lymph nodes. Science 2002;296:1873-6.
33. Steinmetz NF, Mertens ME, Taurog RE, Johnson JE, Commandeur U, Fischer R, et al. Potato virus X as a novel platform for potential biomedical applications. Nano letters 2010;10:305-12.
34. de Gennes PG. Polymers at an interface: a simplified view. Adv Colloid Interface Sci 1987;27:189-209.
35. Shukla S, Ablack AL, Wen AM, Lee KL, Lewis JD, Steinmetz NF. Increased Tumor Homing and Tissue Penetration of the Filamentous Plant Viral Nanoparticle Potato virus X. Mol Pharm 2013;10:33-42.
36. Liu R, Vaishnav RA, Roberts AM, Friedland RP. Humans have antibodies against a plant virus: evidence from tobacco mosaic virus. PloS one 2013;8:e60621.
37. Elliott JA, Winn WC, Jr. Treatment of alveolar macrophages with cytochalasin D inhibits uptake and subsequent growth of Legionella pneumophila. Infection and immunity 1986;51:31-6.
38. Lunov O, Syrovets T, Loos C, Nienhaus GU, Mailander V, Landfester K, et al. Amino-functionalized polystyrene nanoparticles activate the NLRP3 inflammasome in human macrophages. ACS nano 2011;5:9648-57.
39. Vaine CA, Patel MK, Zhu J, Lee E, Finberg RW, Hayward RC, et al. Tuning innate immune activation by surface texturing of polymer microparticles: the role of shape in inflammasome activation. Journal of immunology 2013;190:3525-32.
40. Yazdi AS, Guarda G, Riteau N, Drexler SK, Tardivel A, Couillin I, et al. Nanoparticles activate the NLR pyrin domain containing 3 (Nlrp3) inflammasome and cause pulmonary inflammation through release of IL-1alpha and IL-1beta. Proceedings of the National Academy of Sciences of the United States of America 2010;107:19449-54.
142 CHAPTER 4: STEALTH FILAMENTS
41. Jokerst JV, Lobovkina T, Zare RN, Gambhir SS. Nanoparticle PEGylation for imaging and therapy. Nanomedicine (Lond) 2011;6:715-28.
42. Perry JL, Reuter KG, Kai MP, Herlihy KP, Jones SW, Luft JC, et al. PEGylated PRINT Nanoparticles: The Impact of PEG Density on Protein Binding, Macrophage Association, Biodistribution, and Pharmacokinetics. Nano letters 2012;12:5304-10.
43. Wolfram J, Yang Y, Shen J, Moten A, Chen C, Shen H, et al. The nano-plasma interface: Implications of the protein corona. Colloids and surfaces B, Biointerfaces 2014.
44. Lundqvist M. Nanoparticles: Tracking protein corona over time. Nat Nanotechnol 2013;8:701-2.
45. Tenzer S, Docter D, Kuharev J, Musyanovych A, Fetz V, Hecht R, et al. Rapid formation of plasma protein corona critically affects nanoparticle pathophysiology. Nat Nanotechnol 2013;8:772-81.
46. Dell'Orco D, Lundqvist M, Cedervall T, Linse S. Delivery success rate of engineered nanoparticles in the presence of the protein corona: a systems-level screening. Nanomedicine (Lond) 2012;8:1271-81.
47. Gunawan C, Lim M, Marquis CP, Amal R. Nanoparticle-protein corona complexes govern the biological fates and functions of nanoparticles. Journal of Materials Chemistry B 2014;2:2060-83.
48. Owens DE, 3rd, Peppas NA. Opsonization, biodistribution, and pharmacokinetics of polymeric nanoparticles. International journal of pharmaceutics 2006;307:93- 102.
49. Michel R, Pasche S, Textor M, Castner DG. Influence of PEG architecture on protein adsorption and conformation. Langmuir : the ACS journal of surfaces and colloids 2005;21:12327-32.
50. Agrawal A, Manchester M. Differential uptake of chemically modified cowpea mosaic virus nanoparticles in macrophage subpopulations present in inflammatory and tumor microenvironments. Biomacromolecules 2012;13:3320-6.
51. Mok H, Palmer DJ, Ng P, Barry MA. Evaluation of polyethylene glycol modification of first-generation and helper-dependent adenoviral vectors to reduce innate immune responses. Mol Ther 2005;11:66-79.
52. Nakano K, Bando Y, Tozuka Y, Takeuchi H. Cellular interaction of PEGylated PLGA nanospheres with macrophage J774 cells using flow cytometry. Asian Journal of Pharmaceutical Sciences 2007;2:220-6.
143 CHAPTER 4: STEALTH FILAMENTS
53. Ni F, Jiang L, Yang R, Chen Z, Qi X, Wang J. Effects of PEG length and iron oxide nanoparticles size on reduced protein adsorption and non-specific uptake by macrophage cells. J Nanosci Nanotechnol 2012;12:2094-100.
54. Sheng Y, Yuan Y, Liu CS, Tao XY, Shan XQ, Xu F. In vitro macrophage uptake and in vivo biodistribution of PLA-PEG nanoparticles loaded with hemoglobin as blood substitutes: effect of PEG content. Journal of Materials Science-Materials in Medicine 2009;20:1881-91.
55. Xie J, Xu C, Kohler N, Hou Y, Sun S. Controlled PEGylation of monodisperse Fe3O4 nanoparticles for reduced non-specific uptake by macrophage cells. Advanced Materials 2007;19:3163-+.
56. Quan Q, Xie J, Gao H, Yang M, Zhang F, Liu G, et al. HSA coated iron oxide nanoparticles as drug delivery vehicles for cancer therapy. Molecular pharmaceutics 2011;8:1669-76.
57. Kostarelos K. Carbon nanotubes: Fibrillar pharmacology. Nature materials 2010;9:793-5.
58. Ruggiero A, Villa CH, Bander E, Rey DA, Bergkvist M, Batt CA, et al. Paradoxical glomerular filtration of carbon nanotubes. Proceedings of the National Academy of Sciences of the United States of America 2010;107:12369- 74.
59. Singh R, Pantarotto D, Lacerda L, Pastorin G, Klumpp C, Prato M, et al. Tissue biodistribution and blood clearance rates of intravenously administered carbon nanotube radiotracers. Proceedings of the National Academy of Sciences of the United States of America 2006;103:3357-62.
60. Bruckman MA, Randolph LN, Vanmeter A, Hern S, Shoffstall AJ, Taurog RE, et al. Biodistribution, pharmacokinetics, and blood compatibility of native and PEGylated tobacco mosaic virus nano-rods and -spheres in mice. Virology 2014;449:163-73.
61. Almeida JP, Lin AY, Langsner RJ, Eckels P, Foster AE, Drezek RA. In vivo immune cell distribution of gold nanoparticles in naive and tumor bearing mice. Small 2014;10:812-9.
62. Farkas ME, Aanei IL, Behrens CR, Tong GJ, Murphy ST, O'Neil JP, et al. PET Imaging and biodistribution of chemically modified bacteriophage MS2. Molecular pharmaceutics 2013;10:69-76.
63. Molenaar TJ, Michon I, de Haas SA, van Berkel TJ, Kuiper J, Biessen EA. Uptake and processing of modified bacteriophage M13 in mice: implications for phage display. Virology 2002;293:182-91.
144 CHAPTER 4: STEALTH FILAMENTS
64. Prasuhn DE, Jr., Singh P, Strable E, Brown S, Manchester M, Finn MG. Plasma clearance of bacteriophage Qbeta particles as a function of surface charge. Journal of the American Chemical Society 2008;130:1328-34.
65. Singh P, Prasuhn D, Yeh RM, Destito G, Rae CS, Osborn K, et al. Bio- distribution, toxicity and pathology of cowpea mosaic virus nanoparticles in vivo. Journal of controlled release : official journal of the Controlled Release Society 2007;120:41-50.
66. Kaiser CR, Flenniken ML, Gillitzer E, Harmsen AL, Harmsen AG, Jutila MA, et al. Biodistribution studies of protein cage nanoparticles demonstrate broad tissue distribution and rapid clearance in vivo. International journal of nanomedicine 2007;2:715-33.
67. Arnida, Janat-Amsbury MM, Ray A, Peterson CM, Ghandehari H. Geometry and surface characteristics of gold nanoparticles influence their biodistribution and uptake by macrophages. Eur J Pharm Biopharm 2011;77:417-23.
68. Zhang XD, Wu D, Shen X, Liu PX, Yang N, Zhao B, et al. Size-dependent in vivo toxicity of PEG-coated gold nanoparticles. International journal of nanomedicine 2011;6:2071-81.
69. Mohammad AK, Reineke JJ. Quantitative Detection of PLGA Nanoparticle Degradation in Tissues following Intravenous Administration. Mol Pharm 2013;10:2183-9.
70. Park EJ, Umh HN, Kim SW, Cho MH, Kim JH, Kim Y. ERK pathway is activated in bare-FeNPs-induced autophagy. Archives of Toxicology 2014;88:323-36.
71. Bottini M, Rosato N, Bottini N. PEG-Modified Carbon Nanotubes in Biomedicine: Current Status and Challenges Ahead. Biomacromolecules 2011;12:3381-93.
72. Hamilton RF, Jr., Wu Z, Mitra S, Shaw PK, Holian A. Effect of MWCNT size, carboxylation, and purification on in vitro and in vivo toxicity, inflammation and lung pathology. Part Fibre Toxicol 2013;10:57.
73. Kraft JC, Freeling JP, Wang Z, Ho RJ. Emerging research and clinical development trends of liposome and lipid nanoparticle drug delivery systems. J Pharm Sci 2014;103:29-52.
74. Lin S, Wang X, Ji Z, Chang CH, Dong Y, Meng H, et al. Aspect Ratio Plays a Role in the Hazard Potential of CeO Nanoparticles in Mouse Lung and Zebrafish Gastrointestinal Tract. ACS nano 2014.
145 CHAPTER 4: STEALTH FILAMENTS
75. Baiu DC, Brazel CS, Bao Y, Otto M. Interactions of iron oxide nanoparticles with the immune system: challenges and opportunities for their use in nano-oncology. Curr Pharm Des 2013;19:6606-21.
76. Meng J, Yang M, Jia F, Xu Z, Kong H, Xu H. Immune responses of BALB/c mice to subcutaneously injected multi-walled carbon nanotubes. Nanotoxicology 2011;5:583-91.
77. Raja KS, Wang Q, Gonzalez MJ, Manchester M, Johnson JE, Finn MG. Hybrid virus-polymer materials. 1. Synthesis and properties of PEG-decorated cowpea mosaic virus. Biomacromolecules 2003;4:472-6.
78. Semple SC, Harasym TO, Clow KA, Ansell SM, Klimuk SK, Hope MJ. Immunogenicity and rapid blood clearance of liposomes containing polyethylene glycol-lipid conjugates and nucleic Acid. J Pharmacol Exp Ther 2005;312:1020-6.
79. Shimizu T, Ishida T, Kiwada H. Transport of PEGylated liposomes from the splenic marginal zone to the follicle in the induction phase of the accelerated blood clearance phenomenon. Immunobiology 2013;218:725-32.
80. Croyle MA, Chirmule N, Zhang Y, Wilson JM. "Stealth" adenoviruses blunt cell- mediated and humoral immune responses against the virus and allow for significant gene expression upon readministration in the lung. Journal of Virology 2001;75:4792-801.
81. Lee GK, Maheshri N, Kaspar B, Schaffer DV. PEG conjugation moderately protects adeno-associated viral vectors against antibody neutralization. Biotechnology and Bioengineering 2005;92:24-34.
82. O'riordan CR, Lachapelle A, Delgado C, Parkes V, Wadsworth SC, Smith AE, et al. PEGylation of adenovirus with retention of infectivity and protection from neutralizing antibody in vitro and in vivo. Hum Gene Ther 1999;10:1349-58.
83. Wortmann A, Vohringer S, Engler T, Corjon S, Schirmbeck R, Reimann J, et al. Fully detargeted polyethylene glycol-coated adenovirus vectors are potent genetic vaccines and escape from pre-existing anti-adenovirus antibodies. Molecular therapy : the journal of the American Society of Gene Therapy 2008;16:154-62.
84. De Geest B, Snoeys J, Van Linthout S, Lievens J, Collen D. Elimination of innate immune responses and liver inflammation by PEGylation of adenoviral vectors and methylprednisolone. Hum Gene Ther 2005;16:1439-51.
85. Brown DM, Johnston H, Gubbins E, Stone V. Serum enhanced cytokine responses of macrophages to silica and iron oxide particles and nanomaterials: a comparison of serum to lung lining fluid and albumin dispersions. J Appl Toxicol 2014.
146 CHAPTER 4: STEALTH FILAMENTS
86. Khan HA, Abdelhalim MA, Alhomida AS, Al Ayed MS. Transient increase in IL- 1beta, IL-6 and TNF-alpha gene expression in rat liver exposed to gold nanoparticles. Genet Mol Res 2013;12:5851-7.
87. Meunier E, Coste A, Olagnier D, Authier H, Lefevre L, Dardenne C, et al. Double-walled carbon nanotubes trigger IL-1beta release in human monocytes through Nlrp3 inflammasome activation. Nanomedicine (Lond) 2012;8:987-95.
88. Maruyama K, Yuda T, Okamoto A, Kojima S, Suginaka A, Iwatsuru M. Prolonged circulation time in vivo of large unilamellar liposomes composed of distearoyl phosphatidylcholine and cholesterol containing amphipathic poly(ethylene glycol). Biochim Biophys Acta 1992;1128:44-9.
89. Yang Q, Jones SW, Parker CL, Zamboni WC, Bear JE, Lai SK. Evading Immune Cell Uptake and Clearance Requires PEG Grafting at Densities Substantially Exceeding the Minimum for Brush Conformation. Molecular pharmaceutics 2014.
90. Fang J, Nakamura H, Maeda H. The EPR effect: Unique features of tumor blood vessels for drug delivery, factors involved, and limitations and augmentation of the effect. Adv Drug Deliv Rev 2011;63:136-51.
91. Murahari MS, Yergeri MC. Identification and usage of fluorescent probes as nanoparticle contrast agents in detecting cancer. Curr Pharm Des 2013;19:4622- 40.
92. Sosnovik DE, Nahrendorf M, Weissleder R. Magnetic nanoparticles for MR imaging: agents, techniques and cardiovascular applications. Basic Res Cardiol 2008;103:122-30.
93. Pokorski JK, Breitenkamp K, Liepold LO, Qazi S, Finn MG. Functional virus- based polymer-protein nanoparticles by atom transfer radical polymerization. Journal of the American Chemical Society 2011;133:9242-5.
147 CHAPTER 5: TARGETED FILAMENTS
Chapter 5: PVX-GE11 for cell-specific targeting
The material in this chapter is adapted with permission from: Chariou, P.L.*, Lee,
K.L.*, Wen, A.M., Gulati, N.M., Stewart, P.L., Steinmetz, N.F. Detection and imaging of aggressive cancer cells using and epidermal growth factor receptor (EGFR)-targeted filamentous plant virus-based nanoparticle. Bioconjug. Chem. 2015, 26: 262-269.
Copyright 2015 American Chemical Society.
*These authors contributed equally
As co-first author of this paper, I prepared A647-PVX(-GE11) particles, determined serum stability of particles, performed co-culture uptake studies, and contributed to paper edits. Paul Chariou was my undergraduate trainee, and co-first author, who performed flow cytometry assays staining for EGFR and assaying for PVX uptake.
5.1 Introduction
According to the National Cancer Institute, 13.7 million Americans are currently diagnosed with cancer and 600 000 of them are expected to die this year. Only 68% of patients diagnosed are expected to survive more than 5 years due to poor prognosis and the lack of treatment options. Molecular imaging approaches and targeted drug delivery hold promise for earlier detection of disease and treatment with higher efficacy while reducing side effects, therefore increasing survival rates and quality of life. Of particular interest are nanoscale platform technologies that can be functionalized with multiple functional entities, such as toxic payloads (e.g., chemotherapies) and contrast agents
148 CHAPTER 5: TARGETED FILAMENTS
(for MRI, PET, etc.), while displaying receptor-specific targeting ligands. Advantages arise from theranostic approaches, where a contrast agent-loaded nanoparticle is used to image the disease site, to test for expression profiles and whether the patient qualifies for a particular treatment approach. If the patient tests positive, treatment can begin with nanoparticles loaded with toxic payloads, therefore providing a route toward personalized nanoparticle interventions [1, 2].
Nanomedicine has led to the development of nanocarriers with prolonged systemic circulation that protect the payload and lead to enhanced accumulation in solid tumors based on the enhanced permeability and retention (EPR) effect [3, 4]. Doxil (a liposomal formulation of doxorubicin) and Abraxane (an albumin nanoparticle formulation carrying paclitaxel) increase efficacy of their payloads based on the pathophysiological properties of the target tissue. While passive drug targeting enables tissue accumulation of the carrier and its cargo, cell targeting, entry, and killing may not be achieved. Inefficient cell targeting may promote the development of drug resistance
[5-7], which can lead to recurrence of cancer in a more aggressive form. To overcome this barrier, receptor-targeted nanoparticle formulations are being developed [8, 9].
The tyrosine kinase epidermal growth factor receptor (EGFR) is overexpressed in variety of human malignancies and is considered an important molecular cancer biomarker [10]. EGFR is a 170 kDa transmembrane glycoprotein member of the ErbB family. Upon activation by endogenous ligands of the EGF family, EGFR is internalized mostly via the clathrin-mediated pathway, triggering cell proliferation, cell division, inhibition of apoptosis, and angiogenesis, implicating EGFR in cancer proliferation and growth [11-14]. Several EGFR-targeting strategies are currently under
149 CHAPTER 5: TARGETED FILAMENTS investigation. Both EGF protein and EGFR antibodies have been used to probe EGFR in tumors; however, limitations to these targeting strategies have been identified, leading to varying degrees of success. Full length EGF has a high affinity for EGFR (Kd
= 2 nM), but induces cell proliferation [15, 16], an undesirable effect when targeting cancers. Antibody therapy (e.g., Cetuximab) is promising because it blocks activation of the receptor-associated kinases, inhibits cell growth, and induces apoptosis. It has been approved by the FDA for the treatment of head and neck cancer and is under investigation for the treatment of colorectal and breast cancer [17-20]. Nevertheless, recent research has shown that cancer cell resistance to Cetuximab can be mediated via signaling through the HER2/neu protein [21]. The use of peptide ligands conjugated to functionalized nanoparticles carrying contrast agents, toxic payloads, or combinations thereof is particularly promising [22-25]. The GE11 peptide (YHWYGYTPQNVI), discovered through a phage display peptide library screen, binds EGFR with high selectivity and affinity (Kd = 22 nM) and does not activate EGFR-mediated signaling, making it a promising candidate for EGFR-targeted therapies [26].
In this work, we describe the development of an EGFR-targeted filamentous platform technology using potato virus X (PVX) as a template. PVX is a proteinaceous nanoparticle measuring 515x13 nm; it is composed of 1270 identical coat proteins, each of which can be modified at a solvent-exposed lysine residue using N- hydroxysuccinimide activated probes [27]. We recently demonstrated that the high aspect ratio and elongated shape of PVX increased passive tumor homing in a variety of models, including human tumor xenografts of breast cancer, fibrosarcoma, squamous sarcoma, brain cancer, and colon cancer. Furthermore, tissue penetration of filamentous
150 CHAPTER 5: TARGETED FILAMENTS platforms is enhanced compared to spherical counterparts [27]. These properties make
PVX a promising platform for nanomedical research and technology development. In addition to its shape, PVX, as well as other plant virus-based nanomaterials, offer further benefits compared to synthetic counterparts. Virus-based nanoparticles (VNPs) are genetically encoded, programmed to self-assemble into monodisperse and highly symmetrical biomaterials. Because these materials are genetically encoded, high aspect ratio materials can be obtained with spatial control at the atomic level and with impeccable reproducibility, a level of control not yet achievable with synthetic chemistry. VNPs are biocompatible and biodegradable, and do not cause acute toxicity in vitro or in vivo [28]. Plant-based VNPs can be produced in plants in high yields at a relatively low cost through molecular farming in plants, thus providing a realistic platform for translation and commercialization.
We describe the synthesis and application of GE11-modified PVX filaments for molecularly targeted detection and imaging of EGFR+ cancer cells. Target-specificity of
PVX-GE11 was evaluated using a panel of EGFR-positive human skin epidermoid carcinoma (A-431), colorectal adenocarcinoma (HT-29), and triple negative breast cancer (MDA-MB-231) cell lines, as well as the EGFR-negative ductal breast carcinoma (BT-474) cell line. Furthermore, target-specificity was evaluated in co- culture experiments with cancer cells and macrophages.
151 CHAPTER 5: TARGETED FILAMENTS
5.2 Materials and methods
5.2.1 PVX propagation and purification
Nicotiana benthamiana plants were seeded and grown for 4-6 weeks in humidity controlled growth chambers (15 h day cycle: 25 °C and 65% rel. humidity; night cycle:
23 °C and 70% rel. humidity). Infection was induced through mechanical inoculation using 150 ng μL− 1 purified PVX in 0.1 M potassium phosphate (KP) buffer pH 7.0; leaves were dusted with carborundum (Alfa Aesar). Plants were propagated for 21-
25 days post inoculation and purified using established protocols [27]. Purified PVX particles were stored in KP buffer; concentration and purity was determined using
UV/visible spectroscopy, and PVX particle integrity was determined by transmission electron microscopy (see below).
5.2.2 PVX bioconjugation
GE11 peptide was obtained from the Cleveland Clinic Molecular
Biotechnology Core Facility. GE11 peptide was synthesized with an amino-terminal cysteine residue with intervening GG spacer (CGGYHWYGYTPQNVI).
Fluorescently-labeled, EGFR-targeted PVX were synthesized using a two-step reaction: first, succinimidyl-[(N-maleimidopropionamido)-tetraethyleneglycol] ester
(SM(PEG)4, Pierce) and Alexa Fluor 647 succinimidyl ester (A647-NHS, Life
Technologies) in DMSO were added at a 2-fold molar excess per PVX coat protein to give a final of 10% (v/v) DMSO in KP buffer. After 2 h incubation at room temperature, excess linker and fluorescent molecules were removed using either 10 kDa molecular weight cutoff centrifugal filters (Millipore) or ultracentrifugation (42,000 rpm using 152 CHAPTER 5: TARGETED FILAMENTS
50.2 Ti fixed angle rotor for 3 h over a 40% (w/v) sucrose cushion), prior to immediately proceeding to the second step. GE11 targeting ligand was added using a
20-fold molar excess per PVX coat protein; the reaction was carried out overnight in
10% (v/v) DMSO in KP buffer. Controls were also obtained by omitting the addition of the targeting ligand. Samples were purified by ultracentrifugation over a 40% (w/v) sucrose cushion (42,000 rpm using 50.2 Ti fixed angle rotor for 3 h). Pellets were resuspended in KP buffer and stored in the dark at 4 °C until further processing.
5.2.3 UV/visible spectroscopy
A NanoDrop Spectrophotometer (Thermo Scientific) was used to measure the
UV/visible spectra of native and modified PVX nanoparticles. The amount of A647 fluorophore per PVX was determined based on the ratio of dye:PVX concentration, making use of the Beer-Lambert law and dye- and PVX-specific extinction coefficients:
PVX: ε(260 nm) = 2.97 mL mg−1 cm−1, molecular weight of PVX = 35×106 g mol− 1;
A647: ε(651 nm) = 270,000 M−1 cm−1, molecular weight of A647 = 1300 g mol−1.
5.2.4 Denaturing gel electrophoresis
15 µg of PVX control, A647-PVX-PEG, and A647-PVX-PEG-GE11 were denatured (100°C for 5 min) in 1x LDS loading dye (Life Technologies) to obtain a final volume of 20 μL. PVX proteins, as well as SeeBlue Plus2 ladder (Life
Technologies), were separated for 40 min at 200 V and 120 mA using a 4-12% NuPage precast gel in 1x MOPS buffer (Life Technologies). Gels were photographed before and
153 CHAPTER 5: TARGETED FILAMENTS after staining with Coomassie Blue (0.25% w/v) using the AlphaImager (Biosciences) imaging system under white light.
5.2.5 Transmission electron microscopy
Drops of PVX particles (20 μL, 0.1 mg mL− 1) were added to Formvar carbon film coated copper TEM grids (Electron Microscopy Sciences) for 5 min at room temperature. The grids were washed with deionized water and stained with 2% (w/v) uranyl acetate in deionized water for another 5 min. A Zeiss Libra 200FE transmission electron microscope was used to inspect samples at 200 kV, or a FEI Tecnai G2 Spirit transmission electron microscope at 120 kV.
5.2.6 Particles stability in serum
PVX particles were incubated in FBS at a concentration of 50 μg/mL for 1 h at
37 °C. PVX particles were then purified over a 30% (w/v) sucrose gradient using ultracentrifugation (42,000 rpm using 50.2 Ti fixed angle rotor for 3 h), prior to TEM imaging.
5.2.7 Tissue culture
Cell maintenance:
All cell lines were obtained from ATCC. All culture media reagents were purchased from Invitrogen, unless indicated otherwise. MDA-MB-231 cells (triple
154 CHAPTER 5: TARGETED FILAMENTS negative breast cancer) were cultured in RPMI-1640 medium with L-glutamine
(Fisher). HT-29 cells (colon cancer) were maintained in McCoy’s 5A medium with L- glutamine. A431 cells (epidermal carcinoma) and RAW264.7 cells (murine macrophage) were cultured in high glucose Dulbecco's modified Eagle medium
(DMEM) with L-glutamine. BT-474 cells (breast cancer) were maintained in Hybri-
Care Medium (ATCC). For all cell lines, media were supplemented with 10% (v/v) FBS and 1% (v/v) penicillin–streptomycin. BT-474 cells were additionally supplemented
-1 with 1.5 g L sodium bicarbonate. Cells were grown at 37 °C and 5% CO2.
Flow cytometry:
EGFR staining: MDA-MB-231, HT-29, A431, and BT-474 cells were grown to confluency and collected using an enzyme-free Hank’s based cell dissociation buffer
(Invitrogen). Cells (500,000 cells/200 μL media/well) were added to untreated 96-well v-bottom plates (VWR). EGFR was stained using an anti-EGFR FITC-labeled antibody
(Millipore, 1.25 dilution as per manufacturer’s recommendation) and cells were incubated at 37 °C, 5% CO2 for 60 min. After incubation, cells were spun down at 500 g for 4 min, supernatant was removed, and cells were washed twice in FACS buffer (0.1 mL 0.5M EDTA, 0.5 mL FBS, and 1.25 mL 1M HEPES pH 7.0 in Ca2+ and Mg2+ free
PBS (50 mL total volume)). Cells were fixed in 2% (v/v) paraformaldehyde in FACS buffer at room temperature for 10 min and washed twice and resuspended using FACS buffer. All samples were prepared in triplicates. The BD LSR II flow cytometer was used to analyze samples and 10,000 gated events were recorded. Data were analyzed using FlowJo 8.6.3 software (http://www.flowjo.com/).
155 CHAPTER 5: TARGETED FILAMENTS
EGFR-targeting using PVX-based filaments: MDA-MB-231, HT-29, A431, and
BT-474 cells were grown to confluency and collected using enzyme-free Hank’s-based cell dissociation buffer. Cells (500,000 cells/200 μL media/well) were added to untreated 96-well v-bottom plates. Triplicates of no particles, A647-PVX-PEG, and
A647-PVX-PEG-GE11 were added at a concentration of 100,000 particles/cell and incubated for 2 h at 37 °C, 5% CO2. Following incubation, cells were spun down at 500 g for 4 min and supernatant was removed. Cells were then washed twice in FACS buffer and fixed in 2% (v/v) paraformaldehyde in FACS buffer at room temperature for
10 min. Cells were washed twice after fixing and resuspended in FACS buffer. FACS and data analysis were carried out as described above.
Cell co-culture: HT-29 and RAW264.7 cells were grown to confluency and collected using an enzyme-free Hank’s based cell dissociation buffer. Cell mixtures containing 20:80 ratio of RAW264.7:HT-29 cell were evaluated. Cells were prepared as described above and PVX-based formulations were added at 100,000 particles/cell as described above and incubated for 2 h at 37 °C, 5% CO2. Following incubation, cells were spun down at 500 g for 4 min and supernatant was removed. Cells were then washed twice in FACS buffer. RAW264.7 cells were differentiated from HT-29 cells through staining of CD11b surface markers. Cells were blocked with rat anti-mouse
CD16/CD32 (Fc Block) (BD Biosciences) as follows: a working solution Fc Block was made using 1 μL Fc Block/200 μL FACS buffer. 100 μL of the working solution was added to each well and incubated for 30 min on ice. Following incubation, cells were washed then stained using 1 μL anti-mouse CD11b (BD Biosciences) in 100 μL FACS
156 CHAPTER 5: TARGETED FILAMENTS buffer per well and incubated for 30 min on ice. Cells were washed once and fixed as described previously. FACS and data analysis were carried out as described above.
5.3 Results and discussion
5.3.1 Bioconjugation of targeting and imaging moieties
PVX was produced through farming in N. benthamiana plants using previously established protocols [29] and extracted at yields of 20 mg of pure PVX from 100 grams of infected leaf material. GE11 peptide was synthesized with an amino-terminal cysteine residue with intervening GG spacer for bioconjugation (CGGYHWYGYTPQNVI).
Fluorescently-labeled, EGFR-targeted PVX filaments were obtained using a two-step bioconjugation reaction (Scheme 5.1). Briefly, a bifunctional PEG linker with a 24.6 Å spacer arm (SM(PEG)4) and Alexa Fluor 647 succinimidyl ester (NHS-A647) were conjugated to solvent-exposed lysines on PVX followed by addition of the cysteine- terminated GE11 peptide targeting the maleimide side groups of SM(PEG)4. Nontargeted particles were also synthesized by omitting the second step in order to assess nonspecific cell binding. The PVX-based formulations were subsequently characterized by UV/visible spectroscopy, denaturing gel electrophoresis (SDS-PAGE), and transmission electron microscopy (TEM) to quantify the degree of modification and confirm structural integrity (Figure 5.1).
157 CHAPTER 5: TARGETED FILAMENTS
Scheme 5.1. Bioconjugation of targeted PVX filaments. Bioconjugation of PVX with NHS-A647 and
SM(PEG)4 linkers, and subsequent reaction with GE11 peptide yielding a fluorescent, EGFR-targeted nanofilament.
UV/visible spectroscopy and SDS-PAGE were used to quantify the degree of labeling with A647 fluorophore and GE11 peptide. Based on the Beer-Lambert law and fluorophore- and PVX-specific extinction coefficients, it was determined that approximately 300 dyes per PVX were displayed (Figure 5.1). Because dyes were conjugated in the first reaction step prior to separation of the samples for further modification, the targeted and nontargeted control samples (A647-PVX-GE11 and A647-
PVX) displayed the same number of fluorophores. (It should be noted that the GE11 peptide contributes to the absorbance at 280 nm (Figure 5.1A), and therefore A647 loading was quantified prior to conjugation of GE11).
SDS-PAGE further confirmed covalent attachment of the A647 fluorophores and
GE11 peptides, as indicated by higher molecular weight bands (Figure 5.1B). The PVX coat protein (CP) has a molecular weight of 25 kDa; higher molecular weights indicate 158 CHAPTER 5: TARGETED FILAMENTS the addition of A647 (~1.3 kDa) and GE11 (~1.7 kDa). Band analysis and ImageJ were used to quantify the degree of labeling, and data indicate a ratio of CP:A647-CP:A647-
CP-GE11 of 1:2:2, indicating that 40% (or 500) of the coat proteins are modified with
A647 and 40% of the proteins display GE11 targeting. PVX consists of 1270 identical copies of a CP, each with a single reactive lysine side chain [30]; dual labeling is therefore not expected. There is some discrepancy between the SDS-PAGE analysis and
UV/visible spectroscopy quantification of the fluorophore: SDS gels indicate 500 dyes per PVX while UV/visible data indicate 300 dyes per PVX – the difference is likely explained by the A647 contributing to the staining (the bands are visible under white light without Coomassie Blue staining, not shown), therefore enhancing the band intensity. Gel electrophoresis indicates the presence of dimers and oligomers; these multimeric coat proteins are typically observed in SDS-PAGE after PVX conjugation with peptides using the SM(PEG)4 linker [31]. It may indicate that a small number of coat proteins are interlinked through the bifunctional SM(PEG)4 linker (targeting lysine and potentially cysteine residues). Nonetheless, it is important to note that interparticle cross-linking was not observed in TEM images (Figure 5.1C+D), so any potential cross-linking is likely between coat proteins of the same particle. TEM imaging confirmed that the particles remained structurally intact after two rounds of chemical modification (Figure 5.1C+D).
In addition, we confirmed PVX stability in serum (Figure 5.1E).
159 CHAPTER 5: TARGETED FILAMENTS
Figure 5.1. Characterization of A647-PVX-GE11 particles. A) UV/visible spectra of native and modified PVX nanoparticles and GE11 peptide. B) Particles after electrophoretic separation on 4-12% SDS-PAGE gel run in MOPS buffer visualized with white light after Coomassie staining. M = SeeBlue Plus2 molecular weight standard; the molecular weight in kDa is indicated on the left. 1 = PVX, 2 = A647- PVX-GE11; inset shows lane analysis performed using ImageJ software. TEM images of negatively stained (C) A647-PVX and (D) A647-PVX-GE11. (E) TEM image of negatively stained PVX after 1 h incubation in serum. 160 CHAPTER 5: TARGETED FILAMENTS
5.3.2 Cell binding experiments
To test the affinity of fluorescently-labeled, EGFR-targeted PVX particles for cells expressing EGFR, we conducted a series of flow cytometry experiments using a panel of cancer cell lines that upregulate EGFR to various degrees (Figure 5.2): human skin epidermoid carcinoma, colorectal adenocarcinoma, and triple negative breast cancer cell lines (A-431, HT-29, MDA-MB-231) [32-36]. The EGFR- ductal breast carcinoma
(BT-474) cell line was used to assess nonspecific uptake [34].
An anti-EGFR FITC-labeled antibody and flow cytometry were used to confirm the level of EGFR expression on the cell lines tested (Figure 5.2A+C). In agreement with the literature [32-36], MDA-MB-231, HT-29, and A-431 cells tested positive for
EGFR (with HT-29 and A-431 cells expressing higher levels of EGFR compared to
MDA-MB-231 cells) and BT-474 cells were EGFR-negative.
Next, the cell target-specificity of the A647-PVX-GE11 formulation was
+ evaluated using flow cytometry (Figure 5.2B+C). In EGFR high cells, HT-29 and A-431, targeting of the EGFR-specific A647-PVX-GE11 probe was significantly increased compared to nonspecific PVX–cell interactions. Nonspecific cell interactions have been previously reported for PVX [29], and are frequently observed for synthetic nanoparticle formulations as well [37]. Overall, cell targeting efficiency of the A647-PVX-GE11
+ probe followed a trend, i.e., A647-PVX-GE11 was highest in EGFR high cells (HT-29 and
+ A-431), while moderate and negligible uptake was observed in EGFR low cells (MDA-
MB-231) and EGFR- cells (BT-474).
161 CHAPTER 5: TARGETED FILAMENTS
Figure 5.2. Flow cytometry. Quantification of EGFR expression using (A) an EGFR-specific antibody and (B) quantification of PVX–cell interactions comparing an EGFR-targeted A647-PVX-GE11 and nontargeted A647-PVX formulation. All samples were measured in triplicates and analyzed using FlowJo software. Error bars indicate the standard deviation (* p < 0.05 and ** p < 0.01). (C) Representative dot plots showing the fluorescence overlay of unstained cells (black) with cells incubated with either FITC-labeled EGFR antibody (orange), A647-PVX (red), or A647-PVX-GE11 (green).
Nonspecific cell uptake of nontargeted PVX was significantly lower in the breast cancer cells lines versus the epidermoid carcinoma and colorectal adenocarcinoma cells; this may be explained by different metabolic rates of these cell types. There was no
162 CHAPTER 5: TARGETED FILAMENTS difference in cell uptake comparing the targeted A647-PVX-GE11 versus the nontargeted
A647-PVX formulation using the EGFR- BT-474 cell line, indicating that the targeting ligand confers specificity toward EGFR and does not increase nonspecific cell uptake.
+ Using EGFR low cells, MDA-MB-231, we found that while A647-PVX-GE11 showed increased cell uptake versus A647-PVX, statistical significance was not reached, which may reflect the relatively low EGFR expression level.
Next, we set out to determine cell-specificity in co-culture experiments with cancer cells and macrophages. Understanding the partitioning coefficient of a nanoparticle between the target cancer cell and nontarget macrophage populations is important, because mononuclear phagocytic clearance remains a challenge for successful translation of nanomedicines. Cell mixtures using 20:80 RAW264.7:HT-29 cells were incubated with either A647-PVX or A647-PVX-GE11 and cell interactions were quantified using flow cytometry (Figure 5.3). RAW264.7 cells were differentiated from
HT-29 cells through staining of CD11b surface markers (Figure 5.3A, gates Q1+2). As expected, nontargeted and targeted PVX formulations were taken up by macrophages, but uptake in macrophages is not enhanced upon conjugation of the GE11 targeting ligand
(Figure 5.3). 27% and 35% of macrophages scored positive for A647-PVX and A647-
PVX-GE11, respectively, with no statistically significant differences between the two populations, indicating that the addition of a targeting ligand does not increase nonspecific uptake into macrophages.
163 CHAPTER 5: TARGETED FILAMENTS
Figure 5.3. Evaluation of targeted filaments in a co-culture of macrophages and cancer cells. A) Representative dot plots of co-cultured RAW264.7 macrophages and HT-29 cancer cells at a 20:80 ratio. Macrophages were differentiated from HT-29 cells using CD11b staining (Oregon Green 488 channel); PVX was detected based on the A647 label (A647 channel). Q1 = RAW264.7 cells (CD11b positive) that are PVX-negative; Q2 = RAW264.7 cells (CD11b positive) positive for A647-PVX(-GE11); Q3 = HT-29 cells (CD11b negative) that are PVX-negative; Q4 = HT-29 cells (CD11b negative) positive for A647- PVX(-GE11). B) Percent cell uptake distribution of A647-PVX and A647-PVX-GE11 comparing HT-29– and RAW264.7–PVX interactions. All samples were measured in triplicates and analyzed using FlowJo software, with error bars indicating the standard deviation (* p<0.05 and ** p<0.0005).
For the nontargeted A647-PVX formulation, macrophage interactions are enhanced compared to nonspecific HT-29–PVX interactions (RAW264.7>HT-29 with p
<0.05), which may be explained by the different modes of uptake: macrophages interact with nanomaterials via phagocytosis and cancer cells via endocytosis [38]. This partitioning between macrophages and cancer cells was reversed when the targeted 164 CHAPTER 5: TARGETED FILAMENTS
A647-PVX-GE11 formulation was considered: here, partitioning favored cancer cells. A significantly larger proportion of HT-29 cells (78%) interacted with the A647-PVX-
GE11 particles compared to macrophages (HT-29>>RAW264.7 with p<0.0005); binding to macrophages remained low (35%) and at comparable levels as observed using the nontargeted A647-PVX formulation (27%).
5.4 Conclusion
Overall, the data support that A647-PVX-GE11 targets EGFR+ cells. We have previously shown that fluorescently-labeled PVX is taken up by different cell types [30].
These nonspecific cell interactions may be explained by PVX’s positive surface charge leading to electrostatic attractions with the negatively charged cell membrane; we also showed that this can be overcome through PEGylation [27]. While we did not employ
PEG coatings in this study, our data indicate that display of EGFR-targeting ligand GE11 enables targeting of EGFR+ cancer cells, while not increasing nonspecific cell interactions with EGFR- cancer cells or macrophages.
EGFR-targeted contrast agents and therapeutic formulations are under development; however, all are spherical and this may not be optimal. Mounting evidence suggests advantageous behavior of elongated, filamentous nanomaterials for nanomedical applications. Elongated filaments exhibit increased margination toward the vessel wall, therefore increasing EPR tumor homing and molecular targeting [39-41]. Molecular target recognition is further enhanced because elongated materials present ligands more effectively to the flat vessel wall or target cells compared to their spherical counterparts
[42, 43]. Finally, data indicate that elongated materials have increased immune evasion 165 CHAPTER 5: TARGETED FILAMENTS and reduced macrophage uptake, therefore further contributing to synergistic target enhancement [44]. Therefore, we turned toward the development of an EGFR-targeted filament using the PVX platform technology. This is the first example of a receptor- targeted PVX-based optical imaging agent. While we demonstrate optical detection of
EGFR+ cancer cells here, fluorescent dyes could be exchanged with contrast agents used in clinical imaging, including Gd(DOTA) for magnetic resonance imaging [45] or 64Cu for position emission spectroscopy [46]. Alternatively, toxic payloads such as chemotherapeutic, peptide, or protein drugs could be incorporated to aid therapeutic intervention [47].
Our data are in good agreement with reports using synthetic nanomaterials targeted toward EGFR. For example, poly(inosine/cytosine) GE11 polyplex formulations show promise in nonviral gene therapy targeting EGFR+ disease [48]. Similarly, GE11- targeted liposomes have been developed to target EGFR expressing cells [49, 50]. EGFR- targeted virus-based materials have also been developed and studied; examples include the 30 nm-sized icosahedrons formed by bacteriophage Qβ. Qβ was genetically engineered to express EGF protein in low copy number as a coat protein fusion; the Qβ-
EGF chimera was shown to target EGFR+ cells and promotes autophosphorylation of the
EGF receptor, leading to apoptosis of A-431 cells (a phenomenon specific to A-431 cells)
[51]. A different approach used an EGFR-retargeted measles virus (EGFR-MV) strain engineered with a single chain antibody specific for EGFR for targeted oncolytic viral therapy [52]. We expect presentation of an EGFR-specific targeting peptide on a filamentous particle will exhibit advantageous behavior in vivo.
166 CHAPTER 5: TARGETED FILAMENTS
In summary, this report details the synthesis of fluorescently-labeled, EGFR- targeted filamentous nanoparticles using the PVX platform technology and bioconjugate chemistry. The proteinaceous particles remained stable after two rounds of bioconjugation, and several hundred copies of fluorophores (~300/PVX) and EGFR- specific GE11 peptide ligands (~500/PVX) were incorporated. Serum stability was confirmed, and cell target specificity was demonstrated using a panel of EGFR+ and
EGFR- cancer cells and macrophages. We demonstrate target-specificity and preferred partitioning to cancer cells versus macrophages using the targeted A647-PVX-GE11 formulation. These results offer a critical step toward the development of a scalable nanoparticle platform technology for imaging and/or treatment of EGFR-expressing cancers. Nevertheless, the road toward translation of this technology must be paved by detailed in vivo studies evaluating both the efficacy as well as potential adverse effects.
5.5 Works cited in this chapter
1. Lammers T, Kiessling F, Hennink WE, Storm G. Drug targeting to tumors: Principles, pitfalls and (pre-) clinical progress. J Controlled Release 2012;161:175-87.
2. Portney NG, Ozkan M. Nano-oncology: drug delivery, imaging, and sensing. Anal Bioanal Chem 2006;384:620-30.
3. Matsumura Y, Maeda H. A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs. Cancer Res 1986:6387-92.
4. Maeda H. The enhanced permeability and retention (EPR) effect in tumor vasculature: the key role of tumor-selective macromolecular drug targeting. Adv Enzyme Regul 2001;41:189-207.
5. Moitra K, Lou H, Dean M. Multidrug efflux pumps and cancer stem cells: insights into multidrug resistance and therapeutic development. Clin Pharmacol Ther 2011;89:491-502. 167 CHAPTER 5: TARGETED FILAMENTS
6. Fletcher JI, Haber M, Henderson MJ, Norris MD. ABC transporters in cancer: more than just drug efflux pumps. Nat Rev Cancer 2010;10:147-56.
7. Cho K, Wang X, Nie S, Chen ZG, Shin DM. Therapeutic nanoparticles for drug delivery in cancer. Clin Cancer Res 2008;14:1310-6.
8. Bareford LM, Swaan PW. Endocytic mechanisms for targeted drug delivery. Adv Drug Deliver Rev 2007;59:748-58.
9. Danhier F, Feron O, Préat V. To exploit the tumor microenvironment: Passive and active tumor targeting of nanocarriers for anti-cancer drug delivery. J Controlled Release 2010;148:135-46.
10. Nicholson RI, Gee JMW, Harper ME. EGFR and cancer prognosis. Eur J Cancer 2001;37:9-15.
11. Mickler FM, Mo L, Ruthardt N, Ogris M, Wagner E. Tuning Nanoparticle Uptake: Live-Cell Imaging Reveals Two Distinct Endocytosis Mechanisms Mediated by Natural and Artificial EGFR Targeting Ligand. Nano Lett 2012.
12. Rocha-lima CM, Soares HP, Raez LE, Singal R. EGFR Targeting of Solid Tumors. Cancer Control 2007;14:295-304.
13. Hackel PO, Zwick E, Prenzel N, Ullrich A. Epidermal growth factor receptors: critical mediators of multiple receptor pathways. Curr Opin Cell Biol 1999;11:184-9.
14. Cawley LJMC, Brien PO, Hudson LG. Overexpression of the Epidermal Growth Factor Receptor Contributes to Enhanced Ligand-Mediated Motility in Keratinocyte Cell Lines. Endocrinology 2014;138:121-7.
15. Kitai Y, Fukuda H, Enomoto T, Asakawa Y, Suzuki T, Inouye S, et al. Cell selective targeting of a simian virus 40 virus-like particle conjugated to epidermal growth factor. J Biotechnol 2011;155:251-6.
16. Kickhoefer VA, Han M, Raval-fernandes S, Poderycki MJ, Moniz RJ, Vaccari D, et al. Targeting Vault Nanoparticles to Specific Cell Surface Receptors. ACS Nano 2009;3:27-36.
17. Baselga J. The EGFR as a target for anticancer therapy—focus on cetuximab. Eur J Cancer 2001;37:16-22.
18. Liu B, Fang M, Schmidt M, Lu Y, Mendelsohn J, Fan Z. Induction of apoptosis and activation of the caspase cascade by anti-EGF receptor monoclonal antibodies in DiFi human colon cancer cells do not involve the c-jun N-terminal kinase activity. Brit J Cancer 2000;82:1991-9.
168 CHAPTER 5: TARGETED FILAMENTS
19. Galizia G, Lieto E, Vita F, Orditura M, Castellano P, Troiani T, et al. Cetuximab, a chimeric human mouse anti-epidermal growth factor receptor monoclonal antibody, in the treatment of human colorectal cancer. Oncogene 2007;26:3654- 60.
20. Raff JP, Rajdev L, Malik U, Novik Y, Manalo JM, Negassa A, et al. Phase II Study of Weekly Docetaxel Alone or in Combination with Trastuzumab in Patients with Metastatic Breast Cancer. Clin Breast Cancer 2004;4:420-7.
21. Yonesaka K, Zejnullahu K, Okamoto I, Satoh T, Cappuzzo F, Souglakos J, et al. Activation of ERBB2 signaling causes resistance to the EGFR-directed therapeutic antibody cetuximab. Sci Transl Med 2011;3:99ra86.
22. Chen Z, Deng J, Zhao Y, Tao T. Cyclic RGD peptide-modified liposomal drug delivery system: enhanced cellular uptake in vitro and improved pharmacokinetics in rats. Int J Nanomedicine 2012;7:3803-11.
23. Lee GY, Kim J-H, Oh GT, Lee B-H, Kwon IC, Kim I-S. Molecular targeting of atherosclerotic plaques by a stabilin-2-specific peptide ligand. J Controlled Release 2011;155:211-7.
24. Song S, Liu D, Peng J, Deng H, Guo Y, Xu LX, et al. Novel peptide ligand directs liposomes toward EGF-R high-expressing cancer cells in vitro and in vivo. FASEB J 2009;23:1396-404.
25. Zhang Y, Zhang H, Wang X, Wang J, Zhang X, Zhang Q. The eradication of breast cancer and cancer stem cells using octreotide modified paclitaxel active targeting micelles and salinomycin passive targeting micelles. Biomaterials 2012;33:679-91.
26. Li Z, Zhao R, Wu X, Sun Y, Yao M, Li J, et al. Identification and characterization of a novel peptide ligand of epidermal growth factor receptor for targeted delivery of therapeutics. FASEB J 2005;19:1978-85.
27. Shukla S, Ablack AL, Wen AM, Lee KL, Lewis JD, Steinmetz NF. Increased tumor homing and tissue penetration of the filamentous plant viral nanoparticle Potato virus X. Mol Pharm 2013;10:33-42.
28. Shukla S, Dickmeis C, Nagarajan aS, Fischer R, Commandeur U, Steinmetz NF. Molecular farming of fluorescent virus-based nanoparticles for optical imaging in plants, human cells and mouse models. Biomater Sci 2014;2:784.
29. Lee KL, Uhde-Holzem K, Fisher R, Commendeur U, Steinmetz N. Genetic engineering and chemical conjugation of potato virus X. Methods Mol Biol2014. p. 3-21.
169 CHAPTER 5: TARGETED FILAMENTS
30. Steinmetz NF, Mertens ME, Taurog RE, Johnson JE, Commandeur U, Fischer R, et al. Potato virus X as a novel platform for potential biomedical applications. Nano Lett 2010;10:305-12.
31. Shukla S, Wen AM, Commandeur U, Steinmetz NF. Presentation of HER2 epitopes using a filamentous plant virus-based vaccination platform. J Mater Chem B 2014;2:6249.
32. Kawamotos T, Mendelsohnsq J, Le A, Sato GH, Lazarlt CS, Gill GN. Relation of Epidermal Growth Factor Receptor Concentration to Growth of Human Epidermoid Carcinoma A431 Cells. J Biol Chem 1984;259:7761-6.
33. Wiley HS. Anomalous Binding of Epidermal Growth Factor to A431 Cells Is Due to the Effect of High Receptor Densities and a Saturable Endocytic System. J Cell Biol 1988;107:801-10.
34. Rae JM, Scheys JO, Clark KM, Chadwick RB, Kiefer MC, Lippman ME. EGFR and EGFRvIII expression in primary breast cancer and cell lines. Breast Cancer Res Tr 2004;87:87-95.
35. Wang K, Wang K, Li W, Huang T, Li R, Wang D, et al. Characterizing breast cancer xenograft epidermal growth factor receptor expression by using near- infrared optical imaging. Acta Radiol 2009;50:1095-103.
36. Balin-Gauthier D, Delord J-P, Rochaix P, Mallard V, Thomas F, Hennebelle I, et al. In vivo and in vitro antitumor activity of oxaliplatin in combination with cetuximab in human colorectal tumor cell lines expressing different level of EGFR. Cancer Chemoth Pharm 2006;57:709-18.
37. Kelf T.A S, V.K.A, Sun, J, Kim, E.J, Goldys, E.M, Zvyagin, A.V. Non-specific cellular uptake of surface-functionalized quantum dots. Nanotechnlogy 2010;21:1-14.
38. Hillaireau H, Couvreur P. Nanocarriers' entry into the cell: relevance to drug delivery. Cell Mol Life Sci 2009;66:2873-96.
39. Christian DA, Cai S, Garbuzenko OB, Harada T, Allison L, Minko T, et al. Flexible filaments for in vivo imaging and delivery: persistent circulation of filomicelles opens the dosage window for sustained tumor shrinkage. Molecular pharmaceutics 2009;6:1343-52.
40. Chauhan VP, Popović Z, Chen O, Cui J, Fukumura D, Bawendi MG, et al. Fluorescent nanorods and nanospheres for real-time in vivo probing of nanoparticle shape-dependent tumor penetration. Angew Chem Int Ed 2011;50:11417-20.
170 CHAPTER 5: TARGETED FILAMENTS
41. Decuzzi P, Godin B, Tanaka T, Lee S-Y, Chiappini C, Liu X, et al. Size and shape effects in the biodistribution of intravascularly injected particles. J Controlled Release 2010;141:320-7.
42. Doshi N, Prabhakarpandian B, Rea-Ramsey A, Pant K, Sundaram S, Mitragotri S. Flow and adhesion of drug carriers in blood vessels depend on their shape: a study using model synthetic microvascular networks. J Controlled Release 2010;146:196-200.
43. Lee S-Y, Ferrari M, Decuzzi P. Shaping nano-/micro-particles for enhanced vascular interaction in laminar flows. Nanotechnology 2009;20:495101.
44. Arnida, Janát-Amsbury MM, Ray a, Peterson CM, Ghandehari H. Geometry and surface characteristics of gold nanoparticles influence their biodistribution and uptake by macrophages. Eur J Pharm Biopharm 2011;77:417-23.
45. Bruckman Ma, Jiang K, Simpson EJ, Randolph LN, Luyt LG, Yu X, et al. Dual- modal magnetic resonance and fluorescence imaging of atherosclerotic plaques in vivo using VCAM-1 targeted tobacco mosaic virus. Nano Lett 2014;14:1551-8.
46. Farkas ME, Aanei IL, Behrens CR, Tong GJ, Murphy ST, Neil JPO, et al. PET Imaging and Biodistribution of Chemically Modified Bacteriophage MS2. Molecular pharmaceutics 2013;10:69-76.
47. Zeng Q, Wen H, Wen Q, Chen X, Wang Y, Xuan W, et al. Cucumber mosaic virus as drug delivery vehicle for doxorubicin. Biomaterials 2013;34:4632-42.
48. Abourbeh G, Shir A, Mishani E, Ogris M, Rödl W, Wagner E, et al. PolyIC GE11 polyplex inhibits EGFR-overexpressing tumors. IUBMB life 2012;64:324-30.
49. Tang H, Chen X, Rui M, Sun W, Chen J, Peng J, et al. Effects of Surface Displayed Targeting Ligand GE11 on Liposome Distribution and Extravasation in Tumor. Molecular pharmaceutics 2014;11:3242-50.
50. Song S, Liu D, Peng J, Sun Y, Li Z, Gu J-R, et al. Peptide ligand-mediated liposome distribution and targeting to EGFR expressing tumor in vivo. Int J Pharm 2008;363:155-61.
51. Pokorski JK, Hovlid ML, Finn MG. Cell targeting with hybrid Qβ virus-like particles displaying epidermal growth factor. ChemBioChem 2011;12:2441-7.
52. Paraskevakou G, Allen C, Nakamura T, Zollman P, James CD, Peng KW, et al. Epidermal Growth Factor Receptor ( EGFR )– Retargeted Measles Virus Strains Effectively Target EGFR- or EGFRvIII Expressing Gliomas. Mol Ther 2007;15:677-86.
171 CHAPTER 6: CANCER THERAPY
Chapter 6: PVX-DOX for cancer therapy
6.1 Introduction
Each year, over one million new cases of cancer are diagnosed in the United
States alone [1]. Current cancer therapy options include surgery, hormone therapy, radiation therapy, immunotherapy, and chemotherapy, either alone or in combination.
However, therapy options are limited, especially for patients with advanced disease, and newer treatment options are urgently needed.
Chemotherapy is one of the most widely used therapies for cancer and is the last treatment option for advanced disease. Doxorubicin (DOX), an anthracycline, is a commonly used chemotherapeutic that works via three mechanisms of action: (i) intercalating with DNA, (ii) inhibiting topoisomerase II, and (iii) producing reactive oxygen species [2]. Additionally, unlike other classes of chemotherapies, anthracyclines such as DOX are also advantageous because they cause immunogenic cell death, making them ideal candidates to be combined with immunotherapies [3]. Doxorubicin is FDA- approved to treat a range of cancers, including acute lymphoblastic leukemia, Hodgkin’s lymphoma, breast cancer, ovarian cancer, and small cell lung cancer [4]. However, like all chemotherapies, when administered systemically, it is associated with off-target effects due to interaction with healthy cells that also divide rapidly, including hair cells and the epithelium of the intestinal tract [5].
Recent clinical and preclinical research indicates that the combination of chemo- and immunotherapies can be beneficial, because the therapy regimes can synergize to potentiate the therapy and improve patient outcomes [6, 7]. Immunotherapies are
172 CHAPTER 6: CANCER THERAPY designed to restart or potentiate the cancer-immunity cycle, the body’s defense against cancer. In brief, as a tumor grows, cancer cells undergo apoptosis and release neoantigens that are recognized and taken up by dendritic cells; the dendritic cells then prime T cells to recognize cancer cells expressing these cancer associated neoantigens, the T cells have cytotoxic functions and cell killing leads to further release of neoantigen – the cycle starts over again [8]. However, the tumor microenvironment is naturally immune-suppressive due to the upregulation of suppressive immune cells (i.e. regulatory T cells and myeloid- derived suppressor cells), inhibition of dendritic cell maturation, increased amounts of immunosuppressive molecules (i.e. IL-10), and impairment of antigen presenting machinery, which overall halts the cancer-immunity cycle [9-11]. Therefore immunotherapies set out to restart the cycle and overcome the immune-suppressive phenotype [8]. One subset of immunotherapy is immunostimulatory molecules, such as
IL-2 or virus-like particles, which boost the immune response within the tumor microenvironment [12-14]. The combination of these immune-stimulatory therapies with cytotoxic agents can be particularly powerful: chemotherapy kills tumor cells that release neoantigens and immunotherapy promotes systemic immune stimulation producing T cells with tumor killing function. If the therapy is successful, memory T cells are produced that have potential to protect the patient from recurrence of the disease [15, 16].
Nanotechnology offers an opportunity to combine chemo- and immunotherapy into one delivery system.
Nanoparticles offer advantages over administration of free drug because they are able to deliver multiple molecules simultaneously, for both monotherapy and combination therapy. In addition, nanoparticles are commonly used to encapsulate
173 CHAPTER 6: CANCER THERAPY chemotherapies in order to decrease systemic side effects. They can also be passively and actively targeted to the tumor site using surface modifications [17-20]. Currently, there are seven FDA-approved nanoparticle formulations approved, including Doxil, which is a liposomal formulation of DOX [21]. In addition, there are many synthetic platforms in preclinical and clinical trials being investigated for improved nanoparticle delivery, including polymeric nanoparticles, dendrimers, particle replication in non-wetting templates (PRINT) nanoparticles, carbon nanotubes, and filomicelles [21-25].
Traditionally, nanoparticles have been spherical in shape. However, recent data suggest that high aspect ratio filaments have advantageous properties compared to spherical nanoparticles. Rods have demonstrated improved extravasation and decreased nonspecific uptake by phagocytic cells compared to spherical particles, leading to higher tumor accumulation and penetration [26-34].
However, synthetic particles are difficult to produce at high aspect ratios on the nanoscale. Currently available high aspect ratio particles include filomicelles, carbon nanotubes, and PRINT nanoparticles. However, filomicelles are microparticles [23, 24], carbon nanotubes accumulate in the lungs [22], and PRINT nanoparticles cannot be manufactured below 50 nm [25]. Therefore, I turned toward filamentous viruses for use in cancer therapy. Viral nanoparticles (VNPs) come in a variety of shapes and sizes and are biocompatible, biodegradable, and non-pathogenic in mammals [35]. Additionally,
VNPs can be produced in high quantities, have highly symmetrical structures, and can be modified using chemical and genetic engineering [35-37]. Since they are assembled around an RNA template, plant viruses are monodisperse. VNPs have previously been used to deliver different cargos, including chemotherapeutic molecules for cancer therapy
174 CHAPTER 6: CANCER THERAPY
[38-41]. Additionally, it has been shown that virus-like particles derived from the icosahedral virus cowpea mosaic virus are immunostimulatory, slow tumor growth rate, and prevent recurrence via an immune-mediated mechanism in multiple models of cancer
[12]. The filamentous virus, papaya mosaic virus, was also shown to slow tumor growth and extend survival in a murine model of aggressive melanoma [42]. Here, I am specifically interested in the filamentous plant virus, potato virus X (PVX), for cancer therapy.
PVX is a filamentous plant virus, measuring 515 x 13 nm, and is comprised of
1270 identical coat proteins. It was previously shown to have increased tumor homing and penetration compared to a spherical virus [43]. In this work, I evaluate PVX for cancer therapy, either as an intravenous drug delivery vehicle, as an in situ vaccine for immunotherapy, and as an intratumoral chemo-immunotherapy. Utilizing the hydrophobic nature of DOX, I demonstrate DOX loading to the proteinaceous structure of PVX. PVX vs. PVX-DOX particles as well as physical mixtures of PVX+DOX were then evaluated in a panel of cancer cell lines and a murine model of melanoma.
6.2 Materials and methods
6.2.1 Potato virus X production
Potato virus X was propagated in Nicotiana benthamiana plants and purified as previously reported [44].
175 CHAPTER 6: CANCER THERAPY
6.2.2 Doxorubicin loading onto PVX
PVX (2 mg mL-1 in 0.1 M potassium phosphate buffer (KP), pH 7.0) was incubated with a 5000 molar excess of doxorubicin (DOX) at a 10% (v/v) final concentration of DMSO for 5 days at room temperature, with agitation. PVX-DOX was purified twice over a 30% (w/v) sucrose cushion using ultracentrifugation (212,000 x g for 3 h at 4 °C) and resuspended overnight in 0.1 M KP, pH 7.0. PVX-DOX filaments were analyzed using UV/visible spectroscopy, transmission electron microscopy, and agarose gel electrophoresis.
6.2.3 UV/visible spectroscopy
The number of DOX per PVX filament was determined by UV/visible spectroscopy, using the NanoDrop 2000 spectrophotometer. DOX loading was determined using the Beer-Lambert law and DOX- (11,500 M-1 cm-1 at 495 nm) and
PVX-specific (2.97 mL mg-1 cm-1 at 260 nm) extinction coefficients.
6.2.4 Transmission electron microscopy
Transmission electron microscopy (TEM) was performed after DOX loading to
-1 confirm integrity of PVX-DOX filaments. PVX-DOX samples (0.1 mg mL , in dH2O) were placed on carbon-coated copper grids and negatively stained with 0.2% (w/v) uranyl acetate. Grids were imaged using a Zeiss Libra 200FE transmission electron microscope, operated at 200 kV. TEM imaging was carried out by Amy M. Wen.
176 CHAPTER 6: CANCER THERAPY
6.2.5 Agarose gel electrophoresis
To confirm DOX attachment, PVX-DOX filaments were run in a 0.8% (w/v) agarose gel (in TBE). PVX-DOX and corresponding amounts of free DOX or PVX alone were loaded with 6x agarose loading dye. Samples were run at 100 V for 30 min in TBE.
Gels were visualized under UV light and after staining with 0.25% (w/v) Coomassie blue.
6.2.6 Cell culture
A2780 were a gift from Dr. Analisa DiFeo (Case Western Reserve University).
B16F10 (ATCC) and A2780 were cultured in Dulbecco’s modified Eagle’s media
(DMEM, Life Technologies), supplemented with 10% (v/v) fetal bovine serum (FBS,
Atlanta Biologicals) and 1% (v/v) penicillin-streptomycin (penstrep, Life Technologies).
HeLa (ATCC) were cultured in minimum essential media (MEM, Life Technologies), supplemented with 10% (v/v) FBS, 1% (v/v) penstrep, and 1% (v/v) L-glutamine (Life
Technologies). All cells were maintained at 37 °C, 5% CO2.
6.2.7 PVX-DOX efficacy
Confluent cells were removed with 0.05% (w/v) trypsin-EDTA (Life
3 3 Technologies), seeded at either 2 x 10 cells/100μL/well (B16F10, HeLa) or 5 x 10 cells/100μL/well (A2780) in 96-well plates and grown overnight at 37 °C, 5% CO2. The next day, cells were washed 2 times with PBS and incubated with free DOX or PVX-
DOX corresponding to 0, 0.01, 0.05, 0.1, 0.5, 1, 5, or 10 μM (B16F10, A2780) or 0, 0.1,
0.5, 1, 5, 10, 25, or 50 μM (HeLa) DOX for 24 h, in triplicate. A PVX only control
177 CHAPTER 6: CANCER THERAPY corresponded to the amount of PVX in the highest PVX-DOX sample. Following incubation, cells were washed 2 times to remove unbound DOX or particles. Fresh medium (100μL) was added and cells were returned to the incubator for 48 h. Cell viability was assessed using an MTT proliferation assay (ATCC); the procedure was as the manufacturer suggested.
6.2.8 Tracking of DOX and PVX-delivered DOX in cells
A2780 cells were grown to confluency and removed with 0.05% (w/v) trypsin-
EDTA. Cells were seeded at 1 x 104 cells/500μL/well on coverslips in untreated 24-well plates and grown overnight at 37 °C, 5% CO2. The next day, cells were washed 3 times and incubated with free DOX or PVX-DOX (1.5 μM DOX) for 2, 4, 12, 24, or 48 h.
Following incubation, cells were washed 3 times and fixed in DPBS containing 5% (v/v) paraformaldehyde (Electron Microscopy Sciences) and 0.3% (v/v) gluteraldehyde for 10 min at room temperature. Coverslips were mounted using Fluoroshield (Sigma). Slides were imaged on a Zeiss Axio Observer Z1 motorized FL inverted microscope.
Fluorescence intensity was analyzed using ImageJ 1.47d (http://imagej.nih.gov/ij).
6.2.9 Intracellular tracking of PVX
A2780 cells were grown as described for DOX tracking. The next day, cells were washed 3 times and incubated with PVX (1 x 107 particles/cell) for 24 h. Following incubation, cells were washed 3 times and fixed as described above. To visualize internal targets, cells were permeabilized with 0.2% (v/v) Triton-X100 in DPBS for 2 min at
178 CHAPTER 6: CANCER THERAPY room temperature. Cells were then blocked in 10% (v/v) GS/DPBS for 45 min at room temperature. PVX and endolysosomes were stained using rabbit-anti-PVX antibody
(1:200 in 5% (v/v) GS/DPBS) and mouse-anti-LAMP-1 (1:200 in 5% (v/v) GS/DPBS), respectively, for 1.5 h at room temperature. Primary antibodies were detected with secondary antibody staining: AlexaFluor647-labeled goat-anti-rabbit antibody (Life
Technologies) (1:500 in 5% (v/v) GS/DPBS) and AlexaFluor488-labeled goat-anti-mouse antibody (1:500 in 5% (v/v) GS/DPBS) for 60 min at room temperature. Coverslips were washed 3 times with DPBS in between each step. Following the final wash, coverslips were mounted using Fluoroshield with DAPI. Slides were imaged on an Olympus
FluoView FV100 confocal laser scanning microscope and processed in ImageJ 1.47d
(http://imagej.nih.gov).
6.2.10 Animals
All experiments were conducted in accordance with Case Western Reserve
University’s Institutional Animal Care and Use Committee. C57/Bl6 male mice (Jackson) were used.
6.2.11 Tumor model
B16F10 cells were cultured in DMEM supplemented with 10% (v/v) FBS and 1%
(v/v) penstrep and maintained at 37 °C, 5% CO2. Tumors were induced intradermally into
5 the right flank of C57/Bl6 mice (1.25 x 10 cells/50 μL media). Animals were monitored
179 CHAPTER 6: CANCER THERAPY and tumor volume was calculated as V = 0.5 x a x b2, where a = width of the tumor and b
= length of the tumor. Animals were sacrificed when tumor volume reached >1000 mm3.
6.2.12 Immunotherapy treatment schedule
Eight days post-tumor induction (day 0), mice were randomly assigned to the following groups (n=3): PBS, PVX, or CPMV. Mice were treated intratumorally (20 μL), every 7 days, with 5 mg kg-1 VNP. Mice were sacrificed when tumors reached a volume
>1000 mm3 and lung, liver, spleen, and tumor were collected for analysis.
6.2.13 Chemo-immunotherapy treatment schedule
When tumor reached volumes >20 mm3 (day 0), mice were randomly assigned to the following groups (n=6): PBS, PVX alone, DOX alone, PVX-DOX, or noncoupled
PVX+DOX. PVX+DOX samples were prepared less than 30 min before injections and are considered not bound to each other. Mice were treated intratumorally (20 μL), every other day, with 5 mg kg-1 PVX or PVX-DOX or the corresponding dose of DOX alone
-1 3 (~0.065 mg kg ). Mice were sacrificed when tumors reached a volume >1000 mm .
6.2.14 Immunohistochemistry of tumors
When tumor reached volumes >40 mm3, mice were randomly assigned to the following groups (n=3): PBS or PVX-DOX. Mice were treated intratumorally (20 μL), every 7 days, with 5 mg kg-1 PVX-DOX. Mice were sacrificed when tumors reached a volume >1000 mm3 tumors were collected for analysis. Tumors were frozen in optimal 180 CHAPTER 6: CANCER THERAPY cutting temperature compound (Fisher). Frozen tumors were cut into 12 μm sections.
Sections were fixed in 95% (v/v) ethanol for 20 minutes on ice. Following fixation, tumor sections were permeabilized with 0.2% (v/v) Triton X-100 in PBS for 2 min at room temperature for visualization of intracellular markers. Then, tumor sections were blocked in 10% (v/v) GS/PBS for 60 min at room temperature. PVX and F4/80 were stained using rabbit anti-PVX antibody (1:250 in 1% (v/v) GS/PBS) and rat anti-mouse F4/80 (1:250 in
1% (v/v) GS/PBS) for 1-2 h at room temperature. Primary antibodies were detected using secondary antibody staining: AlexaFluor488-labeled goat-anti-rabbit antibody (1:500 in
1% (v/v) GS/PBS) and AlexaFluor555-labeled goat-anti-rat antibody (1:500 in 1% (v/v)
GS/PBS) for 60 min at room temperature. Tumor sections were washed 3 times with PBS in between each step. Following the final wash, coverslips were mounted using
Fluoroshield with DAPI. Slides were imaged on a Zeiss Axio Observer Z1 motorized FL inverted microscope. Fluorescence intensity was analyzed using ImageJ 1.47d
(http://imagej.nih.gov/ij).
6.3 Results and discussion
6.3.1 Synthesis and characterization of PVX-DOX
PVX was produced in N. benthamiana and purified as previously described [44].
PVX was loaded with DOX by incubating a 5000 molar excess of DOX with PVX for 5 days and purifying excess DOX off using ultracentrifugation. Incubation criteria were optimized: increasing molar excess of DOX resulted in extensive aggregation and further increasing incubation time did not increase loading capacity (data not shown). Lower excess was not tested. I hypothesize that DOX is attached to PVX utilizing hydrophobic 181 CHAPTER 6: CANCER THERAPY interactions. Although the crystal structure of PVX is not known, from the protein sequence, it is apparent that there are numerous nonpolar residues [45]. Specifically, there are phenylalanine, tyrosine, and tryptophan residues, which contain benzene rings.
These amino acids are crucial for protein stability and often form hydrophobic interactions via stacking between them. Additionally, from the cryo-electron microscopy structure of PVX [46], it is evident that PVX has a grooved surface. Since the crystal structure is not known, it is possible that these amino acids are displayed on the surface of the virus or within the grooves. I hypothesize that the ring structure of DOX is able to interact via π-π stacking with the benzene rings of these amino acids. Additionally, it has previously been reported that DOX can interact via π-π stacking with other DOX molecules [47, 48]. Thus, it is also possible that within the grooves, there is additional
DOX-DOX stacking.
182 CHAPTER 6: CANCER THERAPY
Figure 6.1. Synthesis and characterization of PVX-DOX. A) Scheme of DOX loading onto PVX. B) TEM images of negatively stained PVX-DOX. B) UV/visible spectrum of PVX-DOX. D) Agarose gel electrophoresis of PVX, PVX-DOX, and free DOX under UV light (top) and after Coommassie Blue staining (bottom).
DOX was added using 5000 molar excess per PVX and incubated 5 days to obtain maximum loading without aggregation. The reaction was purified twice via ultracentrifugation and characterized by UV/visible spectroscopy, transmission electron microscopy, and agarose gel electrophoresis (Figure 6.1A).
UV/visible spectroscopy was used to determine the number of DOX attached per
PVX. The Beer-Lambert law, in conjunction with PVX- and DOX-specific extinction coefficients, was used to determine the concentrations of both PVX and DOX in solution.
The ratio of DOX to PVX concentration was then used to determine DOX loading. Each
183 CHAPTER 6: CANCER THERAPY
PVX was loaded with 770±130 DOX (Figure 6.1C). Agarose gel electrophoresis was used to confirm that DOX was attached to PVX and not free in solution. DOX is fluorescent when visualized under UV light and PVX was visualized under white light after Coomassie Blue staining. PVX is too large to migrate through the gel in 30 min, and remains confined to the well. Under UV light, no signal was seen in the PVX only lane, while PVX-DOX signal was seen directly within the well. The free DOX control migrated towards the cathode. Imaging of the gels after staining with Coomassie blue confirmed the presence of PVX and PVX-DOX in their respective wells (Figure 6.1D).
TEM imaging confirmed particle integrity following DOX loading (Figure 6.1B). PVX-
DOX stability was confirmed after 1 month of storage at 4 °C; DOX release was not apparent (data not shown).
6.3.2 PVX-DOX efficacy
To determine if doxorubicin maintained its cell killing ability after attachment to
PVX, increasing amounts of PVX-DOX (or corresponding DOX only controls) were evaluated in a panel of cell lines: A2780 (ovarian cancer), B16F10 (melanoma), and
HeLa (cervical cancer) (Figure 6.2). All cell lines tested showed a similar trend: DOX conjugated to PVX maintained cell killing ability, although with decreased efficacy. The calculated IC50 values for free DOX are similar to previously reported values [49-51]. A decrease in efficacy (between 3- to 7- fold difference depending on cell line) after attachment to PVX is not unexpected; similar trends have been reported with synthetic
[52] and virus-based nanoparticles for DOX delivery [41, 53].
184 CHAPTER 6: CANCER THERAPY
Figure 6.2. PVX-DOX efficacy in a panel of cell lines. Increasing doses of free DOX or PVX-DOX were incubated with (A) A2780 (DOX vs. PVX-DOX p<0.05), (B) B16F10 (DOX vs. PVX-DOX p<0.05), or (C) HeLa cells for 24 h. Cell viability was determined using an MTT assay and IC50 values are reported (chart).
6.3.3 Tracking of DOX and PVX-delivered DOX in cells
Although PVX-DOX was still able to kill cells, it did so at decreased efficacy compared to free DOX, which is similar to reports with other nanoparticle delivery systems for chemotherapeutics [41, 52, 53]. This decreased efficacy could be due to decreased uptake of DOX after attachment to PVX because PVX is not taken up as readily as free DOX. Alternately, decreased efficacy could be due to a delayed mechanism of action. Other VNP systems are taken up by the endolysosome, where they are broken down [38, 54, 55]. If a similar mechanism is used to take up PVX, DOX must first be released from PVX and escape the endolysosome before trafficking to the nucleus to exert its mechanism of action.
185 CHAPTER 6: CANCER THERAPY
To determine why PVX-DOX treatment yielded lower efficacy than free DOX after 24 h exposure to treatment, I compared nuclear accumulation of DOX for PVX-
DOX and free DOX in A2780. Cells were plated on coverslips in 24-well untreated plates and 1.5 μM of DOX (either PVX-DOX or free DOX) was added to cells for 2, 4, 12, 24, or 48 h. Cells were imaged using a fluorescence microscope and DOX fluorescence per cell was analyzed. As reported in previous studies, DOX accumulated within the nucleus
[56-58]. Interestingly, DOX fluorescence was only visible within the nucleus, and not within the cytoplasm. However, this is not unexpected because DOX fluorescence intensity is decreased in the cytoplasm due to scattering and absorption by cellular components [59]. Additionally, DOX is more concentrated within the nucleus than when it is dispersed throughout the cytoplasm; increased concentration of DOX is correlated to increases in fluorescence intensity [57, 60, 61]. PVX-delivered DOX exhibited decreased nuclear accumulation of DOX at all time points tested. However, the decrease was only statistically significant (p<0.05) at 24 and 48 h. At 48 h, cell treated with either PVX-
DOX or free DOX were undergoing apoptosis, so DOX fluorescence was decreased for both samples (Figure 6.3). This trend indicates that the decreased efficacy of PVX-DOX compared to free DOX is due to decreased uptake of DOX, not a delayed release.
Differences in DOX uptake could be explained by uptake mechanisms of free DOX versus PVX-delivered DOX. Free DOX is taken up via diffusion across the cell membrane, while VNPs such as PVX are taken up by endocytosis [54, 62]. Thus, PVX- delivered DOX is not as readily taken up as free DOX, resulting in decreased accumulation over time. Interestingly, after 24 h of incubation with DOX, which correlates to the incubation time used for MTT assay, nuclear fluorescence from PVX-
186 CHAPTER 6: CANCER THERAPY delivered DOX was approximately 2/3 that of nuclear fluorescence of free DOX. In comparison, DOX killed A2780 cells 4 times more effectively than PVX-DOX. However, cell viability is measured an additional 48 h after cells are exposed to DOX or PVX-DOX and nuclear accumulation of DOX at 24 h may not have a linear relationship to cell killing 48 h later.
187 CHAPTER 6: CANCER THERAPY
Figure 6.3. Nuclear accumulation of DOX in A2780. Free DOX or PVX-delivered DOX was incubated with cells for increasing time points. DOX accumulation was imaged (A) and quantified (B) using fluorescence microscopy. Scale bar = 20 μm.
188 CHAPTER 6: CANCER THERAPY
6.3.4 Intracellular tracking of PVX
Tracking DOX accumulation in the cell nucleus only gives insight into the movement of DOX. However, to better understand PVX as a drug delivery system, it is important to observe how PVX moves throughout the cell. Other VNP systems are taken up by the endolysosome [38, 54, 55]. It has also previously been shown that PVX is taken up by cancer cells and accumulates in the perinuclear region [63]. However, this study only evaluated PVX intracellular localization out to 3 h. Therefore, based on the time point with the highest DOX nuclear accumulation, the intracellular localization of PVX was also evaluated after 24 h. After 24 h, as expected, PVX was taken up into A2780 cells. However, perinuclear localization was not apparent, nor was any colocalization with the endolysosome. PVX was observed dispersed throughout the cytoplasm (Figure
6.4). Since the PVX antibodies used here can recognize free PVX coat proteins, it is possible that this signal is due to either intact particles, or free coat proteins that escape from the endolysosome after being broken down. Further evaluation including earlier time points will be important to determine a full time course of PVX intracellular localization.
189 CHAPTER 6: CANCER THERAPY
Figure 6.4. PVX tracking in A2780. Cells were incubated with PVX for 24 h and stained for DAPI (blue), LAMP-1 (red), or PVX (green). Scale bar = 20 μm. 190 CHAPTER 6: CANCER THERAPY
6.3.5 Immunotherapy treatment
In addition to use as an intravenous drug delivery platform, PVX-DOX can also be potentially used as a combination therapy for intratumoral chemo-immunotherapy.
This approach builds on recent interest in the development of in situ vaccinations, wherein immunostimulatory agents modulate the immunosuppressive tumor microenvironment and lead to anti-tumor immunity against tumor antigens. It has previously been reported that virus-like particles (VLPs) from cowpea mosaic virus
(CPMV) investigated for in situ vaccination effectively treat primary and metastatic cancer, also preventing recurrence via a systemic immune response [12]. In these studies, empty CPMV (eCPMV) particles were administered into primary tumors (intratumoral injection) or into areas of suspected metastatic disease (intratracheal injection) [12]. A potent response, characterized by increased survival was observed when eCPMV was administered intratumorally in orthotopic models of breast cancer, melanoma, and colon cancer; 50% of the treated mice were cured and remained tumor-free over several months.
In the B16F10 model of melanoma, tumors became rapidly necrotic after administration of the VLP, which suggests that the response was mediated by the innate immune system.
Additionally, mice that saw a complete regression of the tumor were resistant to re- challenge, indicating that in situ vaccination led to protective anti-tumor immunity with immune memory. In an intravenous B16F10 metastatic lung model, eCPMV particles were administered via inhalation. Within 24 h after eCPMV inhalation, the lung microenvironment saw a significant increase in immunostimulatory neutrophils, as well as increases in cytokines and chemokines produced by activated neutrophils. In addition to neutrophils and the cytokines/chemokines that they produced, the antitumor response
191 CHAPTER 6: CANCER THERAPY also required Th-1 cytokines IL-12 and IFNγ, as well as adaptive immunity [12]. This study was the first to report the use of a VLP-based nanoparticle directly cancer therapy immunotherapy, rather than drug delivery, leading to an interest in investigating other
VNP platforms for cancer immunotherapy.
Filamentous VLPs derived from papaya mosaic virus (PapMV) have also been investigated for in situ vaccination. PapMV VLPs showed moderate efficacy in a murine model of melanoma; they slowed tumor growth rate, leading to an extension of survival time [42].
Based on these recent data, I first set out to evaluate the efficacy of PVX alone as an in situ vaccine. B16F10 allografts were induced in C57/Bl6 mice into the intradermal space on the right flank. B16F10 is highly aggressive and poorly immunogenic tumor model used extensively for immunotherapy studies, including evaluation of the immunotherapeutic potential of virus-based therapies [64, 65]. Its low immunogenicity makes it an attractive platform to investigate new immunostimulatory therapies. Eight days post-induction (tumor starting volume = 37±14 mm3), mice were randomized (n=3) and treated weekly intratumorally with PBS or 5 mg kg-1 of PVX or CPMV. Tumor volumes were measured daily and mice were sacrificed when tumors reached >1000 mm3.
Treatment with CPMV or PVX alone significantly slowed tumor growth rate and extended survival time compared to PBS (Figure 6.5). There was no significant difference between CPMV and PVX treatment.
192 CHAPTER 6: CANCER THERAPY
Figure 6.5. Immunotherapy treatment of B16F10 tumors. A) Tumors were induced with an intradermal injection of 125,000 cells/mouse. Mice (n=3) were treated with 5 mg kg-1 of PVX or CPMV (or PBS control) once weekly, starting 8 days post-induction. Arrows = injection days; x = tumors >1000 mm3 and mice were sacrificed. (B) Tumor growth curves shown as relative tumor volume. (C) Survival rates of treated mice.
From the data shown here, it is evident that PVX alone has efficacy; it both slows tumor growth rate and extends survival time versus PBS treated mice. Based on previous studies investigating VNP-based immunotherapies [12, 42], it is likely that this efficacy is due to immunostimulation within the tumor microenvironment. However, further immune studies are necessary to confirm that PVX efficacy is due to a similar mechanism.
193 CHAPTER 6: CANCER THERAPY
6.3.6 Chemo-immunotherapy treatment
To test the hypothesis that a combination chemo-immunotherapy would potentiate the efficacy of PVX alone, DOX-loaded PVX (PVX-DOX) was tested in the same murine model. Additionally, a noncoupled PVX and DOX combination (PVX+DOX) was also tested to determine if the administration of noncoupled PVX and DOX would have an efficacious response. PVX+DOX was made less than 30 min before injection to ensure that the two therapies did not have the time to interact. PBS, PVX alone, and free
DOX were used as controls. When tumors were <40 mm3, mice were treated every other day intratumorally with PVX-DOX, PVX+DOX, or corresponding controls (n=6). All
PVX was administered at 5 mg kg-1 and DOX was administered at 0.065 mg kg-1.
Clinically, doxorubicin is administered at doses of 1-10 mg kg-1, intravenously. Of the administered dose, approximately 5-10% will reach the tumor site, resulting in an intratumoral dose of 0.05-1 mg kg-1; our intratumoral dose is within a clinically relevant range of DOX. Tumor volumes were measured daily and mice were sacrificed when tumors reached >1000 mm3. While there was no statistical difference in tumor growth rate or survival time between PVX-DOX versus PVX as a monotherapy, PVX+DOX did significantly slow tumor growth rate versus PVX (Figure 6.6).
194 CHAPTER 6: CANCER THERAPY
Figure 6.6. Chemo-immunotherapy treatment of B16F10 tumors. A) Injection schedule. Red arrows indicate intratumoral injection (n=6) of PBS, PVX, DOX, PVX-DOX, or PVX+DOX. PVX was administered at a dose of 5 mg kg-1, DOX was administered at a dose of 0.065 mg kg-1. Injections are repeated every other day until day x, when tumors reach >1000 mm3. (B) Tumor growth curves shown as relative tumor volume (time points with n<2 per group were excluded); *p<0.05, **p<0.01, ***p<0.005 vs. PVX (Student’s t-test). (C) Tumor growth curves shown as absolute tumor volume. (D) Survival rates of treated mice.
PVX-DOX did not significantly slow tumor growth rate compared to monotherapy treatments alone. This lack of improvement could be due to multiple factors.
First, I previously showed (sections 6.3.2 and 6.3.3) that attachment of DOX to PVX decreased cell killing efficacy due to decreased uptake of DOX; this may be a contributing factor. Additionally, PVX-DOX particles are not targeted specifically towards cancer cells. As shown in chapter 5, non-targeted particles are preferentially taken up by macrophages compared to tumor cells because of differences in uptake 195 CHAPTER 6: CANCER THERAPY mechanism (phagocytosis versus endocytosis). It is probable that similarly, phagocytic cells, including neutrophils, macrophages, and dendritic cells, within the tumor microenvironment are taking up PVX-DOX. Uptake of PVX-DOX by these cells would cause cell death, leading to a decreased contribution of immunotherapy towards the combination therapy. As a preliminary test of this hypothesis, tumors treated weekly with
PVX-DOX were evaluated using immunohistochemistry. Tumor sections were stained with primary antibodies for PVX and F4/80, a specific cell surface marker for murine macrophages. Staining revealed co-localization of PVX and F4/80 signals, indicating that it is likely that PVX is taken up by murine macrophages (Figure 6.7).
Figure 6.7. Immunohistochemistry of PVX-DOX tumor sections. Tumors treated with PBS (top) or PVX-DOX (rows 2-4) were sectioned and stained with DAPI (blue), F4/80 (red), and PVX (green). Scale bar = 100 μm.
196 CHAPTER 6: CANCER THERAPY
Interestingly, PVX+DOX significantly slowed tumor growth and extended survival compared to PVX (Figure 6.6). Non-coupled PVX+DOX allows PVX and DOX to be taken up independently. Therefore, DOX can be taken up by cancer cells and exert its optimal cell killing efficacy, while PVX is taken up by immune cells to promote immune stimulation. Overall, this combination therapy led to synergistic efficacy.
6.4 Conclusions and future directions
Here, I explored PVX-DOX for potential applications in cancer therapy: as a drug delivery platform (for intravenous administration) and for intratumoral chemo- immunotherapy. I showed that DOX could be successfully attached to PVX using hydrophobic interactions. PVX-DOX successfully killed doxorubicin-sensitive cells, indicating that DOX is still active after attachment to PVX, although at lower efficacy than free DOX. It is important to note that I evaluated bare PVX-DOX particles with no surface modifications in doxorubicin-sensitive cancer cells. However, this model is not the most relevant model of human cancer. A barrier to the use of chemotherapeutics in the clinic is the development of resistance over time. It has previously been shown that delivering chemotherapy molecules using nanoparticles can overcome some mechanisms of resistance, including efflux pumps [66, 67]. Therefore, PVX-DOX should be evaluated in a doxorubicin-resistant cell line to evaluate if it outperforms free DOX. Additionally, chemotherapy is limited by side effects when administered systemically due to interactions with off-target cells. Nanoparticles can decrease these side effects by altering their biodistribution [68, 69]. Therefore, PVX-DOX will be tested versus free DOX in a murine model of cancer. Additionally, adding PEG and/or targeting ligands to improve
197 CHAPTER 6: CANCER THERAPY passive and active targeting, respectively, can further improve the use of PVX-DOX for intravenous drug delivery.
I also demonstrated that PVX alone exhibits an immunotherapeutic response in a model of murine melanoma, but PVX and DOX should be administered as a non-coupled injection to maximize chemo-immunotherapeutic efficacy. To better understand the efficacy (or lack of efficacy for PVX-DOX), it will be important to evaluate the cells and signaling pathways activated after treatment with PVX, PVX-DOX, and PVX+DOX.
Specifically, analyzing tumor tissues using flow cytometry and Luminex panels before treatment and at early and late time points will elucidate the cytokines and cells that become activated with each treatment. Further immunohistochemistry analysis will also provide insights into with which cells PVX is interacting. Additionally, more translationally relevant modes of administration can be evaluated. In the clinic, DOX is administered intravenously, while VLP-based therapies (e.g. T-VEC) are administered intratumorally. Here, after intratumoral injection, PVX+DOX was the most efficacious.
Future studies should evaluate a PVX+DOX treatment in which DOX is administered intravenously and PVX is administered intratumorally.
Although PVX-DOX requires more evaluation and modification before it can be effectively used as an intravenous drug delivery platform or intratumoral chemo- immunotherapy, here, I have laid the groundwork for the development of a promising cancer therapy.
198 CHAPTER 6: CANCER THERAPY
6.5 Works cited in this chapter
1. American Cancer Society. Global Cancer Facts & Figures, 3rd Edition. In: Society AC, editor. 3rd ed. Atlanta: American Cancer Society; 2015.
2. Thorn CF, Oshiro C, Marsh S, Hernandez-Boussard T, McLeod H, Klein TE, et al. Doxorubicin pathways: pharmacodynamics and adverse effects. Pharmacogenet Genomics 2011;21:440-6.
3. Obeid M, Tesniere A, Ghiringhelli F, Fimia GM, Apetoh L, Perfettini JL, et al. Calreticulin exposure dictates the immunogenicity of cancer cell death. Nat Med 2007;13:54-61.
4. Doxorubicin Hydrochloride. National Cancer Institute; 2014.
5. Carelle N, Piotto E, Bellanger A, Germanaud J, Thuillier A, Khayat D. Changing patient perceptions of the side effects of cancer chemotherapy. Cancer 2002;95:155-63.
6. Bagalkot V, Lee IH, Yu MK, Lee E, Park S, Lee JH, et al. A combined chemoimmunotherapy approach using a plasmid-doxorubicin complex. Mol Pharm 2009;6:1019-28.
7. Roy A, Singh MS, Upadhyay P, Bhaskar S. Nanoparticle mediated co-delivery of paclitaxel and a TLR-4 agonist results in tumor regression and enhanced immune response in the tumor microenvironment of a mouse model. Int J Pharm 2013;445:171-80.
8. Chen DS, Mellman I. Oncology meets immunology: the cancer-immunity cycle. Immunity 2013;39:1-10.
9. Michielsen AJ, Hogan AE, Marry J, Tosetto M, Cox F, Hyland JM, et al. Tumour tissue microenvironment can inhibit dendritic cell maturation in colorectal cancer. PLoS One 2011;6:e27944.
10. Monjazeb AM, Zamora AE, Grossenbacher SK, Mirsoian A, Sckisel GD, Murphy WJ. Immunoediting and antigen loss: overcoming the achilles heel of immunotherapy with antigen non-specific therapies. Front Oncol 2013;3:197.
11. Zou W. Immunosuppressive networks in the tumour environment and their therapeutic relevance. Nat Rev Cancer 2005;5:263-74.
12. Lizotte PH, Wen AM, Sheen MR, Fields J, Rojanasopondist P, Steinmetz NF, et al. In situ vaccination with cowpea mosaic virus nanoparticles suppresses metastatic cancer. Nat Nanotechnol 2016;11:295-303.
13. Ott PA, Hodi FS. Talimogene Laherparepvec for the Treatment of Advanced Melanoma. Clin Cancer Res 2016.
199 CHAPTER 6: CANCER THERAPY
14. Rosenberg SA. IL-2: the first effective immunotherapy for human cancer. J Immunol 2014;192:5451-8.
15. Ho RL, Maccubbin D, Zaleskis G, Krawczyk C, Wing K, Mihich E, et al. Development of a safe and effective adriamycin plus interleukin 2 therapy against both adriamycin-sensitive and -resistant lymphomas. Oncol Res 1993;5:373-81.
16. Ho RL, Maccubbin DL, Ujhazy P, Zaleskis G, Eppolito C, Mihich E, et al. Immunological responses critical to the therapeutic effects of adriamycin plus interleukin 2 in C57BL/6 mice bearing syngeneic EL4 lymphoma. Oncol Res 1993;5:363-72.
17. Coxon TP, Fallows TW, Gough JE, Webb SJ. A versatile approach towards multivalent saccharide displays on magnetic nanoparticles and phospholipid vesicles. Org Biomol Chem 2015;13:10751-61.
18. Montet X, Funovics M, Montet-Abou K, Weissleder R, Josephson L. Multivalent effects of RGD peptides obtained by nanoparticle display. J Med Chem 2006;49:6087-93.
19. Venter PA, Dirksen A, Thomas D, Manchester M, Dawson PE, Schneemann A. Multivalent display of proteins on viral nanoparticles using molecular recognition and chemical ligation strategies. Biomacromolecules 2011;12:2293-301.
20. Bazak R, Houri M, Achy SE, Hussein W, Refaat T. Passive targeting of nanoparticles to cancer: A comprehensive review of the literature. Mol Clin Oncol 2014;2:904-8.
21. A.C. A, S. M. Nanoparticles in the Clinic. Bioengineering & Translational Medicine 2016.
22. Hamilton RF, Jr., Wu Z, Mitra S, Shaw PK, Holian A. Effect of MWCNT size, carboxylation, and purification on in vitro and in vivo toxicity, inflammation and lung pathology. Part Fibre Toxicol 2013;10:57.
23. Oltra NS, Swift J, Mahmud A, Rajagopal K, Loverde SM, Discher DE. Filomicelles in nanomedicine - from flexible, fragmentable, and ligand-targetable drug carrier designs to combination therapy for brain tumors. Journal of Materials Chemistry B 2013;1:5177-85.
24. Simone EA, Dziubla TD, Discher DE, Muzykantov VR. Filamentous polymer nanocarriers of tunable stiffness that encapsulate the therapeutic enzyme catalase. Biomacromolecules 2009;10:1324-30.
25. Xu J, Wong DH, Byrne JD, Chen K, Bowerman C, DeSimone JM. Future of the particle replication in nonwetting templates (PRINT) technology. Angew Chem Int Ed Engl 2013;52:6580-9.
200 CHAPTER 6: CANCER THERAPY
26. Arnida, Janat-Amsbury MM, Ray A, Peterson CM, Ghandehari H. Geometry and surface characteristics of gold nanoparticles influence their biodistribution and uptake by macrophages. Eur J Pharm Biopharm 2011;77:417-23.
27. Barua S, Yoo JW, Kolhar P, Wakankar A, Gokarn YR, Mitragotri S. Particle shape enhances specificity of antibody-displaying nanoparticles. Proc Natl Acad Sci U S A 2013;110:3270-5.
28. Chauhan VP, Popovic Z, Chen O, Cui J, Fukumura D, Bawendi MG, et al. Fluorescent nanorods and nanospheres for real-time in vivo probing of nanoparticle shape-dependent tumor penetration. Angew Chem Int Ed Engl 2011;50:11417-20.
29. Decuzzi P, Ferrari M. The adhesive strength of non-spherical particles mediated by specific interactions. Biomaterials 2006;27:5307-14.
30. Gentile F, Chiappini C, Fine D, Bhavane RC, Peluccio MS, Cheng MM, et al. The effect of shape on the margination dynamics of non-neutrally buoyant particles in two-dimensional shear flows. J Biomech 2008;41:2312-8.
31. Lee SY, Ferrari M, Decuzzi P. Shaping nano-/micro-particles for enhanced vascular interaction in laminar flows. Nanotechnology 2009;20.
32. Sharma G, Valenta DT, Altman Y, Harvey S, Xie H, Mitragotri S, et al. Polymer particle shape independently influences binding and internalization by macrophages. J Control Release 2010;147:408-12.
33. Shukla S, Eber FJ, Nagarajan AS, DiFranco NA, Schmidt N, Wen AM, et al. The Impact of Aspect Ratio on the Biodistribution and Tumor Homing of Rigid Soft- Matter Nanorods. Adv Healthc Mater 2015;4:874-82.
34. Toy R, Peiris PM, Ghaghada KB, Karathanasis E. Shaping cancer nanomedicine: the effect of particle shape on the in vivo journey of nanoparticles. Nanomedicine (Lond) 2014;9:121-34.
35. Steinmetz NF. Viral nanoparticles as platforms for next-generation therapeutics and imaging devices. Nanomedicine 2010;6:634-41.
36. Shukla S, Dickmeis C, Nagarajan AS, Fischer R, Commandeur U, Steinmetz NF. Molecular farming of fluorescent virus-based nanoparticles for optical imaging in plants, human cells and mouse models. Biomaterials Science 2014;2:784-97.
37. Bruckman MA, Kaur G, Lee LA, Xie F, Sepulveda J, Breitenkamp R, et al. Surface modification of tobacco mosaic virus with "click" chemistry. Chembiochem 2008;9:519-23.
38. Aljabali AA, Shukla S, Lomonossoff GP, Steinmetz NF, Evans DJ. CPMV-DOX delivers. Mol Pharm 2013;10:3-10.
201 CHAPTER 6: CANCER THERAPY
39. Czapar AE, Zheng YR, Riddell IA, Shukla S, Awuah SG, Lippard SJ, et al. Tobacco Mosaic Virus Delivery of Phenanthriplatin for Cancer therapy. ACS Nano 2016;10:4119-26.
40. Cao J, Guenther RH, Sit TL, Opperman CH, Lommel SA, Willoughby JA. Loading and release mechanism of red clover necrotic mosaic virus derived plant viral nanoparticles for drug delivery of doxorubicin. Small 2014;10:5126-36.
41. Lockney DM, Guenther RN, Loo L, Overton W, Antonelli R, Clark J, et al. The Red clover necrotic mosaic virus capsid as a multifunctional cell targeting plant viral nanoparticle. Bioconjug Chem 2011;22:67-73.
42. Lebel ME, Chartrand K, Tarrab E, Savard P, Leclerc D, Lamarre A. Potentiating Cancer Immunotherapy Using Papaya Mosaic Virus-Derived Nanoparticles. Nano Lett 2016;16:1826-32.
43. Shukla S, Ablack AL, Wen AM, Lee KL, Lewis JD, Steinmetz NF. Increased tumor homing and tissue penetration of the filamentous plant viral nanoparticle Potato virus X. Mol Pharm 2013;10:33-42.
44. Lee KL, Uhde-Holzem K, Fischer R, Commandeur U, Steinmetz NF. Genetic engineering and chemical conjugation of potato virus X. Methods Mol Biol 2014;1108:3-21.
45. Huisman MJ, Linthorst HJ, Bol JF, Cornelissen JC. The complete nucleotide sequence of potato virus X and its homologies at the amino acid level with various plus-stranded RNA viruses. J Gen Virol 1988;69 ( Pt 8):1789-98.
46. Kendall A, McDonald M, Bian W, Bowles T, Baumgarten SC, Shi J, et al. Structure of flexible filamentous plant viruses. J Virol 2008;82:9546-54.
47. Porumb H. The solution spectroscopy of drugs and the drug-nucleic acid interactions. Prog Biophys Mol Biol 1978;34:175-95.
48. Missirlis D, Kawamura R, Tirelli N, Hubbell JA. Doxorubicin encapsulation and diffusional release from stable, polymeric, hydrogel nanoparticles. Eur J Pharm Sci 2006;29:120-9.
49. Shao J, DeHaven J, Lamm D, Weissman DN, Malanga CJ, Rojanasakul Y, et al. A cell-based drug delivery system for lung targeting: II. Therapeutic activities on B16-F10 melanoma in mouse lungs. Drug Deliv 2001;8:71-6.
50. Zhang Q, Xiang G, Zhang Y, Yang K, Fan W, Lin J, et al. Increase of doxorubicin sensitivity for folate receptor positive cells when given as the prodrug N-(phenylacetyl) doxorubicin in combination with folate-conjugated PGA. J Pharm Sci 2006;95:2266-75.
202 CHAPTER 6: CANCER THERAPY
51. Dufes C, Muller JM, Couet W, Olivier JC, Uchegbu IF, Schatzlein AG. Anticancer drug delivery with transferrin targeted polymeric chitosan vesicles. Pharm Res 2004;21:101-7.
52. Yoo HS, Lee KH, Oh JE, Park TG. In vitro and in vivo anti-tumor activities of nanoparticles based on doxorubicin-PLGA conjugates. J Control Release 2000;68:419-31.
53. Ren Y, Wong SM, Lim LY. Folic acid-conjugated protein cages of a plant virus: a novel delivery platform for doxorubicin. Bioconjug Chem 2007;18:836-43.
54. Wen AM, Infusino M, De Luca A, Kernan DL, Czapar AE, Strangi G, et al. Interface of physics and biology: engineering virus-based nanoparticles for biophotonics. Bioconjug Chem 2015;26:51-62.
55. Yildiz I, Lee KL, Chen K, Shukla S, Steinmetz NF. Infusion of imaging and therapeutic molecules into the plant virus-based carrier cowpea mosaic virus: cargo-loading and delivery. J Control Release 2013;172:568-78.
56. Dai X, Yue Z, Eccleston ME, Swartling J, Slater NK, Kaminski CF. Fluorescence intensity and lifetime imaging of free and micellar-encapsulated doxorubicin in living cells. Nanomedicine 2008;4:49-56.
57. Mohan P, Rapoport N. Doxorubicin as a molecular nanotheranostic agent: effect of doxorubicin encapsulation in micelles or nanoemulsions on the ultrasound- mediated intracellular delivery and nuclear trafficking. Mol Pharm 2010;7:1959- 73.
58. Shen F, Chu S, Bence AK, Bailey B, Xue X, Erickson PA, et al. Quantitation of doxorubicin uptake, efflux, and modulation of multidrug resistance (MDR) in MDR human cancer cells. J Pharmacol Exp Ther 2008;324:95-102.
59. Chen NT, Wu CY, Chung CY, Hwu Y, Cheng SH, Mou CY, et al. Probing the dynamics of doxorubicin-DNA intercalation during the initial activation of apoptosis by fluorescence lifetime imaging microscopy (FLIM). PLoS One 2012;7:e44947.
60. Kawai H, Minamiya Y, Kitamura M, Matsuzaki I, Hashimoto M, Suzuki H, et al. Direct measurement of doxorubicin concentration in the intact, living single cancer cell during hyperthermia. Cancer 1997;79:214-9.
61. Karukstis KK, Thompson EH, Whiles JA, Rosenfeld RJ. Deciphering the fluorescence signature of daunomycin and doxorubicin. Biophys Chem 1998;73:249-63.
62. Kamba SA, Ismail M, Hussein-Al-Ali SH, Ibrahim TA, Zakaria ZA. In vitro delivery and controlled release of Doxorubicin for targeting osteosarcoma bone cancer. Molecules 2013;18:10580-98. 203 CHAPTER 6: CANCER THERAPY
63. Steinmetz NF, Mertens ME, Taurog RE, Johnson JE, Commandeur U, Fischer R, et al. Potato virus X as a novel platform for potential biomedical applications. Nano Lett 2010;10:305-12.
64. Baird JR, Byrne KT, Lizotte PH, Toraya-Brown S, Scarlett UK, Alexander MP, et al. Immune-mediated regression of established B16F10 melanoma by intratumoral injection of attenuated Toxoplasma gondii protects against rechallenge. J Immunol 2013;190:469-78.
65. Wang J, Saffold S, Cao X, Krauss J, Chen W. Eliciting T cell immunity against poorly immunogenic tumors by immunization with dendritic cell-tumor fusion vaccines. J Immunol 1998;161:5516-24.
66. Xue X, Liang XJ. Overcoming drug efflux-based multidrug resistance in cancer with nanotechnology. Chin J Cancer 2012;31:100-9.
67. Davis ME, Chen ZG, Shin DM. Nanoparticle therapeutics: an emerging treatment modality for cancer. Nat Rev Drug Discov 2008;7:771-82.
68. Chatterjee K, Zhang J, Honbo N, Karliner JS. Doxorubicin cardiomyopathy. Cardiology 2010;115:155-62.
69. Couvreur P, Kante B, Grislain L, Roland M, Speiser P. Toxicity of polyalkylcyanoacrylate nanoparticles II: Doxorubicin-loaded nanoparticles. J Pharm Sci 1982;71:790-2.
204 CHAPTER 7: CONCLUSIONS
Chapter 7: Conclusions and future directions
7.1 Scope of this work
In the past few decades, the field of nanoparticle drug delivery for cancer therapy has grown. Nanoparticles can be surface modified to improve both active and passive targeting of and large payload delivery to diseased cells while avoiding healthy tissues.
There are seven FDA-approved nanoparticle formulations for cancer therapy, and many formulations of various compositions are in clinical and preclinical studies. Most of the materials under consideration today have been of spherical shape, simply because of chemical synthesis restraints. Despite this, a growing body of data supports that high aspect ratio nanoparticles, such as filomicelles, PRINT nanoparticles, carbon nanotubes, and gold nanorods, exhibit improved in vivo properties for cancer treatment.
Advantageous properties include reduced non-specific uptake by phagocytic cells, increased ligand display and therefore increased target-specificity, improved margination toward the vessel wall and tumor tissue, as well as increased tumor penetration [1-9].
To gain a deeper understanding of the application of high aspect ratio nanoparticles designed for cancer therapy, I developed and studied two high aspect ratio plant virus-based nanoparticle systems for cancer therapy: tobacco mosaic virus (TMV) as a model system for a soft matter, but rigid high aspect ratio nanoparticle and potato virus X (PVX) as a novel filamentous material.
In my initial studies, I investigated the importance of shape, using TMV as a high aspect ratio nanoparticle and cowpea mosaic virus (CPMV) as a sphere-like model system (CPMV has a pseudoT=3 icosahedral symmetry and is as such a sphere-like
205 CHAPTER 7: CONCLUSIONS nanoparticle). I evaluated the diffusion rates of TMV versus CPMV in a 3D tumor model to better understand the effect of shape on diffusion (Chapter 2). I also used TMV in a proof-of-concept study to load cationic photosensitizers via electrostatic interactions towards use as a cancer therapy (Chapter 3).
The central body of my thesis focused on the development of PVX as a cancer therapy. Previous studies with VLPs, including those used for gene delivery, have been limited by the presence of neutralizing antibodies after multiple administrations [10-12].
In fact, immune surveillance is a major barrier for many protein therapeutics and nanoparticle systems, both organic and inorganic. Therefore, I first investigated the formulation of stealth PVX particles using polyethylene glycol (PEG), a polymer frequently used to improve solubility, stability, and biocompatibility of pharmaceutics of protein therapies [13-16]. In particular, I investigated the impact of the PEG chains of different molecular weight and conformation on the stealth properties of PVX (Chapter
4). Once the stealth formulation was established, I went on to active targeting strategies; specifically the targeting of cancer cells overexpressing the epidermal growth factor receptor (EGFR). I modified PVX with an EGFR-specific peptide ligand and achieved targeting of EGFR+ cancer cells (Chapter 5). Lastly, I evaluated a chemotherapy-loaded
PVX for use as a traditional drug delivery vehicle and chemo-immuno combination therapy. Specifically, I loaded PVX with doxorubicin, a commonly used chemotherapy that has also been approved for use as a liposomal formulation. I evaluated the formulation, both in in vitro and in vivo models of cancer (Chapter 6).
The results described in this dissertation contribute to the field of cancer therapy by enhancing the understanding of high aspect ratio viral nanoparticles, and describing
206 CHAPTER 7: CONCLUSIONS the development of a filamentous VNP for cancer therapy. Summary of the findings of the thesis, their implications in the field of cancer therapy, and considerations for continuation of this work are discussed in the following sections.
7.2 Shape matters: tobacco mosaic virus as a model high aspect ratio nanoparticle
The field of nanoparticle delivery has focused predominantly on spherical nanoparticles, although some work has investigated high aspect ratio nanoparticles, such as gold nanorods and carbon nanotubes. As a new direction, in Chapters 2 and 3, I studied the diffusion behavior of the high aspect ratio nanoparticle TMV using a 3D spheroid model.
Three dimensional in vitro tumor spheroids are a model that spans the gap between two dimensional tissue culture and whole animal models. Cells grown in two- dimensional tissue culture exhibit different phenotypes and interactions (cell-cell and cell-matrix), due to the lack of natural 3D architecture [17, 18]. However, whole animal models are not well-controlled environments to dissect targeted questions about, for example, nanoparticle diffusion rates, which makes them unfeasible for testing every potential therapy. 3D tumor spheroids offer a compromise of a controllable system, which more accurately mimics the in vivo environment.
Various spheroid systems have been reported. These include single cell type cultures [18-22] or multicellular spheroids containing tumor and normal cells [23], which can be formed using spontaneous cell aggregation or on a preformed extracellular matrix
(ECM) scaffold. As an alternate strategy, acellular spheroid models can be constructed
207 CHAPTER 7: CONCLUSIONS using only ECM components (e.g. agarose, collagen, etc.) [21, 24]; such a model provides an ideal testbed for a first study of novel therapeutics or nanoparticle systems when studying the diffusive behavior in the ECM. Unlike cellular/multicellular spheroids, this system is a more controlled model to understand interactions of nanoparticles with the scaffold of the tumor microenvironment, without confounding factors such as cell uptake and the presence of small molecules. Here, I investigated TMV in an acellular spheroid model to better understand the diffusion of a high aspect ratio viral nanoparticle in a porous microenvironment (Chapter 2).
TMV is a well-characterized virus, having been studied for numerous biomedical applications, including imaging, drug delivery, and tissue engineering [25-27]. Prior to my work in Chapter 2, however, tissue penetration rates of TMV had not been compared to a spherical virus to elucidate the importance of carrier shape. I compared TMV versus
CPMV and demonstrated that TMV had a more rapid diffusion rate into the model. These results demonstrated that macroscopic diffusion rates determined for particles in solution may not accurately represent diffusion patterns within a porous environment, such as the tumor microenvironment. Additionally, since this study, the experimental model was further expanded using a mathematical modeling to include the impact of altering aspect ratio, surface modifications, spheroid size, and cellular density [28]. Together the experimental and theoretical models provide a framework to gain understanding of the diffusion rates of TMV-based nanoparticles as a function of aspect ratio and surface chemistry. My data support the advantageous nature of TMV compared to spherical viruses; rapid initial diffusion, as demonstrated by the high aspect ratio TMV formulation
208 CHAPTER 7: CONCLUSIONS into the tumor interstitium is thought to be advantageous to avoid washout of recently accumulated nanoparticles.
At the same time, to develop TMV as a cancer therapeutic, in Chapter 3, I investigated the loading of TMV with a photosensitizer using electrostatic interactions.
Prior to my work here, VNPs used for photodynamic therapy (PDT) had been of an icosahedral nature [29-31] and traditional drug loading had been carried out through covalent conjugations. I was interested to test if a high aspect ratio VNP could be also used for photosensitizer delivery without chemical conjugation using a one-step protocol; this would be beneficial in particular for translational studies and scaled-up manufacture.
Our lab had previously shown that glutamic acids on the interior channel of TMV could be targeted for the electrostatic loading of platinum-based chemotherapies, which had an overall charge of 2+ [25]. Here, I investigated a cationic zinc porphyrin-based photosensitizer (Zn-EpPor), which has an overall charge of 3+. I confirmed that the addition of the positive charge did not impact its ability to be loaded onto the interior channel of TMV via electrostatic interactions. On the contrary, the addition of this positive charge significantly improved stability versus a 2+ species: TMV loaded with a platinum-based chemotherapy was stable for less than one day, meaning the drug would diffuse out of the particle and release within one day, while TMV loaded with Zn-EpPor was stable over one month. Moving towards translation, improved stability is important, as producing the VNP fresh for every treatment is neither feasible nor efficient. In future studies it will be interesting to dissect the underlying features for achieving long-term stability in more depth: at this point it is not verified whether the enhanced stability is a charge effect or whether this has to do with the different chemical structure of the therapy
209 CHAPTER 7: CONCLUSIONS employed. For example, a future investigation could study the loading and stability of porphyrin complexes with 1+, 2+, 3+, and 4+ charges.
I also showed that Zn-EpPor-loaded TMV enhanced therapy uptake into cells compared to using free Zn-EpPor, which correlated with improved cell killing efficacy.
Overall, I demonstrated that interior carboxylic acids could be targeted for loading by positively-charged drugs other than the platinum-based molecules that had been initially studied and that these drugs retain their efficacy, thereby opening up this method of TMV modification for a range of other molecules.
Due to its previous extensive characterization, TMV was an ideal candidate to use as a model high aspect ratio nanoparticle for investigation of in vitro diffusion and PDT delivery for cancer therapy. Our lab has also shown that TMV is unique in that it can be modified to have different aspect ratios, as well as thermally transitioned from a rod to a sphere [8, 32]. Altering high aspect ratio and shape results in distinct biodistribution patterns in healthy and tumor-bearing mice, again attesting to the importance of nanoparticle shape in nanomedicine [8, 32]. In healthy mice, spherical nanoparticles from
TMV coat proteins accumulate in the liver and spleen at early time points (4 h), but are then rapidly cleared from the body. In contrast, high aspect ratio TMV rods also accumulate in the liver and spleen, but are retained out to 24 hours [33]. Towards a cancer therapy, the biodistribution of TMV rods of different aspect ratios with different surface coatings were investigated in an immunodeficient mouse model of colon cancer
[8]. Interestingly in this model, PEGylated short TMV particles (TS; AR = 3.5) exhibit higher accumulation in the tumor compared to medium (TM; AR = 7) and long (TL; AR
= 16.5) TMV particles after 24 h. However, when TMV rods were targeted using RGD,
210 CHAPTER 7: CONCLUSIONS
TM particles had the highest accumulation after 24 h, indicating that a balance must be reached between stealth coating for passive tumor targeting, active targeting, and aspect ratio for optimal tumor accumulation [8]. Experimental modeling using TMV has also investigated the impact of aspect ratio, stealth coatings, and active targeting on tumor penetration in a 3D spheroid. From this model, it was predicted that TS stealth filaments have improved penetration versus TM or TL stealth filaments, while targeted TL filaments have decreased penetration compared to stealth TL filaments due to rapid cell uptake [28].
Additionally, murine tumor models have different tumor characteristics, including cell fraction and permeability of vasculature, which can impact nanoparticle accumulation. In a separate study evaluating TMV for cancer therapy, uncoated wild-type
TMV was reported to have good tumor homing in the MDA-MB-231 model of triple negative breast cancer. TMV tumor homing led to efficient delivery of a platinum-based drug candidate and the subsequent improved survival versus free drug alone [25].
In addition to the work shown here (Chapter 3), TMV has recently been demonstrated to deliver platinum drugs targeting breast cancer therapy [25]. In vivo, platinum therapy-loaded TMV had improved tumor efficacy compared to free drug in a model of triple negative breast cancer due. The increased efficacy was explained by increased delivery of the platinum-based drug at the site of disease [25]. In an alternate approach, doxorubicin (DOX) was attached to TMV and its spherical nanoparticle counterparts; these formulations were shown to be effective in vitro as demonstrated using HER-2-positive and triple negative breast cancer cells [34]. The in vivo performance of the materials is being currently investigated.
211 CHAPTER 7: CONCLUSIONS
From these preliminary studies, it is evident that TMV is a promising platform for cancer drug delivery. However, moving towards translation, it is important to further characterize this platform. In addition to standard safety concerns with any nanoparticle- based system, there are concerns that are unique to TMV as a plant virus-based system.
The studies discussed here evaluated TMV in an immunocompromised mouse model of cancer. One potential barrier to translation would be the development of neutralizing antibodies after repeat administration, as with previously reported VLP-based gene therapy systems [10-12]. These antibodies can lead to rapid clearance of the VNP, thereby reducing its efficacy. Repeat injections of a VNP has been shown to lead to increased levels of serum IgM, and then IgG [35]. Using intravital imaging, it was also shown that repeat injections led to formation of VNP clusters throughout the vasculature, characteristic of the formation of VNP-IgM immune complexes [35]. To overcome this barrier, shielding strategies have been investigated to produce “stealth” particles that are not recognized by the immune system. Surface coatings investigated include hydrophilic polymers such as PEG or native proteins such as albumin [36].
Further, while TMV does not infect mammals, it has a range of agricultural hosts
[37]. Further studies investigating the infectivity of TMV cleared from the body will be necessary to determine environmental safety before it moves towards clinical trials.
Alternative strategies could employ the development of disarmed, non-infectious particles or inactivated particles. It is clear that more research and in particular safety studies in non-human primates need to be conducted to launch the technology for clinical investigation. However this holds true for any novel platform technology. The unique shape and engineering design space of TMV offering a high aspect ratio with chemically
212 CHAPTER 7: CONCLUSIONS distinct interior versus exterior environments make it an intriguing platform for further development and investigation for cancer therapy.
7.3 Potato virus X: a filamentous plant virus for cancer therapy
TMV and most synthetic high aspect ratio nanoparticles that have been investigated for biomedical applications are not flexible materials. While there has been some work described with these nanoparticles, the body of work done on filamentous nanoparticles is even more limited. Filomicelles have flexibility but are in the micron size range and cannot be produced with low polydispersity [38-42]. More recently, particle replication in non-wetting templates (PRINT) nanoparticles have been investigated.
However, these cannot yet be synthesized with feature sizes below 50 nm [43]. The filamentous PVX platform technology thus offers a complementary approach to the synthetic nanotechnologies. In Chapters 4-6 I describe my efforts to investigate the PVX platform technology for applications in cancer therapy.
As discussed previously, one of the barriers to using virus-based nanoparticles as drug carriers in clinical studies is the production of neutralizing antibodies against the
VNP after repeat injections. The addition of the hydrophilic polymer PEG is a common strategy to produce “stealth” nanoparticles, which exhibit enhanced circulation time as a result of decreased recognition by immune and non-target cells [13, 14]. Previously, it was shown that modification of the icosahedral virus CPMV with linear PEG chains had decreased antibody production after two injections, over 4 months [44]. This data may indicate that the PEGylated formulation has decreased interaction with immune cells, therefore reducing the adaptive immune response against the carrier. Indeed, a different 213 CHAPTER 7: CONCLUSIONS study showed reduced cell interaction of PEGylated CPMV versus native CPMV, and that the reduction in cell uptake was more profound with increasing PEG chains length
(PEG 1K and 2K were compared in this study) [45]. Here, I investigated the impact of the molecular weight and conformation of the conjugated PEG chains towards the construction of a PVX-based stealth filament (Chapter 4).
In Chapter 4, I demonstrated the first extensive characterization of the in vitro and in vivo properties of PVX as a function of different PEG coatings. Traditionally, therapeutic nanoparticles have been delivered intravenously and are given over multiple doses for the greatest efficacy. However, like other biologics, immune surveillance could be a barrier to success. An intravital microscopy study combined with immunology work- up recently carried out by our laboratory showed that repeat administration of PVX leads to IgM, then IgG production. While the presence of IgG did not affect the circulation time, high IgM titers lead to clustering and rapid clearance of the particles [35]. Further, early uses of viruses for gene therapy were halted after fatalities linked to immune responses against viral carriers [11]. Since then, no virus-based delivery system has been approved for intravenous injection. Therefore, establishment of effective shielding strategies is an important goal, not just for the further development of PVX, but the techniques could be adapted to other plant viral systems or gene delivery vectors. Using various PVX-PEG formulations, I showed that although antibodies were produced against the PVX carrier after multiple injections of PEG-PVX stealth filaments, these antibodies were unable to recognize stealth filaments. All stealth coatings tested showed a decrease in antibody recognition, with no significant difference between the different
214 CHAPTER 7: CONCLUSIONS
PEGs. Therefore my data indicate that PEGylated particles may indeed be a viable option as a delivery vehicle for intravenous injections.
Interestingly, I also found that the pharmacokinetics of PVX could be tailored using different PEG coatings: branched and high molecular weight PEGs had long circulation times, while smaller molecular weight, linear PEGs had shorter circulation times. While there was no difference in antibody recognition between the different PEG tested, branched PEG did significantly extend circulation time versus linear PEG chains of any molecular weight. Branched PEG is synthesized with four NHS groups and based on the spacing between lysine residues on PVX it is possible that a single PEG can crosslink PVX coat proteins within the same particle. This additional attachment may improve shielding in circulation, since it limits PEG movement, leading to the extended circulation times observed. Alternately, branched PEG may impact the stiffness of the
PVX particle due to intraparticle cross-linking. Persistence length is a mechanical property that quantifies the stiffness of a polymer. Preliminary data indicate that PVX- branched PEG has a persistence length two times that of PVX modified with linear PEG chains.
Moving forward, larger or branched PEGs will be better for longer circulation times, to improve passive tumor targeting via the enhanced permeability and retention
(EPR) effect [46]. However, it is important to note that, although branched PEG attachment extends circulation time to approximately 18 h, this is still a shorter circulation time than those previously reported for synthetic nanoparticles [42, 47, 48].
While improved circulation time is advantageous for accumulation via passive targeting, it also leads to increased accumulation within non-target organs [1, 49-55]. Synthetic
215 CHAPTER 7: CONCLUSIONS particles such as gold, polymeric nanoparticles, iron oxide, and carbon nanotubes have been reported as persisting in the liver for months after injection, leading to potential long-term adverse effects [1, 49, 53-55]. Comparatively, PVX is cleared from the organs more rapidly, making it a safer alternative.
Stealth filaments can also be tailored for applications that are more conducive to shorter circulation times, such as receptor-targeted contrast agents, by using short, linear
PEG chains [56, 57]. The attachment of shorter PEG chains led to decreased circulation times versus large or branched PEG chains, but still resulted in effective shielding from the immune system in vitro and in vivo: all stealth filaments, regardless of PEG coating, exhibited decreased interaction with immune cells in vitro and decreased recognition by
PVX-specific antibodies.
While the addition of PEG was originally aimed at the development of a stealth filament, I also found that different PEG coatings led to altered cytokine production, indicating that immunogenicity can be altered for different applications. In particular, stealth filaments constructed using branched PEG lead to increased IL-12 production in bone marrow derived dendritic cells, compared to either native PVX or stealth filaments with linear PEG. This is particularly of interest moving towards using PVX for chemo- immunotherapy (Chapter 6). IL-12 is known to augment Th1 and cytotoxic T lymphocyte responses, which are both important in cancer immunotherapy [58-61]. It is currently being investigated as an adjuvant for vaccines, as well as an immunostimulatory molecule for cancer immunotherapy [59, 60, 62, 63]. In preclinical studies, IL-12 has exhibited a strong anti-tumor response against B16F10, as well as reducing liver metastases after an intrasplenic injection of C26 colon cancer [61, 64, 65].
216 CHAPTER 7: CONCLUSIONS
Overall, in Chapter 4, I determined that stealth filaments of PVX decrease recognition by PVX-specific antibodies. Additionally, altering PEG coatings leads to the ability to tailor the in vivo and in vitro properties of this platform for a variety of applications, including drug delivery, specific immune stimulation, and receptor-targeted contrast agents.
In Chapter 5, I sought to develop PVX for active targeting of aggressive cancer cells. Although the EPR effect is hypothesized to improve passive targeting, it is somewhat controversial due to the heterogeneity of tumors and is not applicable for blood cancers or metastatic sites [46, 66, 67]. Here, I investigated active targeting of aggressive cancer cells using the small peptide GE11, which selectively targets the epidermal growth factor receptor [68]. EGFR is overexpressed on a variety of cancers and is associated with poor prognosis [69]. The monoclonal antibody Cetuximab, which specifically targets EGFR, is currently approved for the treatment of head and neck cancer. However, multiple injections of Cetuximab can lead to resistance over time [70]. Other EGFR- targeting strategies include the full-length EGF or other peptide ligands, typically attached to nanoparticles [71-74]. Here, I showed that PVX could be modified with a targeting peptide using a bifunctional linker, and that PVX-GE11 filaments selectively targeted EGFR+ cells. Importantly, targeted filaments can also selectively target EGFR+ cells in a co-culture of cancer cells and macrophages. Moving towards in vivo applications, the ability of targeted filaments to differentiate between cancer cells and immune cells will be important because the tumor microenvironment is complicated and contains multiple cell types. Using this approach, different targeting ligands can be
217 CHAPTER 7: CONCLUSIONS substituted for GE11 to target receptors on different cancers [75], or for other disease systems, such as cardiovascular disease [26].
One limitation for nanoparticles in general is difficulty in producing large amounts of particles with low variability [76]. As a protein-based nanoparticle, PVX is genetically encoded and the sequence can be genetically modified to introduce functionalities onto the surface of PVX. Our lab previously showed that the N-terminus of PVX could be modified with fluorescent proteins that are expressed on the coat protein.
These coat proteins stably assemble into VNPs [77]. This technique can also be used to scale up production of monodisperse PVX particles displaying small peptide targeting ligands on the surface. In addition to improving scalability, this technique improves the versatility of the PVX platform, by making the lysine residue available for other modifications.
Moving toward therapeutic applications, in Chapter 6, I investigated a PVX-
DOX conjugate for cancer therapy. Doxorubicin (DOX) is a chemotherapy approved for clinical use in a variety of cancers. It has been attached to many different nanoparticles, including synthetic particles and virus-based nanoparticles [78-84]. A liposomal formulation of DOX, Doxil, is clinically available. Other DOX-loaded synthetic nanoparticles are in clinical trials [85, 86]. DOX-loaded VNPs are still in preclinical studies [78, 79, 87-89].
Prior to the work presented here, PVX modifications were done via genetic engineering or by targeting the solvent-exposed lysine residues. Here, I demonstrated a new way to attach molecules to PVX: hydrophobic interactions. Specifically, the ring structures of DOX make it hydrophobic, and I hypothesize that they are interacting via π- 218 CHAPTER 7: CONCLUSIONS
π stacking with hydrophobic residues in the PVX coat protein [90]. π-π stacking has previously been reported between DOX molecules [91, 92]. Through this new loading technique, I achieved approximately 800 DOX per PVX molecule. This correlates to a loading capacity (loading capacity = massDOX/massPVX x 100) of 1.3%. Other VNP-based systems have reported similar loading capacities of 14.1% [93], 6.7% [79], 1.3% [78],
3.1% [89], and 7.5% [87]. It is important to note that higher loading was seen for VNPs loaded via infusion versus chemical conjugation. In comparison, synthetic particles are able to load a higher amount of DOX/particles. Polymer-based nanoparticles have been reported to have 47% (w/w) loading capacity [84], while liposomes, such as DOXIL, have been reported to have loading capacities as high as 48,000 DOX per 100 nm liposome (LC = 30%) [94].
I demonstrated that PVX-DOX could be used as either drug delivery vehicle or combination chemo-immunotherapy. These studies are the first to develop PVX as a cancer therapy, as well as one of the first in which a filamentous nanoparticle modified with chemotherapy drugs is used for cancer therapy. Importantly, DOX retains efficacy after attachment to PVX in a panel of cell lines. However, as with most nanoparticle formulations (both synthetic and virus-based) [79, 87, 95], the efficacy of PVX-DOX is decreased compared to DOX alone. Despite this, the IC50 value for PVX-DOX is comparable to that reported for DOXIL [96], indicating that the decrease in efficacy may not be a barrier to translation.
The reduced efficacy can be due to a variety of factors, including decreased uptake, delayed release from the particle, or accumulation of drug in undesired cell compartments that may render the drug inactive. In the case of PVX-DOX, I found that
219 CHAPTER 7: CONCLUSIONS the 5 times reduced efficacy correlated with decreased uptake of PVX-DOX compared to free DOX. However, monolayer cell culture is not fully predictive of how PVX-DOX will compare to free DOX in an in vivo tumor model. One of the largest limitations against the administration of free drug in the clinic is the side effects seen when it interacts with off-target organs [97, 98]. Nanoparticles are known to decrease these side effects, to improve the safety profile of free drugs [99, 100]. Our lab has also shown that
PVX is more advantageous for tumor homing versus a spherical VNP due to its filamentous nature, leading to higher accumulation of cargo than its spherical counterpart
[101]. It is important to note that PVX-DOX particles did not include any other surface modifications and can still be further optimized to improve cell and tumor killing efficacy.
Targeting ligands can be added to improve cancer cell specific uptake. EGFR-targeting was used in Chapter 5, but there are a variety of other targeting ligand options that have been used on nanoparticles, including RGD (targets αvβ3 integrins) [75, 102], folic acid
(targets folate receptor) [103], or transferrin (targets transferrin receptor) [104].
Additionally, stealth coatings, such as PEG used in Chapter 4 can be used to extend circulation to improve tumor accumulation.
As cancer therapy has progressed, it has become evident that in most cases, monotherapies will not be sufficient as a curative option. Recently, combination therapies of chemotherapies and immunotherapies have been investigated to improve outcomes
[105, 106]. Some classes of chemotherapy, such as anthracyclines, lead to immunogenic cell death, which is characterized by cell death concurrent with the exposure of calreticulin on the surface of dying cells, the release of high-mobility group box 1, and release of ATP. These molecules stimulate dendritic cells to improve antigen presentation
220 CHAPTER 7: CONCLUSIONS to the immune system [107, 108]. Therefore the combination of anthracyclines and immunotherapies has synergistic efficacy.
Other classes of chemotherapy result in immunogenic modulation, in which nonlethal doses of chemotherapy results in altered cell phenotypes that are more susceptible to cytotoxic T lymphocyte killing [109]. Therefore, combining chemotherapy and immunotherapy is a logical option for combination therapies. Indeed, preclinical research indicates that nanoparticle-based platforms that co-deliver both chemotherapeutic drugs (e.g. DOX, paclitaxel) and immunostimulatory molecules (e.g.
CpGs, LPS) exhibited higher antitumor activity compared to either monotherapy alone
[105, 106].
Towards the development of a chemo-immuno combination therapy, I determined that PVX and DOX work synergistically to decrease tumor growth rate and extend survival time in a murine model of melanoma. Interestingly the effect was only seen with the non-coupled PVX+DOX sample, indicating that coupling PVX and DOX together
(PVX-DOX) is inhibiting the synergistic effect. This could be due to the to decreased uptake of DOX after attachment to PVX compared to free DOX; this was observed in the in vitro studies. Decreased uptake of DOX would lead to a limited efficacious response.
Another possibility is that PVX-delivered DOX kills immune cells and not cancer cells.
Within the tumor microenvironment, there are many cell types present, including cancer cells and immune cells. The ultimate goal of PVX-DOX as a chemo-immunotherapy is to kill cancer cells using DOX, while simultaneously activating immune cells, such as neutrophils. For PVX-DOX, the chemotherapy and immunotherapy are coupled, and therefore taken up by the same cell. Preliminary histology data indicate that PVX
221 CHAPTER 7: CONCLUSIONS localized with macrophages not cancer cells, which may explain why synergy of PVX-
DOX was not observed but achieved using PVX+DOX. This is also consistent with my cell uptake studies performed in co-cultures (Chapter 5). I showed the non-targeted PVX
(such as the PVX-DOX sample) is preferentially taken up by macrophages in a co-culture of macrophages and cancer cells due to different mechanisms of uptake: macrophages take up particles via phagocytosis, while cancer cells take up particles via endocytosis.
Since immune cells take up non-targeted PVX-DOX preferentially, the cells needed for immunotherapy are being killed by chemotherapy before they can stimulate the immune system. At the same time, cancer cells are not taking up more DOX due to decreased uptake after attachment to PVX, as previous discussed. However, I expect that when
PVX and DOX are delivered as a non-coupled injection (PVX+DOX), cancer cells take up free DOX, while immune cells take up PVX, leading to a synergistic effect and improved efficacy.
Moving forward, it will be important to understand the immune activation of the treatment groups investigated in Chapter 6, as well as how PVX-DOX interacts with the tumor microenvironment and tumor cells (i.e. cell uptake, DOX release) to design an optimal nanoparticle. Towards the design of a translationally relevant therapy, it will also be important to investigate alternate routes of administration. Clinically, DOX is administered intravenously, while virus-based therapies, such as talimogene laherparepvec, are administered intratumorally [110, 111]. Future studies should investigate the administration of PVX intratumorally, combined with an intravenous injection of free DOX.
222 CHAPTER 7: CONCLUSIONS
In summary, in the studies in Chapters 4-6 I designed and characterized the filamentous PVX platform. From these studies, it is apparent that PVX offers a promising platform for cancer therapy. PVX can be tailored towards drug delivery or immunotherapy by altering surface modifications, making it a versatile platform.
7.4 Future directions
The work summarized contributes to the field of plant-based VNPs for cancer therapy. Here I evaluated the use of high aspect ratio plant virus-based nanoparticles for cancer therapy, with specific focus on TMV and PVX. TMV was used as a model nanoparticle for evaluating shape effects, while PVX was modified with stealth coatings, targeting ligands, and chemotherapeutics for development of a cancer therapy. These studies are a promising first step towards designing a virus-based nanoparticle for cancer therapy, but barriers still exist. As previously discussed, a limitation of VNPs is the production of neutralizing antibodies after repeat injections. Here, I determined that PEG coating decreases recognition by anti-PVX antibodies. However, PEG itself is limited by the presence of anti-PEG antibodies because it is a common polymer found in many biomaterial formulations, including nanoparticles and cosmetics [112-114]. In fact, recent clinical data indicate prevalence of anti-PEG antibodies in the human population and a clinical study of a PEGylated aptamer was stopped after at least one patient experienced life-threatening conditions, which were linked to high anti-PEG titers [112]. Thus, a paradigm shift is needed. For example, nanoparticles could be formulated with less commonly-used polymers to avoid pre-exposure, such as poly(2-oxazoline) [115-117].
223 CHAPTER 7: CONCLUSIONS
Other strategies include the incorporation of “self” peptides and other camouflage coatings, such as CD47 [118] or albumin [36].
Another consideration moving forward is that cancer is a heterogeneous disease; no two cancers are identical. Murine models of cancer are controlled and predictable compared to human disease, so a promising nanoparticle should not only be effective in treating one cancer, it should be a platform that can be easily adapted to address other cancers. This work discussed specific molecules and moieties for therapy. Looking towards the future, each modification shown here for these VNPs can be replaced with others to improve and tailor therapies: cationic photosensitizers can be replaced with other cationic drugs, alternative shielding strategies can be investigated, different targeting peptides can be used, or other chemotherapeutics or immunotherapies can be attached. The steps taken here have evaluated high aspect ratio viral nanoparticles as promising platforms for cancer therapy. The work towards understanding and evaluating these platforms has opened the door for the development of improved and effective high aspect ratio virus-based cancer therapies.
7.5 Works cited in this chapter
1. Arnida, Janat-Amsbury MM, Ray A, Peterson CM, Ghandehari H. Geometry and surface characteristics of gold nanoparticles influence their biodistribution and uptake by macrophages. Eur J Pharm Biopharm 2011;77:417-23.
2. Barua S, Yoo JW, Kolhar P, Wakankar A, Gokarn YR, Mitragotri S. Particle shape enhances specificity of antibody-displaying nanoparticles. Proc Natl Acad Sci U S A 2013;110:3270-5.
3. Chauhan VP, Popovic Z, Chen O, Cui J, Fukumura D, Bawendi MG, et al. Fluorescent nanorods and nanospheres for real-time in vivo probing of
224 CHAPTER 7: CONCLUSIONS
nanoparticle shape-dependent tumor penetration. Angew Chem Int Ed Engl 2011;50:11417-20.
4. Decuzzi P, Ferrari M. The adhesive strength of non-spherical particles mediated by specific interactions. Biomaterials 2006;27:5307-14.
5. Gentile F, Chiappini C, Fine D, Bhavane RC, Peluccio MS, Cheng MM, et al. The effect of shape on the margination dynamics of non-neutrally buoyant particles in two-dimensional shear flows. J Biomech 2008;41:2312-8.
6. Lee SY, Ferrari M, Decuzzi P. Shaping nano-/micro-particles for enhanced vascular interaction in laminar flows. Nanotechnology 2009;20.
7. Sharma G, Valenta DT, Altman Y, Harvey S, Xie H, Mitragotri S, et al. Polymer particle shape independently influences binding and internalization by macrophages. J Control Release 2010;147:408-12.
8. Shukla S, Eber FJ, Nagarajan AS, DiFranco NA, Schmidt N, Wen AM, et al. The Impact of Aspect Ratio on the Biodistribution and Tumor Homing of Rigid Soft- Matter Nanorods. Adv Healthc Mater 2015;4:874-82.
9. Toy R, Hayden E, Shoup C, Baskaran H, Karathanasis E. The effects of particle size, density and shape on margination of nanoparticles in microcirculation. Nanotechnology 2011;22:115101.
10. Kafri T, Morgan D, Krahl T, Sarvetnick N, Sherman L, Verma I. Cellular immune response to adenoviral vector infected cells does not require de novo viral gene expression: implications for gene therapy. Proc Natl Acad Sci U S A 1998;95:11377-82.
11. Thomas CE, Ehrhardt A, Kay MA. Progress and problems with the use of viral vectors for gene therapy. Nat Rev Genet 2003;4:346-58.
12. Thomas CE, Schiedner G, Kochanek S, Castro MG, Lowenstein PR. Preexisting antiadenoviral immunity is not a barrier to efficient and stable transduction of the brain, mediated by novel high-capacity adenovirus vectors. Hum Gene Ther 2001;12:839-46.
13. Allen TM, Hansen C, Martin F, Redemann C, Yau-Young A. Liposomes containing synthetic lipid derivatives of poly(ethylene glycol) show prolonged circulation half-lives in vivo. Biochim Biophys Acta 1991;1066:29-36.
14. Klibanov AL, Maruyama K, Torchilin VP, Huang L. Amphipathic polyethyleneglycols effectively prolong the circulation time of liposomes. FEBS Lett 1990;268:235-7.
15. Larson N, Ghandehari H. Polymeric conjugates for drug delivery. Chem Mater 2012;24:840-53.
225 CHAPTER 7: CONCLUSIONS
16. Liu T, Yuan X, Jia T, Liu C, Ni Z, Qin Z, et al. Polymeric prodrug of bufalin for increasing solubility and stability: Synthesis and anticancer study in vitro and in vivo. Int J Pharm 2016;506:382-93.
17. Kim JB, Stein R, O'Hare MJ. Three-dimensional in vitro tissue culture models of breast cancer-- a review. Breast Cancer Res Treat 2004;85:281-91.
18. Yamada KM, Cukierman E. Modeling tissue morphogenesis and cancer in 3D. Cell 2007;130:601-10.
19. Godugu C, Patel AR, Desai U, Andey T, Sams A, Singh M. AlgiMatrix based 3D cell culture system as an in-vitro tumor model for anticancer studies. PLoS One 2013;8:e53708.
20. Shin CS, Kwak B, Han B, Park K. Development of an in vitro 3D tumor model to study therapeutic efficiency of an anticancer drug. Mol Pharm 2013;10:2167-75.
21. Liang Y, Jeong J, DeVolder RJ, Cha C, Wang F, Tong YW, et al. A cell- instructive hydrogel to regulate malignancy of 3D tumor spheroids with matrix rigidity. Biomaterials 2011;32:9308-15.
22. Chu JH, Yu S, Hayward SW, Chan FL. Development of a three-dimensional culture model of prostatic epithelial cells and its use for the study of epithelial- mesenchymal transition and inhibition of PI3K pathway in prostate cancer. Prostate 2009;69:428-42.
23. Lang SH, Sharrard RM, Stark M, Villette JM, Maitland NJ. Prostate epithelial cell lines form spheroids with evidence of glandular differentiation in three- dimensional Matrigel cultures. Br J Cancer 2001;85:590-9.
24. Lu WD, Zhang L, Wu CL, Liu ZG, Lei GY, Liu J, et al. Development of an acellular tumor extracellular matrix as a three-dimensional scaffold for tumor engineering. PLoS One 2014;9:e103672.
25. Czapar AE, Zheng YR, Riddell IA, Shukla S, Awuah SG, Lippard SJ, et al. Tobacco Mosaic Virus Delivery of Phenanthriplatin for Cancer therapy. ACS Nano 2016;10:4119-26.
26. Bruckman MA, Jiang K, Simpson EJ, Randolph LN, Luyt LG, Yu X, et al. Dual- modal magnetic resonance and fluorescence imaging of atherosclerotic plaques in vivo using VCAM-1 targeted tobacco mosaic virus. Nano Lett 2014;14:1551-8.
27. Wu Y, Feng S, Zan X, Lin Y, Wang Q. Aligned Electroactive TMV Nanofibers as Enabling Scaffold for Neural Tissue Engineering. Biomacromolecules 2015;16:3466-72.
226 CHAPTER 7: CONCLUSIONS
28. Chariou PL, Lee KL, Pokorski JK, Saidel GM, Steinmetz NF. Diffusion and Uptake of Tobacco Mosaic Virus as Therapeutic Carrier in Tumor Tissue: Effect of Nanoparticle Aspect Ratio. J Phys Chem B 2016.
29. Wen AM, Ryan MJ, Yang AC, Breitenkamp K, Pokorski JK, Steinmetz NF. Photodynamic activity of viral nanoparticles conjugated with C60. Chem Commun (Camb) 2012;48:9044-6.
30. Suci PA, Varpness Z, Gillitzer E, Douglas T, Young M. Targeting and photodynamic killing of a microbial pathogen using protein cage architectures functionalized with a photosensitizer. Langmuir 2007;23:12280-6.
31. Stephanopoulos N, Tong GJ, Hsiao SC, Francis MB. Dual-surface modified virus capsids for targeted delivery of photodynamic agents to cancer cells. ACS Nano 2010;4:6014-20.
32. Bruckman MA, Hern S, Jiang K, Flask CA, Yu X, Steinmetz NF. Tobacco mosaic virus rods and spheres as supramolecular high-relaxivity MRI contrast agents. J Mater Chem B Mater Biol Med 2013;1:1482-90.
33. Bruckman MA, Randolph LN, VanMeter A, Hern S, Shoffstall AJ, Taurog RE, et al. Biodistribution, pharmacokinetics, and blood compatibility of native and PEGylated tobacco mosaic virus nano-rods and -spheres in mice. Virology 2014;449:163-73.
34. Bruckman MA, Czapar AE, VanMeter A, Randolph LN, Steinmetz NF. Tobacco mosaic virus-based protein nanoparticles and nanorods for chemotherapy delivery targeting breast cancer. J Control Release 2016;231:103-13.
35. Shukla S, Dorand RD, Myers JT, Woods SE, Gulati NM, Stewart PL, et al. Multiple Administrations of Viral Nanoparticles Alter in Vivo Behavior-Insights from Intravital Microscopy. Acs Biomaterials-Science & Engineering 2016;2:829-37.
36. Pitek AS, Jameson SA, Veliz FA, Shukla S, Steinmetz NF. Serum albumin 'camouflage' of plant virus based nanoparticles prevents their antibody recognition and enhances pharmacokinetics. Biomaterials 2016;89:89-97.
37. Holmes FO. A Comparison of the Experimental Host Ranges of Tobacco-Etch and Tobacco-Mosaic Viruses. Phytopathology 1946;36:643-59.
38. Li Y, Huang Y, Wang Z, Carniato F, Xie Y, Patterson JP, et al. Polycatechol Nanoparticle MRI Contrast Agents. Small 2016;12:668-77.
39. Oltra NS, Swift J, Mahmud A, Rajagopal K, Loverde SM, Discher DE. Filomicelles in nanomedicine - from flexible, fragmentable, and ligand-targetable drug carrier designs to combination therapy for brain tumors. Journal of Materials Chemistry B 2013;1:5177-85. 227 CHAPTER 7: CONCLUSIONS
40. Wang Z, Li Y, Huang Y, Thompson MP, LeGuyader CL, Sahu S, et al. Enzyme- regulated topology of a cyclic peptide brush polymer for tuning assembly. Chem Commun (Camb) 2015;51:17108-11.
41. Simone EA, Dziubla TD, Discher DE, Muzykantov VR. Filamentous polymer nanocarriers of tunable stiffness that encapsulate the therapeutic enzyme catalase. Biomacromolecules 2009;10:1324-30.
42. Geng Y, Dalhaimer P, Cai S, Tsai R, Tewari M, Minko T, et al. Shape effects of filaments versus spherical particles in flow and drug delivery. Nat Nanotechnol 2007;2:249-55.
43. Xu J, Wong DH, Byrne JD, Chen K, Bowerman C, DeSimone JM. Future of the particle replication in nonwetting templates (PRINT) technology. Angew Chem Int Ed Engl 2013;52:6580-9.
44. Raja KS, Wang Q, Gonzalez MJ, Manchester M, Johnson JE, Finn MG. Hybrid virus-polymer materials. 1. Synthesis and properties of PEG-decorated cowpea mosaic virus. Biomacromolecules 2003;4:472-6.
45. Steinmetz NF, Manchester M. PEGylated viral nanoparticles for biomedicine: the impact of PEG chain length on VNP cell interactions in vitro and ex vivo. Biomacromolecules 2009;10:784-92.
46. Fang J, Nakamura H, Maeda H. The EPR effect: Unique features of tumor blood vessels for drug delivery, factors involved, and limitations and augmentation of the effect. Adv Drug Deliv Rev 2011;63:136-51.
47. Longmire M, Choyke PL, Kobayashi H. Clearance properties of nano-sized particles and molecules as imaging agents: considerations and caveats. Nanomedicine (Lond) 2008;3:703-17.
48. Liu Z, Davis C, Cai W, He L, Chen X, Dai H. Circulation and long-term fate of functionalized, biocompatible single-walled carbon nanotubes in mice probed by Raman spectroscopy. Proc Natl Acad Sci U S A 2008;105:1410-5.
49. Bottini M, Rosato N, Bottini N. PEG-modified carbon nanotubes in biomedicine: current status and challenges ahead. Biomacromolecules 2011;12:3381-93.
50. Hamilton RF, Jr., Wu Z, Mitra S, Shaw PK, Holian A. Effect of MWCNT size, carboxylation, and purification on in vitro and in vivo toxicity, inflammation and lung pathology. Part Fibre Toxicol 2013;10:57.
51. Kraft JC, Freeling JP, Wang Z, Ho RJ. Emerging research and clinical development trends of liposome and lipid nanoparticle drug delivery systems. J Pharm Sci 2014;103:29-52.
228 CHAPTER 7: CONCLUSIONS
52. Lin S, Wang X, Ji Z, Chang CH, Dong Y, Meng H, et al. Aspect ratio plays a role in the hazard potential of CeO2 nanoparticles in mouse lung and zebrafish gastrointestinal tract. ACS Nano 2014;8:4450-64.
53. Mohammad AK, Reineke JJ. Quantitative detection of PLGA nanoparticle degradation in tissues following intravenous administration. Mol Pharm 2013;10:2183-9.
54. Park EJ, Umh HN, Kim SW, Cho MH, Kim JH, Kim Y. ERK pathway is activated in bare-FeNPs-induced autophagy. Arch Toxicol 2014;88:323-36.
55. Zhang XD, Wu D, Shen X, Liu PX, Yang N, Zhao B, et al. Size-dependent in vivo toxicity of PEG-coated gold nanoparticles. Int J Nanomedicine 2011;6:2071- 81.
56. Murahari MS, Yergeri MC. Identification and usage of fluorescent probes as nanoparticle contrast agents in detecting cancer. Curr Pharm Des 2013;19:4622- 40.
57. Sosnovik DE, Nahrendorf M, Weissleder R. Magnetic nanoparticles for MR imaging: agents, techniques and cardiovascular applications. Basic Res Cardiol 2008;103:122-30.
58. Ikeda H, Chamoto K, Tsuji T, Suzuki Y, Wakita D, Takeshima T, et al. The critical role of type-1 innate and acquired immunity in tumor immunotherapy. Cancer Sci 2004;95:697-703.
59. Afonso LC, Scharton TM, Vieira LQ, Wysocka M, Trinchieri G, Scott P. The adjuvant effect of interleukin-12 in a vaccine against Leishmania major. Science 1994;263:235-7.
60. Wagner S, Jasinska J, Breiteneder H, Kundi M, Pehamberger H, Scheiner O, et al. Delayed tumor onset and reduced tumor growth progression after immunization with a Her-2/neu multi-peptide vaccine and IL-12 in c-neu transgenic mice. Breast Cancer Res Treat 2007;106:29-38.
61. Colombo MP, Trinchieri G. Interleukin-12 in anti-tumor immunity and immunotherapy. Cytokine Growth Factor Rev 2002;13:155-68.
62. Shukla S, Wen AM, Commandeur U, Steinmetz NF. Presentation of HER2 epitopes using a filamentous plant virus-based vaccination platform. Journal of Materials Chemistry B 2014;2:6249-58.
63. Tugues S, Burkhard SH, Ohs I, Vrohlings M, Nussbaum K, Vom Berg J, et al. New insights into IL-12-mediated tumor suppression. Cell Death Differ 2015;22:237-46.
229 CHAPTER 7: CONCLUSIONS
64. Chiodoni C, Stoppacciaro A, Sangaletti S, Gri G, Cappetti B, Koezuka Y, et al. Different requirements for alpha-galactosylceramide and recombinant IL-12 antitumor activity in the treatment of C-26 colon carcinoma hepatic metastases. Eur J Immunol 2001;31:3101-10.
65. Smyth MJ, Taniguchi M, Street SE. The anti-tumor activity of IL-12: mechanisms of innate immunity that are model and dose dependent. J Immunol 2000;165:2665-70.
66. Prabhakar U, Maeda H, Jain RK, Sevick-Muraca EM, Zamboni W, Farokhzad OC, et al. Challenges and key considerations of the enhanced permeability and retention effect for nanomedicine drug delivery in oncology. Cancer Res 2013;73:2412-7.
67. Schroeder A, Heller DA, Winslow MM, Dahlman JE, Pratt GW, Langer R, et al. Treating metastatic cancer with nanotechnology. Nat Rev Cancer 2012;12:39-50.
68. Li Z, Zhao R, Wu X, Sun Y, Yao M, Li J, et al. Identification and characterization of a novel peptide ligand of epidermal growth factor receptor for targeted delivery of therapeutics. FASEB J 2005;19:1978-85.
69. Nicholson RI, Gee JMW, Harper ME. EGFR and cancer prognosis. Eur J Cancer 2001;37:9-15.
70. Yonesaka K, Zejnullahu K, Okamoto I, Satoh T, Cappuzzo F, Souglakos J, et al. Activation of ERBB2 signaling causes resistance to the EGFR-directed therapeutic antibody cetuximab. Sci Transl Med 2011;3:99ra86.
71. Kitai Y, Fukuda H, Enomoto T, Asakawa Y, Suzuki T, Inouye S, et al. Cell selective targeting of a simian virus 40 virus-like particle conjugated to epidermal growth factor. J Biotechnol 2011;155:251-6.
72. Kickhoefer VA, Han M, Raval-fernandes S, Poderycki MJ, Moniz RJ, Vaccari D, et al. Targeting Vault Nanoparticles to Specific Cell Surface Receptors. ACS Nano 2009;3:27-36.
73. Paraskevakou G, Allen C, Nakamura T, Zollman P, James CD, Peng KW, et al. Epidermal Growth Factor Receptor ( EGFR )– Retargeted Measles Virus Strains Effectively Target EGFR- or EGFRvIII Expressing Gliomas. Mol Ther 2007;15:677-86.
74. Pokorski JK, Hovlid ML, Finn MG. Cell targeting with hybrid Qβ virus-like particles displaying epidermal growth factor. ChemBioChem 2011;12:2441-7.
75. Shukla S, DiFranco NA, Wen AM, Commandeur U, Steinmetz NF. To Target or Not to Target: Active vs. Passive Tumor Homing of Filamentous Nanoparticles Based on Potato virus X. Cellular and Molecular Bioengineering 2015;8:433-44.
230 CHAPTER 7: CONCLUSIONS
76. Desai N. Challenges in development of nanoparticle-based therapeutics. AAPS J 2012;14:282-95.
77. Shukla S, Dickmeis C, Nagarajan AS, Fischer R, Commandeur U, Steinmetz NF. Molecular farming of fluorescent virus-based nanoparticles for optical imaging in plants, human cells and mouse models. Biomaterials Science 2014;2:784-97.
78. Aljabali AA, Shukla S, Lomonossoff GP, Steinmetz NF, Evans DJ. CPMV-DOX delivers. Mol Pharm 2013;10:3-10.
79. Lockney DM, Guenther RN, Loo L, Overton W, Antonelli R, Clark J, et al. The Red clover necrotic mosaic virus capsid as a multifunctional cell targeting plant viral nanoparticle. Bioconjug Chem 2011;22:67-73.
80. Yildiz I, Tsvetkova I, Wen AM, Shukla S, Masarapu MH, Dragnea B, et al. Engineering of Brome mosaic virus for biomedical applications. Rsc Advances 2012;2:3670-7.
81. Kattan J, Droz JP, Couvreur P, Marino JP, Boutan-Laroze A, Rougier P, et al. Phase I clinical trial and pharmacokinetic evaluation of doxorubicin carried by polyisohexylcyanoacrylate nanoparticles. Invest New Drugs 1992;10:191-9.
82. Zhu Q, Jia L, Gao Z, Wang C, Jiang H, Zhang J, et al. A tumor environment responsive doxorubicin-loaded nanoparticle for targeted cancer therapy. Mol Pharm 2014;11:3269-78.
83. Aryal M, Vykhodtseva N, Zhang YZ, McDannold N. Multiple sessions of liposomal doxorubicin delivery via focused ultrasound mediated blood-brain barrier disruption: a safety study. J Control Release 2015;204:60-9.
84. Sanson C, Schatz C, Le Meins JF, Soum A, Thevenot J, Garanger E, et al. A simple method to achieve high doxorubicin loading in biodegradable polymersomes. J Control Release 2010;147:428-35.
85. Reynolds JG, Geretti E, Hendriks BS, Lee H, Leonard SC, Klinz SG, et al. HER2- targeted liposomal doxorubicin displays enhanced anti-tumorigenic effects without associated cardiotoxicity. Toxicol Appl Pharmacol 2012;262:1-10.
86. Zagar TM, Vujaskovic Z, Formenti S, Rugo H, Muggia F, O'Connor B, et al. Two phase I dose-escalation/pharmacokinetics studies of low temperature liposomal doxorubicin (LTLD) and mild local hyperthermia in heavily pretreated patients with local regionally recurrent breast cancer. Int J Hyperthermia 2014;30:285-94.
87. Ren Y, Wong SM, Lim LY. Folic acid-conjugated protein cages of a plant virus: a novel delivery platform for doxorubicin. Bioconjug Chem 2007;18:836-43.
231 CHAPTER 7: CONCLUSIONS
88. Phumyen A, Jantasorn S, Jumnainsong A, Leelayuwat C. Doxorubicin-conjugated bacteriophages carrying anti-MHC class I chain-related A for targeted cancer therapy in vitro. Onco Targets Ther 2014;7:2183-95.
89. Cao J, Guenther RH, Sit TL, Opperman CH, Lommel SA, Willoughby JA. Loading and release mechanism of red clover necrotic mosaic virus derived plant viral nanoparticles for drug delivery of doxorubicin. Small 2014;10:5126-36.
90. Huisman MJ, Linthorst HJ, Bol JF, Cornelissen JC. The complete nucleotide sequence of potato virus X and its homologies at the amino acid level with various plus-stranded RNA viruses. J Gen Virol 1988;69 ( Pt 8):1789-98.
91. Porumb H. The solution spectroscopy of drugs and the drug-nucleic acid interactions. Prog Biophys Mol Biol 1978;34:175-95.
92. Missirlis D, Kawamura R, Tirelli N, Hubbell JA. Doxorubicin encapsulation and diffusional release from stable, polymeric, hydrogel nanoparticles. Eur J Pharm Sci 2006;29:120-9.
93. Zeng Q, Wen H, Wen Q, Chen X, Wang Y, Xuan W, et al. Cucumber mosaic virus as drug delivery vehicle for doxorubicin. Biomaterials 2013;34:4632-42.
94. Abraham SA, Waterhouse DN, Mayer LD, Cullis PR, Madden TD, Bally MB. The liposomal formulation of doxorubicin. Methods Enzymol 2005;391:71-97.
95. Yoo HS, Lee KH, Oh JE, Park TG. In vitro and in vivo anti-tumor activities of nanoparticles based on doxorubicin-PLGA conjugates. J Control Release 2000;68:419-31.
96. Zhao Y, Alakhova DY, Kim JO, Bronich TK, Kabanov AV. A simple way to enhance Doxil(R) therapy: drug release from liposomes at the tumor site by amphiphilic block copolymer. J Control Release 2013;168:61-9.
97. Carelle N, Piotto E, Bellanger A, Germanaud J, Thuillier A, Khayat D. Changing patient perceptions of the side effects of cancer chemotherapy. Cancer 2002;95:155-63.
98. Morstyn G, Campbell L, Souza LM, Alton NK, Keech J, Green M, et al. Effect of granulocyte colony stimulating factor on neutropenia induced by cytotoxic chemotherapy. Lancet 1988;1:667-72.
99. Chatterjee K, Zhang J, Honbo N, Karliner JS. Doxorubicin cardiomyopathy. Cardiology 2010;115:155-62.
100. Couvreur P, Kante B, Grislain L, Roland M, Speiser P. Toxicity of polyalkylcyanoacrylate nanoparticles II: Doxorubicin-loaded nanoparticles. J Pharm Sci 1982;71:790-2.
232 CHAPTER 7: CONCLUSIONS
101. Shukla S, Ablack AL, Wen AM, Lee KL, Lewis JD, Steinmetz NF. Increased tumor homing and tissue penetration of the filamentous plant viral nanoparticle Potato virus X. Mol Pharm 2013;10:33-42.
102. Montet X, Funovics M, Montet-Abou K, Weissleder R, Josephson L. Multivalent effects of RGD peptides obtained by nanoparticle display. J Med Chem 2006;49:6087-93.
103. Teow Y, Valiyaveettil S. Active targeting of cancer cells using folic acid- conjugated platinum nanoparticles. Nanoscale 2010;2:2607-13.
104. Yhee JY, Lee SJ, Lee S, Song S, Min HS, Kang SW, et al. Tumor-targeting transferrin nanoparticles for systemic polymerized siRNA delivery in tumor- bearing mice. Bioconjug Chem 2013;24:1850-60.
105. Roy A, Singh MS, Upadhyay P, Bhaskar S. Nanoparticle mediated co-delivery of paclitaxel and a TLR-4 agonist results in tumor regression and enhanced immune response in the tumor microenvironment of a mouse model. Int J Pharm 2013;445:171-80.
106. Bagalkot V, Lee IH, Yu MK, Lee E, Park S, Lee JH, et al. A combined chemoimmunotherapy approach using a plasmid-doxorubicin complex. Mol Pharm 2009;6:1019-28.
107. Martins I, Tesniere A, Kepp O, Michaud M, Schlemmer F, Senovilla L, et al. Chemotherapy induces ATP release from tumor cells. Cell Cycle 2009;8:3723-8.
108. Kepp O, Galluzzi L, Martins I, Schlemmer F, Adjemian S, Michaud M, et al. Molecular determinants of immunogenic cell death elicited by anticancer chemotherapy. Cancer Metastasis Rev 2011;30:61-9.
109. Hodge JW, Garnett CT, Farsaci B, Palena C, Tsang KY, Ferrone S, et al. Chemotherapy-induced immunogenic modulation of tumor cells enhances killing by cytotoxic T lymphocytes and is distinct from immunogenic cell death. Int J Cancer 2013;133:624-36.
110. Ott PA, Hodi FS. Talimogene Laherparepvec for the Treatment of Advanced Melanoma. Clin Cancer Res 2016.
111. Segeren CM, Sonneveld P, van der Holt B, Baars JW, Biesma DH, Cornellissen JJ, et al. Vincristine, doxorubicin and dexamethasone (VAD) administered as rapid intravenous infusion for first-line treatment in untreated multiple myeloma. Br J Haematol 1999;105:127-30.
112. Ganson NJ, Povsic TJ, Sullenger BA, Alexander JH, Zelenkofske SL, Sailstad JM, et al. Pre-existing anti-polyethylene glycol antibody linked to first-exposure allergic reactions to pegnivacogin, a PEGylated RNA aptamer. J Allergy Clin Immunol 2016;137:1610-3 e7. 233 CHAPTER 7: CONCLUSIONS
113. Verhoef JJ, Carpenter JF, Anchordoquy TJ, Schellekens H. Potential induction of anti-PEG antibodies and complement activation toward PEGylated therapeutics. Drug Discov Today 2014;19:1945-52.
114. Wang X, Ishida T, Kiwada H. Anti-PEG IgM elicited by injection of liposomes is involved in the enhanced blood clearance of a subsequent dose of PEGylated liposomes. J Control Release 2007;119:236-44.
115. Wyffels L, Verbrugghen T, Monnery BD, Glassner M, Stroobants S, Hoogenboom R, et al. muPET imaging of the pharmacokinetic behavior of medium and high molar mass Zr-labeled poly(2-ethyl-2-oxazoline) in comparison to poly(ethylene glycol). J Control Release 2016;235:63-71.
116. Luxenhofer R, Han Y, Schulz A, Tong J, He Z, Kabanov AV, et al. Poly(2- oxazoline)s as polymer therapeutics. Macromol Rapid Commun 2012;33:1613-31.
117. He Z, Schulz A, Wan X, Seitz J, Bludau H, Alakhova DY, et al. Poly(2- oxazoline) based micelles with high capacity for 3rd generation taxoids: preparation, in vitro and in vivo evaluation. J Control Release 2015;208:67-75.
118. Rodriguez PL, Harada T, Christian DA, Pantano DA, Tsai RK, Discher DE. Minimal "Self" peptides that inhibit phagocytic clearance and enhance delivery of nanoparticles. Science 2013;339:971-5.
234 APPENDIX I: NANODIAMONDS FOR CANCER THERAPY
Appendix I: Plasmonic nanodiamonds for cancer therapy
The material in this chapter is adapted with permission from: Rehor, I.*, Lee,
K.L.*, Chen, K., Hajek, M., Havlik, J., Lokajova, J., Masat, M., Slegerova, J., Shukla, S.,
Heidari, H., Bals, S., Steinmetz, N.F., Cigler, P. Plasmonic Nanodiamonds – Targeted
Core-shell Type Nanoparticles for Cancer Cell Thermoablation. Adv. Healthcare Mater.
2015, 4: 460-468. Copyright 2015 John Wiley and Sons
*Both authors contributed equally
As co-first author on this paper, I performed cell uptake (flow cytometry and confocal microscopy) and cytotoxicity assays and contributed to writing and editing the paper. Ivan Rehor, a collaborator from the Cigler laboratory, synthesized and characterized the particles and performed laser ablation studies.
A1.1 Introduction
Plasmonic nanostructures (PNs) of various shapes and composition [1, 2] have garnered scientific interest in recent years due to their unique optical properties, which allow their use for construction of therapeutic and theranostic nanoparticles, bioprobes, and sensors [1, 3]. For a nanosized noble metal particle, the de Broglie wavelength of the valence electrons is of the same order of magnitude as the size of the particle, and quantum size effects may appear. The valence electrons then start to oscillate at a collective oscillation frequency, giving rise to characteristic plasmon resonance bands [3,
4]. Because of these properties, PNs gained interest in the fields of biotechnology and biomedicine. Through nanostructure design, their plasmonic absorption wavelength can
235 APPENDIX I: NANODIAMONDS FOR CANCER THERAPY be finely tuned to fall in the near-infrared tissue imaging window (650-900 nm) where light can penetrate up to a few centimeters into the tissue. The huge absorption and scattering of PNs enables their use as a contrast agent for optical imaging of tissues, e. g., in optical coherence tomography [5, 6] or photoacoustic [7] imaging. Furthermore, the absorbed light is transformed into heat, allowing for use of PNs in cancer therapy [8-10].
The heat may be used for thermal ablation of tumors or to control the release of therapeutics, which are usually but not exclusively [11], attached to the PN surface [12].
Merging diagnostic imaging ability with a therapeutic function in one so-called
“theranostic” agent is indeed promising, as evidenced by numerous recent publications addressing the topic [13, 14].
Here, we describe preparation of a plasmonic gold nanoshell (GNS) around silica- encapsulated diamond nanocrystals. Nanodiamonds (NDs) are highly biocompatible materials with applications in nanomedicine [15] and bioimaging [16-19]. Previous studies on gold plasmonic structures connected with diamonds were performed primarily on macroscopic substrates [20-25]. They have focused mainly on the photophysics of fluorescent nitrogen-vacancy centers embedded in a diamond crystal lattice. Creation of a well-defined plasmonic system on a single diamond nanoparticle in solution is limited by the colloidal instability of NDs in aqueous buffers [26]. To date, only direct covalent attachment of gold nanoparticles to NDs has been achieved [27, 28], and materials with unordered structural morphology [29, 30] have been prepared.
We took advantage of our recently published methodology for silica coating of
NDs [31], which enables the creation of a well-defined plasmonic material based on a
ND dielectric core coated with a GNS. We chose the core-shell design because it
236 APPENDIX I: NANODIAMONDS FOR CANCER THERAPY provides an extremely high plasmonic absorption cross section, as well as the possibility to tune the position of the absorption maximum within the near-infrared region, where light is minimally absorbed and scattered by the tissue. As a first step to demonstrate the utility of this newly constructed biomaterial in nanomedical applications, we show that it can be stabilized and rendered biocompatible by addition of PEG-containing ligands bearing bioorthogonally reactive alkyne groups, followed by decoration with synthetically modified transferrin (Tf). We use these particles to target cancer cells, which overexpress the Tf-receptor, and thermally ablate them by irradiation with a near- infrared pulse laser.
A1.2 Materials and methods
A1.2.1 Chemicals and solvents
The NHS ester of lipoic acid was prepared according to a previously published procedure [32]. H2N-PEG(5000)-alkyne was purchased from Iris Biotech. Alexa Fluor
647-azide was purchased from Invitrogen. Tetrakis(hydroxymethyl)phosphonium chloride (THPC) and tetraethyl orthosilicate (TEOS) were purchased from Sigma-Aldrich.
HAuCl4 was purchased from Alfa Aesar.
A1.2.2 UV-Vis
UV-Vis spectra were recorded with a Specord 210 (Analytik Jena) spectrometer in the 400-1000 nm range at room temperature with an optical path of 1 cm.
237 APPENDIX I: NANODIAMONDS FOR CANCER THERAPY
A1.2.3 DLS
For stability studies in buffers, DLS was recorded with a Zetasizer Nano ZS system (Malvern Instruments) at 25 °C. The particle concentration was 0.3 mg mL-1 (10
μg ND mL-1).
A1.2.4 Electron microscopy
To prepare the samples, a drop of diluted colloidal solution was placed on a carbon-coated copper grid and left to dry. Bright field TEM experiments were performed with a JEOL JEM-1011 electron microscope operated at 60 kV and equipped with a
Veleta side-mounted camera. Carbon coated grids (Pyser) were used in all cases.
Nanodiamond samples were prepared according to a previously published procedure [33].
Other samples were prepared as follows: a 3 μL droplet of particle dispersion (0.2 mg mL-1) was placed on the grid and gently removed with a piece of tissue after 1 min incubation. Tilt series of 2-dimensional projection images for electron tomography were acquired using a FEI Tecnai G2 transmission electron microscope operated at 200 kV in scanning transmission electron microscopy (STEM) mode. The high angle annular dark field (HAADF-STEM) image series was acquired using a single tilt tomography holder
(Fischione 2020) over the angular range (-64°,+76°) with step increments of 2°. The alignment and 3D reconstruction were carried out with FEI Inspect3D software.
Quantification of the 3D data was performed using MATLAB codes.
238 APPENDIX I: NANODIAMONDS FOR CANCER THERAPY
A1.2.5 Synthesis of LA-PEG-alkyne
Synthesis was performed according to published procedures [34, 35], with some modifications. Briefly, H2N-PEG(5000)-alkyne (50 mg, 10 μmol) was dissolved in DCM
(1 mL) and mixed with LA-NHS (50 mg, 165 μmol). The reaction mixture was stirred overnight, then washed 3 times with 10 mL hot water. The combined aqueous fractions were purified using a Millipore Ultracel 3K separation tube (washed 3 times with 20 mL water). TLC: MeOH:triethylamine 100:5 (Rf = 0.5), visualized with Co(NCS)x and
KMnO4 (no reaction with ninhydrin). H2N-PEG(5000)-alkyne Rf < 0.3, visualized with
1 ninhydrin and Co(NCS)x (no reaction with KMnO4). H NMR (400 MHz, CDCl3): δ 1.6
(m, 2H), 1.7-1.9 (m, 3H), 2.0 (m, 1H), 2.1 (t, 1H, J = 3.6 Hz), 2.2-2.4 (m, 4H), 2.5 (t, 2H,
J = 7.2 Hz), 2.6 (m, 3H), 3.2-3.3 (m, 2H), 3.5-3.9 (m, ~240H), 3.9 (t, 1H, J = 5.2).
A1.2.6 Transferrin-azide (Tf-azide)
Preparation (see Figure A1.3A). We used a procedure similar to that previously described for preparation of transferrin-alkyne [36]. NaIO4 solution was slowly added to a cooled solution of human holo transferrin (30 mg, 390 nmol; 2 mg/mL) in acetate buffer (0.1 M, pH 5.5) to a final NaIO4 concentration of 1 mM. The mixture was incubated on ice in the dark for 30 min. The solution containing Tf-aldehyde was concentrated six times in an ultrafiltration cell (from 70 ml to 5 ml). HEPES buffer (0.1
M, pH 7.2) was used to refill the volume. Tf-aldehyde was incubated with 3- aminooxypropyl-1-azide (16.2 mg, 140 µmol) in HEPES buffer with dimethyl sulfoxide
(DMSO, 20%, total volume of 17 mL) for 5 h at room temperature with gentle mixing.
Removal of excess 3-aminooxypropyl-1-azide was performed by ultrafiltration (70 ml to
239 APPENDIX I: NANODIAMONDS FOR CANCER THERAPY
5 ml, repeated six times) in HEPES buffer (0.1 M, pH 8). The solution was freeze-dried to obtain Tf-azide.
Characterization. The presence of a reactive azide group in Tf-azide was tested by reaction with fluorescein-alkyne. All solutions were aqueous except the fluorescein- alkyne stock, which was prepared in DMSO. The solutions were mixed to achieve the following final concentrations: 0.02 mM Tf-azide, 0.075 mM fluorescein-alkyne, 0.17 mM CuSO4⋅5H2O, 0.33 mM tris(3-hydroxypropyltriazolylmethyl)amine (THPTA), and 5 mM sodium ascorbate. The solutions of CuSO4⋅5H2O and THPTA were premixed (in a
1:2 molar ratio) before adding to the reaction mixture. The reaction mixture was well- sealed after adding sodium ascorbate, mixed, and reacted for 2 h with no stirring. The reaction product (Tf-Fl) was analyzed by SDS-PAGE. All samples (Tf, Tf-azide, and Tf-
Fl) were of the same molecular weight, and only Tf-Fl can be seen under UV-lamp.
A1.2.7 Nanoshell preparation
Silica encapsulation. NDs were solubilized in a manner similar to that described in commonly used procedures [16, 37]. Briefly, NDs were treated with a mixture of
HNO3 and H2SO4 (85 °C, 3 days), washed with 2 M NaOH and 2 M HCl, washed five times with water, and freeze-dried. Prior to use, the particles were dissolved in water
(2 mg mL-1) and sonicated with a probe (Cole-Parmer, 750 W) for 30 min. The resulting transparent colloid was filtered using a 0.2 μm PVDF microfilter to provide a colloidal solution of ND particles. A modified version [31] of previously described general procedure [38] was used to coat NDs with silica shells. Polyvinylpyrrolidone (96 mg, 9.6
μmol) was dissolved in water (204 mL) and sonicated for 10 min in an ultrasonic bath. 240 APPENDIX I: NANODIAMONDS FOR CANCER THERAPY
ND colloid (6 mL, 2 mg mL-1) was added, and the mixture was stirred for 24 h. The colloid was then concentrated via centrifugation in two steps. In the first step (40,000 rcf,
1 h), the volume was reduced to approximately 12 mL. The second centrifugation step
(30,000 rcf, 30 min) was performed in microvials and reduced the solvent volume to approximately 0.4 mL. Sedimented NDs were resuspended in ethanol (12 mL) in a round bottom flask and sonicated in an ultrasonic bath for 2-4 min. TEOS (112 mg, 539 μmol) was added. After 2 min of vigorous stirring, ammonia solution (25%, 500 μL) was added, and the reaction mixture was stirred for 14 h, affording silica-coated particles ND@Sil.
The product was purified by centrifugation (14,000 rcf, 5 min) with ethanol (12 mL, 4x) and MeCN (12 mL, 2x) and was dissolved in 6 mL MeCN. ND@Sil particles were stored in the freezer (–18 °C) as a stable colloid for several months without changes in particle characteristics (confirmed with TEM and DLS) or reactivity.
GNS formation. GNSs were prepared according to modified published procedures
[39-41]. First, seeding gold colloid was prepared. Water (45 mL) was mixed with NaOH
(5 mL, 0.1 M). Tetrakis(hydroxymethyl)phosphonium chloride (THPC, 67.2 μmol in 1 mL water) was added. After exactly 5 min, HAuCl4 solution (2 mL of a 1% w/w solution in water, 59 μmol) was added in one portion under vigorous stirring. The mixture was stirred for 10 min, and the resulting gold colloid was aged at 4 °C for 2 weeks without purification. ND@Sil particles (1 mL MeCN dispersion, 2 mg ND content, 8 mg total
ND@Sil weight) were mixed with 2-week aged gold colloid (30 mL). The pH of the mixture was adjusted to 3. The mixture was gently stirred for 20 min and then kept at
4 °C for 16 h. ND@Sil particles coated with gold seeds were isolated by centrifugation
(2500 rcf, 1 h) and washed twice with water (30 mL, isolated at 2500 rcf, 1 h). The
241 APPENDIX I: NANODIAMONDS FOR CANCER THERAPY
volume of the dispersion was adjusted to 5 mL. K2CO3 (200 mg, 1.44 mmol) was dissolved in 800 mL water, and HAuCl4 solution (12 mL of a 1% w/w solution in water,
3.5 μmol) was added. The solution was stirred for 24 h in the dark. Then, the pH of the solution was adjusted to 9. Gold-seeded ND@Sil dispersion (1.25 mL; 0.5 mg ND content) was added to 200 mL solution. A stream of CO was bubbled through the solution for 2 min under vigorous stirring. The color changed from transparent to red, purple, and then dark blue. Gold encapsulated ND@Sil (ND@Au) were separated by centrifugation at 20 rcf overnight and concentrated to 2.5 mL. The total mass of ND@Au particles obtained in one run was on average 15 mg, i.e., 30-fold the mass of ND and 7.5- fold the mass of ND@Sil.
PEGylation of ND@Au. LA-PEG-alkyne (10.3 mg, 2 μmol) was added to
ND@Au dispersion (15 mg; corresponds to 0.5 mg ND) in 2.5 mL water. The mixture was sonicated in an ultrasonic bath for 20 min, then stirred overnight and sonicated for another 20 min. PEG-coated ND@Au particles were separated by centrifugation (5x, 500 rcf, 20 min), yielding ND@Au-PEG.
Modification with transferrin and Alexa Fluor 647. A solution of Cu-catalyst was prepared in a separate vial by mixing CuSO4·5H2O (20 μL of a 25 mM solution) and
THPTA ligand (20 μL of a 50 mM solution). The click reactions were performed similarly to those described in the literature [42] by mixing reactants in a 0.6 mL vial.
ND@Au-PEG solution (1.5 mg particles in 125 μL) was mixed with a DMSO solution of
Alexa Fluor 647-azide (1.7 μL of a 5.88 mM solution). Other components were added in the following order and quantities: aminoguanidine hydrochloride (12 μL of a 100 mM solution) and Cu-catalyst solution (3.2 μL) (see above). Water was added to adjust the
242 APPENDIX I: NANODIAMONDS FOR CANCER THERAPY total reaction volume to 200 μL. Sodium ascorbate (12 μL of a 100 mM solution) was added. The vials were well-sealed and kept for 2 h without stirring or shaking. Modified nanoparticles were isolated in near-quantitative yield by centrifugation (500 rcf, 20 min) and washed with water (2x, 1 mL) and PBS (1x, 1 mL) to yield gold-coated NDs tagged with Alexa Fluor 647 (ND@Au-nT). ND@Au-nT was further modified with Tf-azide in a similar manner. Solid Tf-azide (0.2 mg) was added to nanoparticles, and other components were added in the same quantities and order as in the previous reaction step.
After centrifugal separation, ND@Au-Tf was obtained.
A1.2.8 Stability experiments
A colloidal aqueous solution of ND@Au-PEG (1.5 mg mL-1, 100 μL) was added to buffer (900 µL). The following buffers were used: PBS (pH = 7.5), 0.15 M NaCl, 0.3
M NaCl, and cell growth medium (RPMI-1640 + serum). The samples were stored at
25 °C, and the aggregation state was examined with DLS 2 h, 1 day, and 1 month after mixing. Ten minutes before each measurement, samples were sonicated in a bath for 20 s.
A1.2.9 Cell studies
Flow cytometry. SKBR3 cells were cultured in McCoy’s 5A media supplemented with 10% (v/v) fetal bovine serum (FBS), 1% (v/v) penicillin-streptomycin, and 1% (v/v)
L-glutamine at 37 °C and 5% CO2 (all reagents were obtained from Invitrogen). Cells
(100,000 cells/500 μL media/well) were added to untreated 24-well plates and incubated overnight. The following day, triplicates of i) culture medium alone (negative control), ii)
243 APPENDIX I: NANODIAMONDS FOR CANCER THERAPY gold-coated NDs tagged with Alexa Fluor 647 (ND@Au-nT), and iii) gold-coated NDs tagged with Alexa Fluor 647 and transferrin (ND@Au-Tf) were added. Particle concentrations were approximately 1 x 1012 particles/mL (1.2 mg particles per 1 mL, 300
μL final volume; 3 x 106 particles/cell) in culture medium. Particles were incubated with cells for 3 h. (Competition binding assays were also carried out: ND@Au-Tf were incubated with free Tf added to the medium in a molar excess of 5:1 or 20:1.) Following incubation, cells were washed three times with 0.09% (w/v) saline to remove non-bound particles remaining in solution. Cells were removed using 200 μL enzyme-free Hank’s based cell dissociation buffer (Gibco), added to untreated 96-well v-bottom plates, and centrifuged at 500 g for 4 min. The supernatant was removed. Cells were then washed in
FACS buffer (0.1 mL of 0.5 M EDTA, 0.5 mL FBS, and 1.25 mL of 1 M HEPES, pH 7.0, in 50 mL Ca2+- and Mg2+-free PBS). Washing was repeated twice. Cells were fixed in 2%
(v/v) paraformaldehyde in FACS buffer at room temperature for 10 min and washed twice. Samples were analyzed using a BD LSR II flow cytometer, with a total of 10,000 gated events collected per sample. FlowJo 10.0.00003 software was used for data analysis.
Confocal microscopy. SKBR3 cells (25,000 cells/500 μL media/well) were added to coverslips, which were placed in untreated 24-well plates and incubated overnight. The following day, duplicates of i) no particles, ii) ND@Au-nT, and iii) ND@Au-Tf were added at concentrations of approximately 1 x 1012 particles/mL (1.2 mg particles per 1 mL, 300 μL final volume, 1.2 x 107 particles/cell) and incubated with the cells for 16 h.
Following incubation, cells were washed three times with 0.09% (w/v) saline to remove excess particles. Samples were fixed in 5% (v/v) paraformaldehyde and 0.3% (v/v)
244 APPENDIX I: NANODIAMONDS FOR CANCER THERAPY gluteraldehyde in Dulbecco’s PBS (Fisher) for 10 min at room temperature. Cells were blocked in 5% (v/v) goat serum (Invitrogen) for 90 min at room temperature. Cell membranes were stained using 1 μg mL-1 wheat germ agglutinin-A555 (Invitrogen) and
1% (v/v) goat serum in Dulbecco’s PBS for 45 min at room temperature. Cell nuclei were stained using 4’6’-diamidino-2-phenylindole (DAPI) (MP Biomedicals) diluted 1:9500 in
Dulbecco’s PBS for 15 min at room temperature. In between each step, cells were washed three times with Dulbecco’s PBS. Slides were mounted using Permount mounting media (Fisher). Confocal analysis was performed using the Olympus FV1000 laser scanning confocal microscope and a 40x objective. Images were analyzed using
ImageJ 1.43u software.
XTT cell proliferation assay. SKBR3 cells (25,000 cells, 200 μL media per well) were added to 96-well plates and incubated at 37 °C under 5% CO2 overnight. The following day, ND@Au-Tf particles were added at a concentration of approximately 1 x
1012 particles/mL (1.2 mg particles per 1 mL, 75 μL final volume, 3 x 106 particles/cell); control cells with no added particles were also set up. Following a 3 or 24 h incubation, wells were washed three times with 0.09% (w/v) saline to remove unbound NDs. Then,
200 μL fresh media was added, and cells were returned to incubate for an additional 24 h.
An XTT cell proliferation assay (ATCC) was used to assess cellular viability; the protocol was performed according to the manufacturer’s instructions. A Tecan Infinite
200 plate reader was used to measure the absorbance, and percent viability was determined by normalizing to the cell-only control.
Laser ablation experiments. Human cervical adenocarcinoma (HeLa) cells
(ATCC® CCL-2™) were cultured in RPMI 1640, Dutch modification medium
245 APPENDIX I: NANODIAMONDS FOR CANCER THERAPY supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS), antibiotics (200 g/mL streptomycin and 200 U/mL penicillin G) and 2 mM glutamine (all purchased from
Sigma-Aldrich) at 37 °C in a humidified atmosphere containing 5% CO2. Cells were subcultured as needed (2-3 times a week in a subcultivation ratio of 1:5 to 1:8) and harvested from flasks using 0.25% (w/v) trypsin-EDTA. HeLa cells were seeded into a white wall clear bottom 384-well plate in 30 µL at a density of 3000 cells/well. Then, cells were grown for 48 h under standard conditions (37 °C and 5% CO2) before adding
ND@Au-Tf nanoparticles at a final amount of approximately 2 x 1011 (240 μg of particles per 1 mL) in tetraplicates. After 24 h of treatment with or without nanoparticles, the medium was removed, and the cells were thoroughly washed with fresh medium
(three times). Then, the medium in each well was replaced with PBS, and selected wells were illuminated immediately (Ti:Sapphire laser – 75 MHz, 750 nm, pumped at 532 nm by ND:YAG, laser power of 2.1 W, spot diameter of 2.7 mm, irradiation time of 60 sec).
After irradiation, PBS was replaced with fresh complete growth medium (30 μL), and the plate was incubated for an additional 24 h before viability measurement. Cell viability was determined using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega,
Madison, WI, USA), which is based on quantification of the ATP present in cell lysates, according to the manufacturer's protocol. Briefly, the 384-well plate and its contents were equilibrated at room temperature for 30 min. CellTiter-Glo® reagent was prepared by reconstituting the lyophilized enzyme/substrate mixture with CellTiter-Glo® buffer equilibrated to room temperature. After an equal volume of CellTiter-Glo® reagent was added to the wells (30 μL), the plate was shaken for 2 min on an orbital shaker (500
RPM) to induce cell lysis. Luminescence was recorded after an additional 10 min
246 APPENDIX I: NANODIAMONDS FOR CANCER THERAPY incubation in the dark using the multimode microplate reader Tecan Infinite M1000
(Tecan Austria GmbH, Grödig, Austria). Blank wells (containing medium without cells) were measured for luminescence and deducted from the values obtained from experimental wells (background luminescence). The viability values of treated cells were expressed as percentages of the values obtained for the corresponding control cells.
Values (of the luminescent signal) represent the mean ± S.D. of four replicates.
A1.3 Results and discussion
A1.3.1 Preparation and characterization of particles
Creation of GNSs with a diamond core was achieved via multistep encapsulation, as depicted in Figure A1.1A. Commercially available ND particles (Figure A1.1B) are of irregular shape (circularity ~0.67) with sharp edges and often appear elongated in one dimension (needle–like). Their size distribution is broad, ranging from several nm to more than 50 nm in diameter. Therefore, before the GNS is generated on the ND surface, the particle shape needs to be normalized to spherical, and the size distribution should be narrowed. We achieved this through encapsulation of NDs in a silica shell, approximately
20 nm thick, using a method we described earlier [31]. The formation of the desired architecture was confirmed at each step by transmission electron microscopy (TEM), as shown in Figure A1.1B-E. After coating with silica (Figure A1.1C), the particles became more spherical (circularity ~0.87), and their diameter increased to 66 ± 10 nm. These pseudospherical silica-coated NDs (ND@Sil) are suitable for encapsulation with a GNS, according to a procedure introduced by Halas and collaborators [39, 40]. First, small gold nanoparticles (2-3 nm in diameter) were electrostatically anchored onto the silica particle 247 APPENDIX I: NANODIAMONDS FOR CANCER THERAPY surface (Figure A1.1D). These assemblies were exposed to a reductive environment containing gold(III) ions, which served as nucleation centers for GNS growth (Figure
A1.1E). The growth of shells ended after several tens of seconds, yielding a deep blue solution containing GNS-coated NDs (ND@Au).
248 APPENDIX I: NANODIAMONDS FOR CANCER THERAPY
Figure A1.1. Preparation of particles. A) Schematic representation of the preparation of GNSs with a diamond core. First, a silica shell is created on diamond particles, followed by formation of a GNS upon – reduction of [AuCl4] promoted by adsorbed gold nanoparticle seeds. The GNS is modified with a lipoic acid-PEG conjugate, which is terminated with an alkyne. Using click chemistry, Alexa Fluor 647 dye and azide-modified transferrin (the targeting protein) are attached in consecutive steps. (B-D) TEM microphotographs of (B) diamond particles, (C) silica-coated diamond particles (ND@Sil), (D) silica- coated diamond particles with gold seeds, and (E) GNSs a with diamond core (ND@Au). The magnification is the same for all microphotographs, and the scale bar corresponds to 100 nm. 249 APPENDIX I: NANODIAMONDS FOR CANCER THERAPY
To investigate the structure and thickness of these GNSs in detail, we analyzed individual ND@Au particles using HAADF-STEM electron tomography. This technique yields images in which the intensity approximately scales with the square of the atomic number of the elements present in the region of interest. Due to the limited dynamic range of the HAADF detector, maintaining similar intensities for Au and silica in the projection images is not feasible because of the large differences in atomic number. We therefore focused on 3-dimensional reconstruction of GNSs. In Figure A1.2 A-C, 2- dimensional projections of a GNS imaged at different angles are presented. The 3- dimensional reconstruction resulting from the electron tomography experiment is presented in Figure A1.2 D and E. The shell thickness is mostly homogenous. We evaluated the average shell thickness as 12.6 ± 0.3 nm, and the total internal surface of the GNS was 32,582 nm2. The intermittent presence of small holes is likely caused by incomplete filling of the spaces between individual seeds with gold.
250 APPENDIX I: NANODIAMONDS FOR CANCER THERAPY
Figure A1.2. Characterization of particles. (A-C) 2D HAADF-STEM projections of a ND-silica particle coated with a GNS (ND@Au) obtained at different tilt angles. The diamond core and silica coating are not visible due to the limited dynamic range of the image detector. (D) A 3D representation of the reconstructed nanoshell. (E) A slice through the 3D reconstruction of the GNS demonstrating the homogeneity of shell thickness. (F) A histogram indicating the measured thicknesses of the shell based on electron tomography reconstruction. The average shell thickness was 12.6 ± 0.3 nm. (G) Absorption spectrum of ND@Au in water at 15 μg/mL concentration (which corresponds to a ND concentration of 0.5 μg mL-1).
251 APPENDIX I: NANODIAMONDS FOR CANCER THERAPY
The formation of the GNS is reflected in absorption spectra by a characteristic broad plasmonic band with an absorption maximum at 675 nm (Figure A1.2G). The position of the maximum corresponds to values published for silica particles coated with
GNSs of similar sizes and thicknesses [41].
A1.3.2 Introduction of protective and bioorthogonally reactive PEG coating
The application of GNS-based materials in living systems requires their protection against ionic-strength-induced aggregation/precipitation in buffers and biological liquids, as well as against opsonization. Polyethylene glycol (PEG) is an effective polymeric bio- nanointerface, shielding particles against these factors, rendering them “stealth” to the immune system, and prolonging their circulation in the body [43]. In addition to these attributes, PEG can serve as heterobifunctional linker to connect nanoparticles with attached moieties. For functionalization of ND@Au, we utilized mid-size PEG (5 kDa) terminated with lipoic acid at one end and an aliphatic alkyne at the other (Figure
A1.3A). Lipoic acid serves as an instant anchoring group, possessing stronger and more stable interaction with gold than thiols [34]. Of the available bioconjugation techniques, we chose Cu(I)-catalyzed Huisgen alkyne-azide cycloaddition (click reaction) because of its high orthogonality with other reactive groups in biomolecules and excellent conjugation yields in aqueous solution even at very dilute concentrations [44].
252 APPENDIX I: NANODIAMONDS FOR CANCER THERAPY
Figure A1.3. Structure of ND@Au-PEG conjugate and its colloidal stability in aqueous solutions with high ionic strength. A) Composition of the particle surface architecture after modification and attachment of Tf. B) Hydrodynamic radii of ND@Au-PEG in various solutions after 1 h (hatched), 1 week (white) and 1 month (black), showing no aggregation. C) Photograph of naked (ND@Au, left) and PEG-coated (ND@Au-PEG, right) particles dispersed in PBS (20 min after mixing; 0.2 mg mL-1 concentration). The precipitating ND@Au particles are already partially sedimented on the bottom of the vial, while the remaining large aggregates unevenly scatter the laser beam. The ND@Au-PEG particles form a stable colloidal solution, which evenly and strongly scatters the laser beam.
253 APPENDIX I: NANODIAMONDS FOR CANCER THERAPY
To analyze the effect of PEG protection on the colloidal stability of particles, we performed comparative stability tests of PEG-modified GNSs (ND@Au-PEG) and unmodified ND@Au. We exposed the particles to different aqueous solutions with high ionic strength and monitored the hydrodynamic radii over time by dynamic light scattering. While PEG-protected ND@Au-PEG exhibited unlimited colloidal stability in
PBS, physiological solution (0.15 M NaCl) and cell growth media (RPMI media + serum) (Figure A1.3B), naked ND@Au particles immediately agglomerated and precipitated from the buffers (Figure A1.3C), with the exception of cell growth media.
The particles remained stable in media, most likely due to formation of a protein corona by adsorption of proteins from serum.
A1.3.3 Modification of particles with Alexa Fluor 647 and transferrin
For cancer cell targeting experiments, we selected human holo-transferrin (Tf), a glycoprotein that is internalized into cells via clathrin-mediated endocytosis upon binding to Tf receptors (TfR). TfR are expressed in negligible numbers on non-dividing cells, but are highly upregulated on rapidly dividing cancer cells, reaching expression levels of up to 105 TfR per cell [45]. This makes Tf a suitable targeting ligand to direct nanomaterials, such as ND@Au, to cancer cells. This general approach has been successfully demonstrated for various nanoparticles [46], such as virus-like particles [47], liposomes
[48] and nanodiamonds [49, 50]. To ensure protein reactivity for the click bioconjugation strategy, we introduced azide groups to the protein. As previously described [36], Tf offers a favorable pathway for selective derivatization: a reactive aldehyde can be produced by mild periodate cleavage of 1,2-diols on sialic acid (N-acetyl neuraminic
254 APPENDIX I: NANODIAMONDS FOR CANCER THERAPY acid) moieties present in the Tf glycosylation pattern. We derivatized the obtained aldehydes with aminooxypropylazide, a “clickable” heterobifunctional linker that forms a physiologically stable aldoxime (for structures, see Figure A1.3A). Compared to ligation via amino or thiol groups, this approach results in better control over protein attachment points, because one Tf molecule contains only four sialic acids residues at well-defined and sterically accessible positions [36].
To obtain fluorescent particles, which enable quantification of targeting by flow cytometry and analysis of the particles’ subcellular localization by confocal microscopy, we first reacted the alkyne-bearing ND@Au-PEG particles with Alexa Fluor 647-azide, yielding fluorescent ND@Au-nT conjugate. Although gold plasmonic systems can quench emission from fluorescent dyes [51], the dye in this case remained fluorescent and was observable with both flow cytometry and confocal microscopy. Linear PEG with a molecular weight of 5,000 Da has a Flory dimension of ~6.0 nm in solution [52]; it is therefore anticipated that the PEG spacer placed between the plasmon surface and fluorophore will effectively shield the quenching effects. The number of Alexa Fluor 647 molecules per particle, however, was difficult to estimate because of the strong interference of particles’ plasmonic properties and the extinction and emission fluorescence bands of the dye. As a next step, we attached azide-modified Tf to unreacted alkyne groups of ND@Au-nT under similar conditions, providing ND@Au-Tf.
A1.3.4 Targeting of cancer cells
Target-specificity and cellular uptake of particles was evaluated in TfR- expressing SKBR3 cells (a human breast cancer cell line) using flow cytometry 255 APPENDIX I: NANODIAMONDS FOR CANCER THERAPY
(quantitative) and confocal microscopy (qualitative). Flow cytometry indicated that both types of particles, ND@Au-nT and ND@Au-Tf, bound to the cells, with the Tf-targeted preparation ND@Au-Tf showing enhanced interactions. Our data indicate that 26% of the cell population was targeted by ND@Au-Tf, while non-specific uptake was attributed to 18% of the cells, as indicated by ND@Au-nT-cell interactions (p<0.05, Figure A1.4).
Cell targeting properties were found to be reproducible in other cell lines, such as HeLa cells (not shown).
Figure A1.4. SKBR3 cell interactions with particle determined by flow cytometry. Upper histogram: gray, cells only; red, ND@Au-Tf; orange, ND@Au-nT. Bottom graph: Statistical analysis showing percent cellular uptake (positive cells shown on histogram by gate) for each sample. Error bars indicate standard deviation. Experiments were conducted in triplicate, and 10,000 gated events were analyzed. Student’s t- test indicates significant differences comparing targeted ND@Au-Tf and non-targeted ND@Au-nT formulations (p < 0.05). 256 APPENDIX I: NANODIAMONDS FOR CANCER THERAPY
Figure A1.5. Nanodiamond-SKBR3 cell interactions determined by flow cytometry – competition binding assay. Left: Histograms: gray, cells only; red, ND@Au-Tf; orange, ND@Au-nT; blue, competition with 5-fold molar excess of free Tf; purple, competition with 20-fold molar excess of free Tf. Right: Statistical analysis showing percent cellular uptake (positive cells shown on histogram by gate) for each sample. Error bars indicate mean ± standard deviation. Experiments were conducted in triplicate, and 10,000 gated events were analyzed.
Competition binding assays using free Tf ligand further confirmed the target- specificity of the ND@Au-Tf conjugate. Competition with a molar excess of 5:1 or 20:1
Tf:ND@Au-Tf particles resulted in reduced cell uptake, with levels comparable to those observed for non-targeted ND@Au-nT. This indicates that the ND@Au-Tf particles indeed target the cells, and binding can be attributed to specific interactions between TfR and ND@Au-Tf (Figure A1.5).
Next, we sought to investigate the cellular fates of ND@Au-Tf and ND@Au-nT, specifically addressing whether the ND formulations would be taken up into cells. Cell membranes and nuclei were stained, and Z-stacked (0.3 micron/steps) confocal images were recorded. Data indicate that after a 3 h incubation period, both ND@Au-Tf and
257 APPENDIX I: NANODIAMONDS FOR CANCER THERAPY
ND@Au-nT formulations were bound to cell membranes (data not shown). Interestingly, after a 16 h incubation period, ND@Au-Tf appeared intracellularly, whereas ND@Au- nT particles co-registered with the cell membrane (Figure A1.6), indicating that although both formulations are able to bind to cellular membranes, only ND@Au-Tf are internalized. Because our cell binding studies showed that ND@Au-Tf particles specifically bind TfR, ND@Au-Tf internalization is likely mediated by TfR endocytosis.
Co-localization studies were carried out, further indicating that non-targeted ND@Au-nT are co-localized with the cell membrane with Mander’s coefficient M2 = 0.99. In stark contrast, only a fraction of the targeted ND@Au-Tf formulation remained bound to the cell membrane (M2 = 0.21), while the remainder translocated inside the cell. Our flow cytometry and confocal imaging results are in agreement, and the data support ND@Au-
Tf targeting of TfR on cancer cells, leading to receptor-mediated internalization.
258 APPENDIX I: NANODIAMONDS FOR CANCER THERAPY
Figure A1.6. SKBR3 cell interactions with particles observed by confocal microscopy. The particles were incubated with SKBR3 cells for 16 h, fixed, stained, and imaged. ND@Au-Tf (A+C) and ND@Au- nT (B+D) are pseudo-colored in green (imaged based on the Alexa Fluor 647 label), nuclei are shown in blue (stained with DAPI), and cell membranes are shown in red (stained with WGA-A555); the scale bar is 30 μm. C+D shows 3D reconstruction of single cells: the top panel shows all channels, the middle panel depicts co-localization of the particles and WGA signals (M = Mander’s coefficient of co-localization determined using ImageJ software), and the bottom panel shows ND signals.
259 APPENDIX I: NANODIAMONDS FOR CANCER THERAPY
A1.3.5 Toxicity study
The toxicity of the ND@Au-Tf particles themselves was investigated in SKBR3 cells, using an XTT cell proliferation assay. After 3 or 24 h incubation with ND@Au-Tf particles, we did not observe significant differences in cell viability compared to non- treated cells, indicating that ND@Au-Tf particles are not cytotoxic (Figure A1.7).
Figure A1.7. Viability of SKBR3 cells after exposure to ND@Au-Tf. ND@Au-Tf (red) were incubated with SKBR3 cells for 3 or 24 h. Cell viability (%) was evaluated using an XTT assay and compared to a cell-only control (gray). Toxicity is not indicated. Although reduced cell viability was indicated for cells exposed to ND@Au-Tf for a 3-h time frame, the toxic effects were not statistically significant, nor were toxic effects observed at longer exposure times (24 h).
A1.3.6 Laser ablation
The ability of GNSs to kill cancer cells upon red laser irradiation was demonstrated in vitro. HeLa cells were incubated with Nd@Au-Tf, and after successive washing, irradiated with a Ti:Sapphire pulse laser. After a one-minute irradiation, cells were incubated for 24 h, and their viability was estimated using luciferase assay (Figure
A1.8). Exposing HeLa cells to ND@Au-Tf did not affect their viability, supporting the
260 APPENDIX I: NANODIAMONDS FOR CANCER THERAPY non-toxicity of these particles. Laser irradiation of cells also had no influence on their viability. Only cells exposed to both nanoparticles and laser were affected and—after only one minute of irradiation—completely killed. The laser power we used (37 W/cm2) was of the same order of magnitude as values described in the literature for in vitro experiments [9, 10, 53].
Figure A1.8. Laser ablation of HeLa cells incubated with ND@Au-Tf nanoparticles. Cell viability was estimated by luciferase assay with 24 h delay after 1 min irradiation with 37 W/cm2 intensity. The viability of cells treated with ND@Au-Tf and laser was ~0.15%.
A1.4 Conclusions
In summary, we synthesized a novel plasmonic nanomaterial consisting of a diamond core coated with a silica layer and encapsulated with a thin GNS. PEG chains were attached to the surface of GNSs using lipoic acid as an anchor. The other ends of the
PEG chains were further functionalized with Alexa Fluor 647 and modified transferrin via click chemistry. Transferrin-labeled ND-based GNSs (ND@Au-Tf) target transferrin receptors, which are overexpressed on many cancer cell types. We found that ND@Au- 261 APPENDIX I: NANODIAMONDS FOR CANCER THERAPY
Tf bind and are internalized into human SKBR3 breast cancer cells and HeLa cervical cancer cells. Cell viability assays were also conducted using XTT assay; toxic effects were not observed after 3 or 24 h incubation periods. We also demonstrated the ability of the prepared GNSs to kill cancer cells upon red laser irradiation in vitro. HeLa cells were completely killed after a one-minute irradiation with a pulse 750-nm pulse laser (37
W/cm2), while no harm was caused to cells by irradiation itself or by the presence of
GNSs without irradiation.
This work lays the foundation for the multi-step synthetic route leading to ND core plasmonic nanoparticles and is the first stepping stone toward translational research.
Future work will focus on targeted therapeutic studies of plasmonic NDs in vivo.
Specifically, studies will set out to gain and understanding on the therapeutic efficacy in the context of biodistribution and overall biocompatibility of the materials. At the same time, studies will asses the photophysical interactions of the fluorescent nitrogen-vacancy
(NV) centers in NDs with the gold plasmonic shell.
It is well recognized that cancer nanotechnology holds great promise in modern medicine, and several nanoparticles have advanced into clinical application [54-56].
While the development pipeline with new nanomaterials-based technologies is moving rapidly, the fundamental understanding of the nanomaterial’s in vivo fate; i.e., the biodistribution, clearance or persistence, pharmacokinetics and pharmacodynamics, is often lacking. However, detailed understanding of the biological behaviour, the interplay of tissue-targeting and immune surveillance in the context of imaging sensitivity and therapeutic efficacy, is imperative for rapid clinical viability and success of plasmonic nanostructures [57]. The combined knowledge of the biological properties and
262 APPENDIX I: NANODIAMONDS FOR CANCER THERAPY diagnostic/therapeutic potential will help identify a suitable niche application harnessing the unique properties of the proposed material.
A1.5 Works cited in this appendix
1. Khlebtsov NG, Dykman LA. Optical properties and biomedical applications of plasmonic nanoparticles. Journal of Quantitative Spectroscopy & Radiative Transfer 2010;111:1-35.
2. Jones MR, Osberg KD, Macfarlane RJ, Langille MR, Mirkin CA. Templated techniques for the synthesis and assembly of plasmonic nanostructures. Chem Rev 2011;111:3736-827.
3. Daniel MC, Astruc D. Gold nanoparticles: assembly, supramolecular chemistry, quantum-size-related properties, and applications toward biology, catalysis, and nanotechnology. Chem Rev 2004;104:293-346.
4. Henry AI, Bingham JM, Ringe E, Marks LD, Schatz GC, Van Duyne RP. Correlated Structure and Optical Property Studies of Plasmonic Nanoparticles. Journal of Physical Chemistry C 2011;115:9291-305.
5. Agrawal A, Huang S, Wei Haw Lin A, Lee MH, Barton JK, Drezek RA, et al. Quantitative evaluation of optical coherence tomography signal enhancement with gold nanoshells. J Biomed Opt 2006;11:041121.
6. Kah JC, Chow TH, Ng BK, Razul SG, Olivo M, Sheppard CJ. Concentration dependence of gold nanoshells on the enhancement of optical coherence tomography images: a quantitative study. Appl Opt 2009;48:D96-D108.
7. Chen YS, Frey W, Kim S, Kruizinga P, Homan K, Emelianov S. Silica-coated gold nanorods as photoacoustic signal nanoamplifiers. Nano Lett 2011;11:348-54.
8. Stern JM, Stanfield J, Kabbani W, Hsieh JT, Cadeddu JA. Selective prostate cancer thermal ablation with laser activated gold nanoshells. J Urol 2008;179:748-53.
9. Lu W, Xiong C, Zhang G, Huang Q, Zhang R, Zhang JZ, et al. Targeted photothermal ablation of murine melanomas with melanocyte-stimulating hormone analog-conjugated hollow gold nanospheres. Clin Cancer Res 2009;15:876-86.
263 APPENDIX I: NANODIAMONDS FOR CANCER THERAPY
10. Huang X, El-Sayed IH, Qian W, El-Sayed MA. Cancer cell imaging and photothermal therapy in the near-infrared region by using gold nanorods. J Am Chem Soc 2006;128:2115-20.
11. Wu G, Mikhailovsky A, Khant HA, Fu C, Chiu W, Zasadzinski JA. Remotely triggered liposome release by near-infrared light absorption via hollow gold nanoshells. J Am Chem Soc 2008;130:8175-7.
12. Bardhan R, Mukherjee S, Mirin NA, Levit SD, Nordlander P, Halas NJ. Nanosphere-in-a-Nanoshell: A Simple Nanomatryushka. Journal of Physical Chemistry C 2010;114:7378-83.
13. Bardhan R, Lal S, Joshi A, Halas NJ. Theranostic nanoshells: from probe design to imaging and treatment of cancer. Acc Chem Res 2011;44:936-46.
14. Gobin AM, Lee MH, Halas NJ, James WD, Drezek RA, West JL. Near-infrared resonant nanoshells for combined optical imaging and photothermal cancer therapy. Nano Lett 2007;7:1929-34.
15. Chow EK, Zhang XQ, Chen M, Lam R, Robinson E, Huang H, et al. Nanodiamond therapeutic delivery agents mediate enhanced chemoresistant tumor treatment. Sci Transl Med 2011;3:73ra21.
16. Vaijayanthimala V, Cheng PY, Yeh SH, Liu KK, Hsiao CH, Chao JI, et al. The long-term stability and biocompatibility of fluorescent nanodiamond as an in vivo contrast agent. Biomaterials 2012;33:7794-802.
17. Hui YY, Cheng CL, Chang HC. Nanodiamonds for optical bioimaging. Journal of Physics D-Applied Physics 2010;43.
18. Slegerova J, Hajek M, Rehor I, Sedlak F, Stursa J, Hruby M, et al. Designing the nanobiointerface of fluorescent nanodiamonds: highly selective targeting of glioma cancer cells. Nanoscale 2015;7:415-20.
19. Slegerova J, Rehor I, Havlik J, Raabova H, Muchova E, Cigler P. Nanodiamonds as Intracellular Probes for Imaging in Biology and Medicine. Intracellular Delivery Ii: Fundamentals and Applications 2014;7:363-401.
20. Lim TS, Fu CC, Lee KC, Lee HY, Chen K, Cheng WF, et al. Fluorescence enhancement and lifetime modification of single nanodiamonds near a nanocrystalline silver surface. Phys Chem Chem Phys 2009;11:1508-14.
21. Schietinger S, Barth M, Aichele T, Benson O. Plasmon-enhanced single photon emission from a nanoassembled metal-diamond hybrid structure at room temperature. Nano Lett 2009;9:1694-8.
264 APPENDIX I: NANODIAMONDS FOR CANCER THERAPY
22. Hui YY, Lu YC, Su LJ, Fang CY, Hsu JH, Chang HC. Tip-enhanced sub- diffraction fluorescence imaging of nitrogen-vacancy centers in nanodiamonds. Applied Physics Letters 2013;102.
23. Barth M, Schietinger S, Schroder T, Aichele T, Benson O. Controlled coupling of NV defect centers to plasmonic and photonic nanostructures. Journal of Luminescence 2010;130:1628-34.
24. Chen GX, Liu Y, Song M, Wu BT, Wu E, Zeng HP. Photoluminescence Enhancement Dependent on the Orientations of Single NV Centers in Nanodiamonds on a Gold Film. Ieee Journal of Selected Topics in Quantum Electronics 2013;19.
25. Chi YZ, Chen GX, Jelezko F, Wu E, Zeng HP. Enhanced Photoluminescence of Single-Photon Emitters in Nanodiamonds on a Gold Film. Ieee Photonics Technology Letters 2011;23:374-6.
26. Rehor I, Mackova H, Filippov SK, Kucka J, Proks V, Slegerova J, et al. Fluorescent Nanodiamonds with Bioorthogonally Reactive Protein-Resistant Polymeric Coatings. Chempluschem 2014;79:21-4.
27. Zhang BL, Fang CY, Chang CC, Peterson R, Maswadi S, Glickman RD, et al. Photoacoustic emission from fluorescent nanodiamonds enhanced with gold nanoparticles. Biomedical Optics Express 2012;3:1662-9.
28. Ismaili H, Workentin MS. Covalent diamond-gold nanojewel hybrids via photochemically generated carbenes. Chemical Communications 2011;47:7788- 90.
29. Cheng LC, Chen HM, Lai TC, Chan YC, Liu RS, Sung JC, et al. Targeting polymeric fluorescent nanodiamond-gold/silver multi-functional nanoparticles as a light-transforming hyperthermia reagent for cancer cells. Nanoscale 2013;5:3931-40.
30. Liu YL, Sun KW. Plasmon-enhanced photoluminescence from bioconjugated gold nanoparticle and nanodiamond assembly. Applied Physics Letters 2011;98.
31. Rehor I, Slegerova J, Kucka J, Proks V, Petrakova V, Adam MP, et al. Fluorescent Nanodiamonds Embedded in Biocompatible Translucent Shells. Small 2014;10:1106-15.
32. Howarth M, Liu WH, Puthenveetil S, Zheng Y, Marshall LF, Schmidt MM, et al. Monovalent, reduced-size quantum dots for imaging receptors on living cells. Nature Methods 2008;5:397-9.
33. Rehor I, Cigler P. Precise estimation of HPHT nanodiamond size distribution based on transmission electron microscopy image analysis. Diamond and Related Materials 2014;46:21-4. 265 APPENDIX I: NANODIAMONDS FOR CANCER THERAPY
34. Mei BC, Susumu K, Medintz IL, Delehanty JB, Mountziaris TJ, Mattoussi H. Modular poly(ethylene glycol) ligands for biocompatible semiconductor and gold nanocrystals with extended pH and ionic stability. Journal of Materials Chemistry 2008;18:4949-58.
35. Susumu K, Mei BC, Mattoussi H. Multifunctional ligands based on dihydrolipoic acid and polyethylene glycol to promote biocompatibility of quantum dots. Nature Protocols 2009;4:424-36.
36. Banerjee D, Liu AP, Voss NR, Schmid SL, Finn MG. Multivalent Display and Receptor-Mediated Endocytosis of Transferrin on Virus-Like Particles. Chembiochem 2010;11:1273-9.
37. Mohan N, Chen CS, Hsieh HH, Wu YC, Chang HC. In Vivo Imaging and Toxicity Assessments of Fluorescent Nanodiamonds in Caenorhabditis elegans. Nano Letters 2010;10:3692-9.
38. Graf C, Vossen DLJ, Imhof A, van Blaaderen A. A general method to coat colloidal particles with silica. Langmuir 2003;19:6693-700.
39. Brinson BE, Lassiter JB, Levin CS, Bardhan R, Mirin N, Halas NJ. Nanoshells Made Easy: Improving Au Layer Growth on Nanoparticle Surfaces. Langmuir 2008;24:14166-71.
40. Oldenburg SJ, Averitt RD, Westcott SL, Halas NJ. Nanoengineering of optical resonances. Chemical Physics Letters 1998;288:243-7.
41. Rasch MR, Sokolov KV, Korgel BA. Limitations on the Optical Tunability of Small Diameter Gold Nanoshells. Langmuir 2009;25:11777-85.
42. Hong V, Presolski SI, Ma C, Finn MG. Analysis and Optimization of Copper- Catalyzed Azide-Alkyne Cycloaddition for Bioconjugation. Angewandte Chemie- International Edition 2009;48:9879-83.
43. Prokop A, Davidson JM. Nanovehicular intracellular delivery systems. Journal of Pharmaceutical Sciences 2008;97:3518-90.
44. Presolski SI, Hong VP, Finn MG. Copper-Catalyzed Azide-Alkyne Click Chemistry for Bioconjugation. Curr Protoc Chem Biol 2011;3:153-62.
45. Daniels TR, Delgado T, Helguera G, Penichet ML. The transferrin receptor part II: Targeted delivery of therapeutic agents into cancer cells. Clinical Immunology 2006;121:159-76.
46. Daniels TR, Bernabeu E, Rodriguez JA, Patel S, Kozman M, Chiappetta DA, et al. The transferrin receptor and the targeted delivery of therapeutic agents against cancer. Biochimica Et Biophysica Acta-General Subjects 2012;1820:291-317.
266 APPENDIX I: NANODIAMONDS FOR CANCER THERAPY
47. Huang RK, Steinmetz NF, Fu CY, Manchester M, Johnson JE. Transferrin- mediated targeting of bacteriophage HK97 nanoparticles into tumor cells. Nanomedicine 2011;6:55-68.
48. Iinuma H, Maruyama K, Okinaga K, Sasaki K, Sekine T, Ishida S, et al. Intracellular targeting therapy of cisplatin-encapsulated transferrin-polyethylene glycol liposome on peritoneal dissemination of gastric cancer. International Journal of Cancer 2002;99:130-7.
49. Weng MF, Chang BJ, Chiang SY, Wang NS, Niu H. Cellular uptake and phototoxicity of surface-modified fluorescent nanodiamonds. Diamond and Related Materials 2012;22:96-104.
50. Chang BM, Lin HH, Su LJ, Lin WD, Lin RJ, Tzeng YK, et al. Highly Fluorescent Nanodiamonds Protein-Functionalized for Cell Labeling and Targeting. Advanced Functional Materials 2013;23:5737-45.
51. Kang KA, Wang JT, Jasinski JB, Achilefu S. Fluorescence Manipulation by Gold Nanoparticles: From Complete Quenching to Extensive Enhancement. Journal of Nanobiotechnology 2011;9.
52. Degennes PG. Polymers at an Interface - a Simplified View. Advances in Colloid and Interface Science 1987;27:189-209.
53. Bernardi RJ, Lowery AR, Thompson PA, Blaney SM, West JL. Immunonanoshells for targeted photothermal ablation in medulloblastoma and glioma: an in vitro evaluation using human cell lines. Journal of Neuro-Oncology 2008;86:165-72.
54. Farokhzad OC, Langer R. Impact of Nanotechnology on Drug Delivery. Acs Nano 2009;3:16-20.
55. Ferrari M. Cancer nanotechnology: Opportunities and challenges. Nature Reviews Cancer 2005;5:161-71.
56. Chow EKH, Ho D. Cancer Nanomedicine: From Drug Delivery to Imaging. Science Translational Medicine 2013;5.
57. Webb JA, Bardhan R. Emerging advances in nanomedicine with engineered gold nanostructures. Nanoscale 2014;6:2502-30.
267 APPENDIX II: VIRUS-BASED VACCINES
Appendix II: Virus-based nanoparticles as vaccines
The material in this chapter is adapted with permission from: Lee, K.L., Twyman,
R.M., Fiering, S., Steinmetz, N.F. Virus-based nanoparticles as platform technologies for modern vaccines. WIREs Nanomed. Nanobiotechnol. 2016. doi: 10.1002/wnan.1383
Copyright 2016 John Wiley and Sons.
A2.1 Introduction
Vaccines are designed to elicit a strong immune response and to provide long- lasting protective immunity by generating neutralizing antibodies, activating cellular immunity and inducing immune memory [1, 2]. The earliest reports of vaccination were subjects protected against smallpox by exposure to powders from infected scabs.
However, Edward Jenner presented the first formal description of a vaccine in 1798, when he observed that milkmaids previously infected with the less virulent Cowpox virus were no longer susceptible to smallpox. In 1967, the World Health Organization (WHO) oversaw a worldwide smallpox eradication program, which was completed by 1980 [3].
Since then, eradication programs have been established for other diseases such as polio, measles, mumps, rubella, and malaria [4, 5]. Vaccines have also been developed against other prevalent infectious diseases, such as hepatitis B, rabies, anthrax, and cholera [6-9].
The development of vaccines has achieved an immense socioeconomic impact by reducing the burden of erstwhile pandemic diseases responsible for widespread morbidity and mortality. Even so, several major pathogens cannot yet be controlled by vaccines including Human immunodeficiency virus (HIV) and hemorrhagic fever viruses, such as
268 APPENDIX II: VIRUS-BASED VACCINES those responsible for the recent Ebola outbreak affecting West African countries. More recently vaccines have also been developed against non-infectious diseases including cancer and chronic disorders.
A2.2 Virus-based nanoparticles as platform technologies
Nanoparticle-based vaccines have been developed using a diverse range of materials (Figure A2.1), including synthetic particles (e.g. gold, polymers or lipid micelles) and biological particles (e.g. nucleic acids and proteins, including viruses) [10,
11]. We consider two broad types of particles in the latter category: virus-based nanoparticles (VNPs) that feature a modified capsid encapsulating the virus genome, and virus-like nanoparticles (VLPs) that comprise protein components alone.
Figure A2.1. Nanoparticles as vaccination platforms. 269 APPENDIX II: VIRUS-BASED VACCINES
Virus-based materials have many beneficial properties. Their proteinaceous, highly-ordered, multivalent structures, when combined with an appropriate adjuvant, often elicit robust cellular and humoral immune responses [12]. They display antigens in a repetitive array (which promotes B cell crosslinking and subsequent activation) and pathogen-associated molecular patterns (PAMPs) that induce stronger and longer-lasting antigen-specific immune responses than soluble antigens [13-15]. The single-stranded viral RNA (ssRNA) found in VNPs is also a PAMP, and this is a natural ligand for Toll- like receptors 7 and 8 that induce cytokine expression [16][17-19]. The size range of virus particles (20–500 nm) means they are efficiently taken up by antigen presenting cells (APCs), including dendritic cells (DCs) and other phagocytes, thus stimulating T cells [20, 21].
Figure A2.2. Categories of viral vaccines. UV = ultraviolet; VNP = viral nanoparticle; VLP = virus-like particle.
Viral vaccines can be divided into four categories (Figure A2.2): live-attenuated, inactivated, subunit vaccines, and native or recombinant VNP/VLP structures. The latter are considered safer because there is no risk of virulence, yet stronger than inactivated viruses or subunit vaccines because they induce a robust immune response without 270 APPENDIX II: VIRUS-BASED VACCINES multiple doses [22, 23]. Native VLPs lack the viral genome but are otherwise identical to the infectious virus, making them highly immunogenic but unable to replicate. These are particularly suitable when the native virus replicates and causes disease in humans. VNPs retain the genome and are therefore easier to produce by relying on natural virus replication. This format is particularly suitable when the native virus does not replicate in humans, i.e. bacteriophage and plant viruses. Recombinant VLP/VNP formats add an important further layer of advantages because they can be engineered to present antigenic epitopes of a counterpart virus or any other disease-associated antigen. VLPs and VNPs can be manufactured in heterologous production systems, including plants, mammalian cells, yeast and bacteria [24].
A2.3 Chemical and genetic engineering of virus-based scaffolds
Viruses comprise many identical copies of one or more coat proteins arranged in helical or icosahedral symmetry to form a capsid that encapsulates the genome. The structure of many virus capsids has been solved at atomic resolution, allowing site- specific modification and the multivalent display of antigenic epitopes on particular surface loops or the N/C-terminal region of the coat protein. Epitopes and/or other immunostimulatory molecules can be introduced by chemical engineering
(bioconjugation) of particular residues (Figure A2.3) or genetic engineering of the coat protein sequence (Figure A2.4).
271 APPENDIX II: VIRUS-BASED VACCINES
A2.3.1 Chemical conjugation strategies
Antigenic peptide sequences can be added to a virus coat protein by chemical modification strategies that target five of the 20 naturally occurring amino acids: lysine
(amine functional group), glutamic and aspartic acid (carboxylate functional group), cysteine (thiol functional group), and tyrosine (hydroxyl functional group). Lysine residues contain a highly nucleophilic amine that reacts with isothiocyanate or N- hydroxysuccinimide (NHS) esters. Amines can also be covalently attached to carboxylate groups through the formation of an amide (peptide) bond, facilitated by a carboxylate- selective coupling agent such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).
Glutamic and aspartic acid residues contain carboxylate groups that can be modified using EDC to react with amine-functionalized peptides resulting in the formation of a stable amide bond, in the mirror image of the reaction described above. Cysteine residues contain thiol groups that can be reacted with haloacetyls or maleimides. Finally, tyrosine residues contain a phenolic hydroxyl group, which can be modified using diazonium coupling strategies, although this is more complex than the other reactions listed above.
272 APPENDIX II: VIRUS-BASED VACCINES
Figure A2.3. Chemical conjugation strategies (bionconjugation). NHS = N-hydroxysuccinimde; EDC = 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide.
273 APPENDIX II: VIRUS-BASED VACCINES
As well as these direct bioconjugation strategies, bifunctional linkers can be used to introduce additional functionalities that are not naturally found in virus coat proteins.
Bio-orthogonal reactions, including ‘click chemistries’ such as Cu(I)-catalyzed azide- alkyne cycloaddition are particularly useful because the kinetics of the reaction are much more efficient than standard coupling. Ligation handles can be introduced via the bioconjugation of an azide or alkyne-NHS ester to a lysine side chain, or through the incorporation of non-natural amino acids in vitro. The diverse chemistries used to engineer viruses have been reviewed in detail [25].
A2.3.2 Genetic engineering strategies
Unlike synthetic nanoparticles, VNPs can be modified not only chemically but also genetically, i.e. the nucleic acid sequence encoding the coat protein can be changed to exchange particular amino acids or introduce additional contiguous amino acids to form linear epitopes. Three major approaches are used to insert additional peptides into the virus coat protein, resulting in a coat protein fusion or chimera: direct fusion, linker fusion, and the “protein overcoat” strategy. In the direct fusion approach, the foreign peptide is linked directly to the N-terminus [26-28] or C-terminus [28-31] of the coat protein or inserted into flexible surface loops presented on the capsid surface [29, 32].
Although the external surface is usually chosen for the presentation of native antigens recognized by B cells, the internal surface may be more suitable in some applications that involve the presentation of processed peptides [33, 34]. In contrast, linker fusion involves the inclusion of a short sequence of amino acids (e.g. multiple glycine residues) between the foreign peptide and the end of the coat protein to allow flexibility. Finally, the
274 APPENDIX II: VIRUS-BASED VACCINES
“protein overcoat” strategy places the Foot and mouth disease virus (FMDV) 2A sequence between the foreign peptide and coat protein sequences, causing an inconsistent ribosomal skip during translation. The outcome is a mixture of native coat proteins and fusion proteins, which is useful if the inserted sequence is so large that its presence on every copy of the coat protein would prevent virus assembly [35].
Figure A2.4. Genetic engineering strategies for the display of epitopes on viral coat proteins.
A2.4 VNP and VLP vaccines and immunotherapies
The first vaccines were developed against infectious diseases and likewise the first VLP and VNP vaccines were developed as strategies to contain the disease caused by the corresponding native form of the virus. However, as chemical and genetic engineering strategies have become more sophisticated, VLPs and VNPs have been adapted into platform technologies for the presentation of more diverse antigens, including abnormal self-proteins that can be used to treat chronic diseases and cancer.
Several key vaccines based on VLPs or chimeric VNPs are summarized in Table A2.1.
275 APPENDIX II: VIRUS-BASED VACCINES
Table A2.1. Key VLP/VNP-based vaccines approved or under development.
Vaccine Target Platform Composition Stage of References Development HIV AP205 gp41 epitope Animal [36] studies HIV PVX gp41 epitope Animal [27] studies HIV BPV-1 CCR5 peptide Animal [37] studies HIV Qβ CCR5 peptides NHP studies [38] HIV Canarypox env, gag, pol Testing in [39-43] genes + humans AIDSVAX (gp120) HIV/SIV RABV SIV envelope Animal [44] studies HIV/SIV Virosomes gp41 epitopes NHP studies [45] Ebola Ebola VP40 and GP Animal [46, 47] studies Ebola Ebola GP, NP, and NHP studies [48] VP40 Ebola Ebola EBOVΔVP30 NHP studies [49] Ebola rVSV GP Testing in [50] humans Ebola RABV GP NHP Studies [51, 52] Sudan virus and RABV GP Animal [52] Marburg virus studies Influenza Influenza HA, NA, and Animal [53] (pandemic) M1 from H1N1 studies Influenza Influenza HA and NA Animal [54] (pandemic) from H7N9; studies M1 from H5N1 Influenza Influenza HA and NA Testing in [55] (pandemic) from H1N1; humans M1 from H5N1 Influenza Influenza H5 and H1 Testing in [56] (pandemic) humans Influenza Influenza HA, NA, and Animal [57] (universal M1 from H5N1 studies vaccine) Influenza HBc HA Animal [58] (universal studies vaccine) Influenza Dd M1 Cell studies [59] (universal 276 APPENDIX II: VIRUS-BASED VACCINES vaccine) Influenza PapMV M2e Animal [60] (universal studies vaccine) Influenza IBDV HA and M2 Animal [61] (universal from H1N1 studies vaccine) Influenza PapMV NP Animal [62] (universal studies vaccine) Influenza PVX NP Animal [63] (universal studies vaccine) Influenza P22 NP Animal [34] (universal studies vaccine) Influenza sHSP n/a Animal [64, 65] (universal studies vaccine) Leukemia TMV TACA Animal [66] studies Leukemia Qβ TACA Animal [67] studies Melanoma TMV p15e and Trp2 Animal [68] studies Lymphoma PVX Id Animal [69] studies Ductal BPV MUC-1 Animal [70] adenocarcinoma studies HPV HPV-16, -18 L1 Clinically Cervarix ® (prophylactic) available HPV HPV-6, -11, - L1 Clinically Gardasil® (prophylactic) 16, -18 available HPV MS2 L2 from HPV- Animal [71, 72] (prophylactic) 16 and -31 studies HPV PP7 L2 from HPV- Animal [72] (prophylactic) 1, -16, and -18 studies HPV HPV-18 L1 L2 from HPV- Animal [73] (prophylactic) VLP 18, -45, and -59 studies HPV HPV-16 E7 Testing in [74] (therapeutic) humans HPV HPV-16 L1 E6 and E7 Animal [75] (therapeutic) VLP studies HBV HBV HBsAg Clinically Recombivax HB (prophylactic) available HBV HBc HBx-derived Animal [76]
277 APPENDIX II: VIRUS-BASED VACCINES
(therapeutic) cytotoxic T studies lymphocyte epitopes and PADRE HER-2+ breast IRIV P4, P6, and P7 Testing in [77] cancer humans HER-2+ breast Influenza GPI-HER-2 Animal [78] cancer studies + HER-2 breast MPyV HER-21-683 Animal [79] cancer studies HER-2+ breast T7 p66 Animal [80] cancer studies HER-2+ breast PVX P4 Animal [81] cancer studies Nicotine Qβ Nicotine Testing in [82] addiction humans Nicotine dAd5 AM1 Animal [83] addiction studies Cocaine dAd5 GNC Animal [84] addiction studies Cocaine dAd5 GNE Animal [85] addiction studies Hypertension Qβ Angiotensin II Testing in [28] peptide (8 aa) humans Alzheimer’s HPV-16 Aβ peptides Animal [86] disease studies Alzheimer’s Qβ Aβ1-9 Animal [86] disease studies Alzheimer’s Qβ Aβ1-6 Testing in [87, 88] disease humans Alzheimer’s HBcΔ Aβ1-15 Animal [89] disease studies Alzheimer’s BPV1 Aβ1-9 Animal [90] disease studies
A2.4.1 Infectious diseases
HIV
HIV (Figure A2.5) is unusual in that it primarily attacks the immune system and therefore destroys the very cells whose function is to neutralize it. By disabling the immune system, HIV not only achieves a successful infection but it also renders the body
278 APPENDIX II: VIRUS-BASED VACCINES susceptible to other adventitious pathogens, i.e. acquired immunodeficiency syndrome
(AIDS). There is no cure for HIV/AIDS. More than 35 million people are currently infected with HIV, two thirds of the infected population living in sub-Saharan Africa [91].
The current best treatment option is highly active antiretroviral therapy, a cocktail of drugs consisting of a non-nucleoside reverse transcriptase inhibitor and two nucleoside analog reverse transcriptase inhibitors [92]. Early HIV vaccine candidates based on inactivated or attenuated viruses were ineffective or unsafe [93, 94]. More recent vaccine development strategies have focused on eliciting both humoral and cell-mediated responses targeting HIV envelope proteins.
Recombinant VLPs and VNPs displaying full-length HIV envelope proteins, individual glycoproteins, glycoprotein precursors or fragments thereof, include carriers such as Flock House virus (FHV) [95], Hepatitis B virus (HBV) [96], papillomaviruses
[37], bacteriophages Qβ, AP205 [36] and MS2 [97], and plant viruses such as Tobacco mosaic virus (TMV) [98, 99] and Potato virus X (PVX) [27]. The membrane-proximal external region (MPER) of gp41 can be recognized and neutralized by monoclonal antibodies thus providing a good target for a vaccine candidate [36]. Accordingly, a set of gp41 peptides chemically conjugated to VLPs derived from phage AP205 elicited high- titer, peptide-specific antibodies in mice. Depending on the peptide, sera were able to neutralize a highly-sensitive laboratory strain of HIV-1 and a less-sensitive primary isolate, but not a clade C primary isolate. Some sera exhibited antibody-dependent cell- mediated cytotoxicity (ADCC) in infected cells, indicating that ADCC epitopes are most likely located in the distal region of gp41 [36]. The highly conserved epitope of gp41
(ELDKWA) has been genetically fused to the N-terminus of PVX, among other
279 APPENDIX II: VIRUS-BASED VACCINES platforms [27, 100, 101]. Sera from mice immunized with these chimeric virus particles contained high IgG titers specific for HIV-1 MN gp160-derived synthetic peptide (H66), and were able to neutralize HIV-1. Additionally, human DCs pulsed with the vaccine triggered the proliferation of peripheral blood lymphocytes in vivo [27]. Multiple gp41 epitopes such as a trimeric recombinant gp41 (rgp41), which contains several conserved gp41epitopes, were conjugated to influenza virosomes. Vaccinated rhesus monkeys were challenged intravaginally 13 times with a heterologous simian HIV (SHIV). All subjects vaccinated through intramuscular or intranasal route were protected from challenge, whereas only 50% of the intramuscular-only group was protected [45].
Figure A2.5. Structure of HIV-1. Structural proteins in blue, viral enzymes in magenta, accessory proteins in green, and viral RNA in yellow. (1) Envelope glycoprotein spike, comprised of transmembrane glycoprotein gp41 and external envelope glycoprotein gp41; (2) lipid membrane; (3) capsid; (4) matrix; (5) viral RNA (Adapted from Johnson GT, Goodsell DS, Autin L, Forli S, Sanner MF, Olson AJ. 3D molecular models for whole HIV-1 virions generated with cellPACK. Faraday Discuss 2014, 169:23-44 – Published by The Royal Society of Chemistry). 280 APPENDIX II: VIRUS-BASED VACCINES
Other HIV vaccine development approaches include the targeting of host cell receptors such as the C-C chemokine receptor CCR5 co-receptor used by macrophage strains of HIV-1 [102-106]. Bovine papillomavirus type 1 (BPV-1) VLPs were engineered to express CCR5 peptides. Vaccinated mice produced high antibody titers against CCR5, and functional studies demonstrated that sera displaced the native CCR5 ligand in a competition assay. Most importantly, sera from immunized mice neutralized
HIV-1 in cells transfected with a human-mouse chimeric receptor of CCR5 [37]. CCR5 peptides have also been conjugated to bacteriophage Qβ. Two peptides, representing the
N-terminus (EC1) or second extracellular loop (ECL2) of macaque CCR5 (mCCR5), were conjugated to Qβ and administered to rhesus macaques. Animals immunized with
Qβ-EC1 and Qβ-ECL2 produced high titers of anti-CCR5 antibodies. When vaccinated animals were challenged with SIV, the viral load was lower than in non-vaccinated controls [38].
Another promising strategy is the combination of ALVAC-HIV, a vaccine based on Canarypox virus (vCP1521), and AIDSVAX (VaxGen), which consists of gp120 from two different HIV strains [39, 43]. Unlike the vaccines discussed above, the ALVAC
Canarypox virus vector contains the HIV env, gag, and pol genes [40]. The two treatments were tested together in a clinical trial in Thailand (RV 144). Vaccinated subjects showed a 31% lower rate of HIV infection compared to the placebo group [43].
Despite this, ALVAC-HIV alone has shown promise as a pediatric HIV therapy, involving infants born to HIV-positive mothers. Low levels of binding antibodies were detected in one subject as expected because the subjects did not receive a gp120 boost
[40]. The addition of recombinant glycoprotein subunit vaccine (rgp120) to ALVAC-HIV
281 APPENDIX II: VIRUS-BASED VACCINES resulted in higher levels of HIV-specific serum antibodies in infants that were distinguishable from maternal antibodies. Additionally, 50% of subjects who received both ALVAC-HIV and rgp120 generated neutralizing antibodies against homologous strains of HIV [41].
Ebola virus disease and related diseases
Ebola virus disease is caused by four different filoviruses of the genus Ebolavirus:
Bundibugyo ebolavirus, Sudan ebolavirus, Tai Forest ebolavirus, and the eponymous
Ebola virus (formerly Zaire ebolavirus) which is the most dangerous and prolific (Figure
A2.6). The ease of infection [107, 108] and lack of clinically approved treatment produces mortality rates of up to 90% [109].
VLP/VNP vaccines against Ebola virus are currently in the development pipeline, using either complete Ebola VLPs or specific components such as the viral matrix protein
(VP40), nucleoprotein (NP) and glycoprotein (GP) displayed on other viruses.
Recombinant VLPs containing Ebola virus VP40 and GP were constructed using a baculovirus system. The VLPs yielded high levels of GP-specific antibodies in mice, particularly the IgG2a subtype which is needed to achieve protective immunity.
Additionally, VLPs induced the secretion of IL-6, IL-10, IL-12, and TNFα from DCs, confirming their adjuvant and immunostimulatory properties. Serum from vaccinated mice was also able to block the infection of JC53 cells by a pseudotyped virus [47]. The immunization of rodents with VLPs comprising the viral envelope (including GP, NP, and VP40) has also conferred protection against Ebola challenge [48]. Furthermore,
282 APPENDIX II: VIRUS-BASED VACCINES immunized cynomolgus macaques were completely protected when challenged with
Ebola virus [48]. VLPs based on recombinant Vesicular stomatitis virus (rVSV) expressing Ebola virus glycoproteins were shown to protect mice and non-human primates against a lethal challenge with homologous Ebola virus after a single injection
[110, 111]. Post-exposure protection was also conferred [112]. The rVSV-EBOV vaccine showed a good safety profile in non-human primates and pigs [113, 114] and was able to prevent the disease when administered during an outbreak in a ring vaccination strategy
[50].
Other examples include the application of an inactivated Rabies virus fused to
Ebola virus GP (INAC-RV-GP) resulting in a strong, multivalent humoral response against both viruses in mice and non-human primates, protecting the animals against both diseases [51, 115]. The titer of neutralizing antibodies was increased further by the addition of an adjuvant and resulted in 100% protection from a lethal challenge [52]. The inactivated Rabies virus platform has also been expanded to express the GP from other filoviruses, including Sudan ebolavirus and Marburg virus [52].
283 APPENDIX II: VIRUS-BASED VACCINES
Figure A2.6. Structure of Ebola virus. (Courtesy of David S. Goodsell and RCSB PDB).
284 APPENDIX II: VIRUS-BASED VACCINES
Influenzavirus
Seasonal influenza epidemics cause up to 500,000 deaths every year [116-118] as well as regular pandemics, which can result in millions of deaths in a relatively short time span [117, 119]. Seasonal influenza epidemics are typically caused by human influenzaviruses (Figure A2.7) that have acquired mutations, whereas pandemics occur when the influenzaviruses cross species barriers [120, 121]. Seasonal vaccines are typically based on whole-inactivated viruses or live-attenuated viruses, both of which achieve good protection and significantly improve public health [122]. However, these vaccines are based on hemagglutinin (HA) and neuraminidase (NA), the major targets of the immune system [122]. Epitopes on both proteins are highly prone to genetic drift, leading to the rapid emergence of influenza strains that are not genetically similar to the strains covered by the vaccine [53, 123-125]. Seasonal vaccines must therefore be updated every year, and vaccines against pandemic strains must be developed in response to the pathogen, a reactive strategy that places millions of people at risk. Preclinical and clinical research has focused on the development of a proactive universal influenza vaccine based on more conserved epitopes such as the matrix proteins (M1 and M2) and nucleoprotein (NP) [126, 127]. Other strategies include the development of multivalent
VLPs with HA and/or NA epitopes from diverse strains, combined with immunostimulatory molecules.
Recombinant influenzavirus VLPs based on HA, NA, and M1 have recently been used to develop heterotypic vaccines [53-55, 57]. Studies in mice and ferrets indicate that
H1N1 VLP vaccination protects against challenges with the homologous subtype (H1N1) as well as a heterologous subtype (H5N1), and intranasal administration elicited higher
285 APPENDIX II: VIRUS-BASED VACCINES
IgG and IgA titers than intramuscular vaccination [53]. These VLP vaccines were well- tolerated in a phase II study [55]. Other VLPs have been developed based on the highly pathogenic avian influenza (HPAI) H5N1 and avian-origin influenza A (H7N9) [54, 57].
The H5N1 VLP was composed of HA, NA, and M1 from H5N1, whereas the H7N9 vaccine was composed of HA and NA from H7N9 and M1 from H5N1 [54, 57]. Mice vaccinated with H5N1 VLPs were challenged with homologous or heterologous (H5N8) strains and all survived. Ferrets immunized with H7N9 VLPs plus adjuvant produced high titers of H7N9-specific neutralizing antibodies, and viral loads in the lungs and viral shedding were both reduced compared to controls [54].
Figure A2.7. Structure of influenza A virus. Three viral proteins are on the outer surface of virus particles: haemagglutinin (HA), neuraminidase (NA), and M2. Influenza virus matrix protein M1 associates inside the membrane, and the viral genome is packaged into the particle as a ribonucleoprotein in complex with nucleocapsid protein (NP) and viral polymerases (PA, PB1, PB2). (From Tao YJ, Zheng W. Visualizing the Influenza Genome. Science 2012, 338: 1545-1546. Reprinted with permission from AAAS).
286 APPENDIX II: VIRUS-BASED VACCINES
Influenzavirus epitopes have also been expressed on the surface of VLPs based on
HPV core protein (HBc) [58], Infectious bursal disease virus (IBDV) [61], PVX [63],
Papaya mosaic virus (PapMV) [60, 62], Adenovirus[59], and Simian virus 40 (SV40)
[128]. Several of these platforms were used to display HA, NA, and matrix proteins, similar to the influenzavirus VLPs.
Influenza vaccines can also be developed to promote the formation of inducible bronchus-associated lymphoid tissue (iBALT), which plays a role in adaptive immunity in the lungs, similar to the role of the spleen in primary adaptive immunity [129]. Small heat shock protein (sHSP) cages are structurally similar to VLPs, and when administered to the lung they promote the formation of iBALT, which includes B cells, follicular DCs, and CD4+ and CD8+ T cells. Mice treated with sHSP cages were protected from primary influenzavirus infection, as well as from a secondary infection from a different strain of the virus [65]. The sHSP cages also induced antibody class-switching when mice were challenged with influenzavirus, increasing the amount of IgA and IgG present in the lung
[64].
Seasonal influenza vaccines are usually produced in eggs, or in avian or mammalian cell cultures. However, pandemic strains that develop when viruses cross the species barrier (particularly the avian-to-human barrier) are more difficult to produce in avian cells and alternative production platforms are required. One particularly attractive option is to produce the influenzavirus VLPs in plants. The H5 and H1 proteins have each been successfully expressed in Nicotiana benthamiana plants and can self-assemble into
VLPs. The molecular farming of VLPs has several advantages: in planta production greatly reduces the risks associated with human viruses because plants do not support the
287 APPENDIX II: VIRUS-BASED VACCINES replication of human viruses, and the process is highly scalable. For example, Figure
A2.8 shows the Medicago production facility. Mice immunized with plant-derived
H5-VLPs were protected from homologous and heterologous viral challenge [130].
Furthermore, when the plant-derived VLPs were tested in a phase I clinical trial, none of the subjects developed allergy or hypersensitivity symptoms, and the IgG and IgE responses to plant-derived epitopes returned to baseline after 6 months. Additionally, there was no IgE response to the glycan motif MMXF, which is associated with allergenicity [56], confirming the safety profile of the vaccine.
Figure A2.8. Production facility at Medicago. The photograph was provided courtesy of Medicago Inc.
288 APPENDIX II: VIRUS-BASED VACCINES
A2.4.2 Cancer
Cancer is one of the leading causes of death worldwide, with 14 million new cases diagnosed every year and over 8 million cancer-related deaths. Although cancer includes a diverse spectrum of diseases with different causes, sites of origin, and clinical outcomes, they are all defined by six hallmarks: sustained proliferative signaling, evasion of growth suppressors, promotion of invasion and metastasis, limitless replicative potential, induction of angiogenesis, and resistance to programmed cell death[131]. Some cancers are caused by viral infections and can thus be prevented with vaccines. The first
VLP vaccine offering protection against a cancer-causing virus (Hepatitis B virus, HBV) was approved in 1981 for infants. Two vaccines against Human papillomavirus (HPV) have been approved more recently for the prevention of cervical cancer, or oropharyngeal cancers. In addition to these FDA-approved vaccines, numerous VLP vaccine candidates are being investigated for the prevention or treatment of lymphoma, leukemia, melanoma, and breast cancer.
VLP-based cancer vaccines can also be developed to enhance the tumor-antigen specific T-cell response and elicit antibodies against tumor-specific surface antigens. In addition to papillomaviruses [70], such vaccines have been developed using bacteriophages [67] and plant viruses [66, 68, 69] as delivery platforms because the native viruses do not infect or replicate in human cells so the virus genome can be left intact. The coat proteins of TMV and bacteriophage Qβ have been modified to present the tumor-associated carbohydrate antigen (TACA), which normally has low immunogenicity. TMV-TACA generated much higher titers of antigen-specific antibodies than the soluble form of the antigen, whereas Qβ-TACA elicited a stronger
289 APPENDIX II: VIRUS-BASED VACCINES humoral response to the TACA than the soluble form or TACA attached to other nanoparticles [67]. The resulting IgG antibodies also reacted strongly in vitro against cells expressing the antigen [67].
Tolerance against self-peptides with low immunogenicity can be broken using
VLP/VNP-based immunotherapy platforms. For example, the immunogenicity of melanoma T-cell epitopes p15e and tyrosinase-related protein-2 (Trp2) can be increased by presenting them together on a single bivalent TMV particle, improving cellular immunity and conferring protection against tumor challenge [68]. PVX has been modified with the idiotypic (Id) immunoglobulin from B-cell lymphomas, a weak tumor antigen. When administered to mice, Id-PVX induced high titers of anti-Id antibodies, which prolonged survival after lymphoma challenge [69].
In addition to traditional vaccines using VLP/VNP-based platforms, recent work has investigated the use of in situ vaccination to manipulate identified tumors to counteract local immunosuppression, resulting in systemic anti-tumor immunity. We have shown that VLPs from the cowpea mosaic virus (CPMV) devoid of RNA, LPS, or any other recognized immune adjuvant, stimulate a potent immune-mediated anti-tumor response when introduced into the tumor microenvironment after tumor establishment.
Efficacy was demonstrated in the setting of primary tumors and metastatic disseminated disease using mouse models of melanoma, breast, ovarian, and colon cancer. Importantly, the resulting effect was systemic and durable, resulting in immune-memory that protected mice from re-challenge [132].
The FDA-approved vaccines for HPV are based on papillomavirus VLPs.
However, these VLPs can also be modified to express other tumor antigens, such as 290 APPENDIX II: VIRUS-BASED VACCINES human mucin-1 (MUC-1), which is a marker of ductal adenocarcinoma. BPV-1 particles modified with a MUC-1 epitope were administered to mice, which were later challenged with a MUC-1+ lymphoma cell line. T cells were strongly induced in the vaccinated mice and their tumors grew more slowly, resulting in a smaller tumor mass at the end of the study [70]. We will consider HPV vaccines and hepatocellular carcinoma vaccines in more detail in the following sections because commercial vaccines are already available.
We will also discuss HER-2+ breast cancer vaccines.
Prophylactic vaccines to protect against cervical cancer caused by HPV
Cervical cancer is the fourth most common cancer in women and more than
500,000 new cases are diagnosed each year [133, 134]. HPV, usually sexually transmitted, is implicated in 90% of cervical cancers [135, 136]. Among more than 150 known strains of HPV, up to 20 are designated high risk because they cause almost all cervical cancers
[137]. The two highest-risk strains are HPV-16 and HPV-18, which are responsible for
70% of all cases [138, 139]. There are currently two FDA-approved prophylactic vaccines for HPV: a bivalent vaccine (Cervarix) that protects against strains 16 and 18, and a quadrivalent vaccine (Gardasil) that protects against strains 6, 11, 16, and 18. Both vaccines are based on VLPs composed of the HPV L1 coat protein (Figure A2.9) [71] combined with adjuvants to further boost the immune system. Both vaccines have proven efficacious after a three-dose schedule. However, the L1 protein is not conserved across all serotypes, so these vaccines only offer protection against the specific serotypes within each formulation [140-142].
291 APPENDIX II: VIRUS-BASED VACCINES
Figure A2.9. Human papillomavirus 16 L1 capsid. (viperdb.scripps.edu).
The development of VLPs based on the more conserved L2 coat protein would offer increased cross-protection against multiple serotypes, and the next generation of
HPV vaccines is likely to be based on this principle [72]. L2 is naturally shielded from the immune system [143] but vaccination with L2 can nevertheless protect against a range of HPV serotypes [144-146]. The first vaccines based on L2 were limited in efficacy by low antibody titers and the need to sufficiently protect against all high-risk serotypes [147, 148] so VLPs have been considered as a strategy to overcome these limitations.
Bacteriophage MS2 has been used to express L2 epitopes from HPV strains 16 and 31, individually or as a bivalent formulation. MS2-16L2 was previously shown to confer protection against 11 HPV serotypes but not HPV31 [71]. Mice immunized with
292 APPENDIX II: VIRUS-BASED VACCINES the individual constructs (MS2-16L2 or MS2-31L2) were protected against some strains, whereas the bivalent formulation (MS2-16/31L2) elicited high antibody titers across a panel of HPV serotypes and strongly neutralized all HPV pseudoviruses [72]. In a complementary approach, bacteriophage PP7 was modified to express L2 from HPV strains 16 and 18 (which are closely related) and 1 (which is more distant) individually and in pairwise combinations (PP7-18L2, PP7-18/1L2 and PP7-16/18L2). Mice immunized with PP7-18/1L2 only produced antibodies against HPV1 peptides, whereas
PPV-18/16L2 elicited antibodies that bound strongly to HPV16 and HPV18, as well as
HPV1, HPV5, and HPV6. Only mice vaccinated with PPV-18/16L2 were able to neutralize an HPV-6 pseudovirus, a heterologous serotype [72].
The high-risk HPV strains 18, 45, and 59 have been targeted by inserting a cross- neutralizing epitope from HPV45 L2 into a surface loop of HPV18 L1 and creating VLPs from the chimeric construct (18L1-45RG1). L2-specific antibodies from vaccinated rabbits reacted against HPV strains 39, 45, 68, and 70 (which are members of the same clade as HPV45 and HPV18). Additionally, when mice were passively immunized with immune sera from rabbits, they were protected against a challenge with HPV strains 18,
39, 45, and 68[73].
Vaccines to treat cancers caused by HPV
Although prophylactic vaccines have been successful, they are unable to treat established tumors. The development of HPV therapeutic vaccines has focused on the E6 and E7 oncoproteins, which are necessary for tumor development and are expressed in all
293 APPENDIX II: VIRUS-BASED VACCINES cervical cancer cells [149]. For example, HPV16L1 was genetically modified to express the HPV16 E7 protein. In mice, these recombinant VLPs induced L1-specific antibodies, as well as cytotoxic T cells that recognized L1 and E7 [150-153]. In a phase I trial, patients with proven ectocervical CIN 2/3 lesions who were also HPV16 mono-infected, were treated with HPV16L1E7 VLPs. Approximately 50% of vaccinated patients exhibited a 50% reduction in lesion size following the final vaccination [74]. Further improvements of this therapeutic strategy include the incorporation of T-cell epitopes from HPV16 E6 and E7. Preclinical studies in mice showed an 85% reduction in tumor size when immunized with such recombinant VLPs [75].
Vaccines targeting hepatocellular carcinoma caused by HBV
Liver cancer causes 600,000 deaths per year and there are 700,000 new cases, approximately 500,000 of whom are male, making it the fourth most common form of cancer in men [133]. Among these cases, 95% are classified as hepatocellular carcinoma
[154], which is associated with risk factors such as alcoholism, hepatitis B, hepatitis C, and liver cirrhosis [155, 156]. HBV (Figure A2.10) is responsible for about 50% of all primary hepatocellular carcinomas. A vaccine against HBV has been available since 1981, and is on the World Health Organization’s List of Essential Medicines. This vaccine is a
VLP comprising the HBV surface antigen (HBsAg), which is administered as two or three injections within one year. The vaccine provides lasting immunity against HBV by producing anti-HBV antibodies [157, 158].
294 APPENDIX II: VIRUS-BASED VACCINES
Figure A2.10. Human Hepatitis B virus. (viperdb.scripps.edu).
The prophylactic HBV vaccine has greatly reduced the incidence of hepatocellular carcinoma but a therapeutic vaccine is needed to treat established disease.
One of the key targets is the HBV X protein (HBx), a regulatory protein that promotes carcinogenesis and is expressed at high levels in hepatocellular carcinoma [76, 159]. The
HBc has therefore been genetically modified to express dominant HBx-derived cytotoxic
T-cell epitopes, as well as the universal Th-cell epitope, pan-HLA DR-binding epitope
(PADRE). These chimeric proteins self-assemble into VLPs, and mice vaccinated with this formulation inhibited tumor growth up to 30 days after tumor challenge, while also eliciting a strong T-cell response [76].
295 APPENDIX II: VIRUS-BASED VACCINES
Vaccines targeting HER-2+ breast cancer
Breast cancer is the most common form of cancer in women, as well as a minor form of cancer in men, with over one million cases diagnosed each year [133]. There are five molecular subtypes of breast cancer: normal-like, luminal A, luminal B, HER-2+, and triple negative [160]. Each is defined by the expression of different receptors and the prognosis varies accordingly. HER-2+ tumors overexpress HER-2/neu/ERBb2. They do not express hormone receptors and they are associated with aggressive tumors, high rates of metastasis, and an overall poor prognosis [161]. FDA-approved treatments include the
HER2-specific antibodies trastuzumab and pertuzumab, which are used for passive immunotherapy and require repetitive administration [162, 163]. Passive immunization requires prolonged therapeutic delivery, cannot be developed as a prophylactic, and does not induce cellular immune responses [77, 78]. To overcome these challenges, HER-2+ breast cancer research is now focused on active immunotherapy that elicits long-lasting cellular and humoral responses.
VLPs that display full-length HER-2 or specific immunogenic epitopes have been tested in clinical trials [77-81]. Peptides derived from the extracellular domain of HER-2 were incorporated into immunopotentiating reconstituted influenzavirus virosomes
(IRIVs) [164-166]. Three immunogenic peptides that are known to elicit B cell responses were tested: P4 (378-394), P6 (545-560), and P7 (610-623) [167]. The clinical trial revealed that 80% of vaccinated patients produced peptide-specific antibodies, and HER-
2-specific IgG was elicited in 70% of the patients after immunization. A cellular immune response was also observed following vaccination, involving the increased secretion of
IL-2, TNFα, and IFNγ [77]. In a different approach, enveloped influenzavirus VLPs were
296 APPENDIX II: VIRUS-BASED VACCINES modified to incorporate glycosylphosphatidylinositol (GPI)-anchored HER-2 (GPI-HER-
2-VLP). Preclinical studies showed strong anti-D2F2/E2 (HER-2+) serum IgG responses in mice, with comparable levels of IgG1, IgG2a, and IgG2b in the serum of vaccinated animals, indicating a balanced Th1 and Th2 response. In contrast, the vaccination of mice with soluble GPI-HER-2 predominantly elicited a Th2 response, whereas Th1-biased responses are needed to induce a potent anti-tumor reaction. When mice were challenged with HER-2+ cells, those vaccinated with GPI-HER-2 VLPs showed a slower tumor growth rate compared to those administered GPI-HER-2 alone. Sixty-seven percent of the mice vaccinated with GPI-HER-2 VLPs remained tumor free [78].
VLPs have also been genetically modified to express HER-2 peptides. The internal face of the murine polyomavirus (MPyV) major capsid protein (VP1) can bind to the minor capsid protein (VP2) [168]. VP2 was genetically modified to express the N- terminal domain of HER-2, which contains the extracellular and transmembrane domains
(VP2Her21-683). VP1 and VP2Her21-683 were produced in a baculovirus vector to obtain
+ Her21-683PyVLPs. Immunized mice were challenged with HER-2 D2F2/E2 cells, and
87% of the vaccinated mice did not develop tumors. Similar results were obtained using transgenic BALB-neuT mice, which overexpress the rat HER-2 oncogene. The mice did not produce HER-2-specific antibodies, but did induce HER-2-specific T cells [79].
A T7 bacteriophage was genetically modified to express a H-2kd-restricted cytotoxic T lymphocyte (CTL) epitope (p66) derived from rat HER-2 to investigate whether a CTL response is required for cancer immunotherapy. Preclinical studies showed that splenocytes from mice immunized with T7-p66 produced a higher IFNγ response than controls. Interestingly, splenocytes from mice vaccinated with a mixture of
297 APPENDIX II: VIRUS-BASED VACCINES unconjugated T7 and p66 did not yield a strong IFNγ response when challenged with p66, indicating that the CTL peptide must be attached to T7 in order to elicit the CTL response.
Splenocytes from mice vaccinated with T7-p66 were also able to lyse target cells pulsed with p66-peptide in vitro. Healthy mice vaccinated with T7-p66 rejected HER-2+ TUBO cells, with five of the six mice remaining tumor-free 42 days after challenge. Furthermore, therapeutic vaccination with T7-p66 slowed the growth of pre-implanted tumors, eventually resulting in the full regression of HER-2+ tumors [80].
We have recently worked on the development of a HER-2+ breast cancer vaccine using the plant virus PVX. The chemical conjugation of PVX with the P4 B-cell epitope, which contains amino acids 387–394 from the extracellular domain of HER-2, elicited higher titers of HER-2-specific antibodies in mice than soluble P4 alone. These antibodies selectively recognized HER-2+ breast cancer cells [81].
A2.4.3 Addiction (cocaine and nicotine)
Addictive substances, such as nicotine and cocaine (Figure A2.11), affect the body by interacting with the nervous system. Nicotine and cocaine both modulate dopamine levels in the brain, thereby affecting the reward pathway. Vaccines against these addictive substances can help to reduce the severity of withdrawal symptoms and prevent relapse. However, small molecules tend to have low immunogenicity, so VLP platform technologies are required to elicit potent and long-lasting immune responses against the drugs [169-171]. VLPs displaying nicotine or cocaine as multivalent arrays have been shown to elicit potent humoral responses, yielding drug-specific antibodies that prevent the substances from crossing the blood-brain barrier to exert their effects 298 APPENDIX II: VIRUS-BASED VACCINES
[172-174]. Nicotine is the addictive component of tobacco, and tobacco use is the leading preventable cause of disease, disability, and death in the industrialized world [172].
Several vaccines against nicotine are currently undergoing clinical trials, including
NicVaxTM, NIC002, SEL-068, Ta-NIC, and IP18-KLH, but similar approaches are also under development for cocaine addition.
NIC002 is a VLP vaccine in which bacteriophage Qβ is chemically modified to display nicotine (NicQb) [82, 172, 174]. Preclinical studied in mice demonstrated the development of nicotine-specific antibodies [172]. Importantly, when challenged with nicotine, vaccinated mice showed a higher concentration of nicotine remaining in the blood and a corresponding reduction of nicotine levels in the brain, compared to non- vaccinated mice [172]. In phase I trials, NicQb was immunogenic, well tolerated and efficacious in patients with high antibody titers [82]. In an alternative approach, a nicotine analog was chemically linked to disrupted serotype-5 adenovirus (dAd5). The dAd5 VLP lacks the E1 and E3 proteins, allowing the particle to circumvent any pre- existing Ad5 immunity, which is prevalent in the population [83]. Sera from vaccinated mice contained high titers of anti-nicotine antibodies for a prolonged duration, resulting in lower concentrations of nicotine in the brain than naïve mice, inversely related to the levels of nicotine in the serum [83].
Figure A2.11. Chemical structures of nicotine (left) and cocaine (right). 299 APPENDIX II: VIRUS-BASED VACCINES
The dAd5 VLP has also been conjugated with the cocaine analog. Vaccinated mice challenged with cocaine were found to have 41% less cocaine in the brain than naïve mice. Locomotor activity in vaccinated mice challenged with cocaine was the same as in non-challenged mice treated with PBS, confirming that the vaccine reduced the cognitive impact of cocaine [84]. Similar strategies are being developed using alternative cocaine haptens [85].
A2.4.4 Chronic diseases
Chronic diseases are persistent or even life-long conditions that require regular therapeutic intervention as a form of disease management. The prevalence of chronic diseases is increasing globally due to the ageing population and various dietary and lifestyle factors, which means that such diseases represent a significant and increasing public health burden in almost every country. Where chronic diseases are caused by malfunctioning self-proteins, vaccination can be used to induce the generation of autoantibodies. This approach has been tested in numerous disease models, including rheumatoid arthritis [175] [176-180], osteoporosis [177, 181], experimental autoimmune encephalitis [179], myocarditis [182], and obesity [183]. Many of these diseases are currently treated by passive immunotherapy, i.e. the regular administration of therapeutic antibodies targeting pathogenic self-proteins, which is expensive and restricts the patient.
We will discuss hypertension and Alzheimer’s disease as case studies for the alternative active immunization approach. The reader is recommended to consult further review articles for information about the development of vaccines against other chronic diseases[184-186].
300 APPENDIX II: VIRUS-BASED VACCINES
Hypertension
Hypertension (high blood pressure) is an underlying risk factor that promotes the development of cardiovascular disease, which can lead to life threatening events such as heart attack and stroke. Although hypertension can be regulated with drugs, many hypertensive individuals never receive a diagnosis and therapeutic compliance tends to be poor even in diagnosed hypertensive patients. Another risk factor is the so-called morning pressure surge, a steep increase in blood pressure prior to waking, before medication can be taken. Active immunotherapy could overcome many of these challenges by inducing long-lasting immune responses targeting key regulators of blood pressure.
Angiotensin I and II could be the first targets of hypertension immunotherapy.
These are small, soluble regulatory peptides (10 and 8 amino acids in length, respectively), which do not elicit a strong immune response in their native state. As described above, the immunogenicity of small molecules can be increased by exploiting
VLP technology. A vaccine candidate has therefore been developed in which angiotensin
II is chemically coupled to bacteriophage Qβ VLPs (AngQb). Preclinical studies in a rat model of hypertension showed that vaccination produced high titers of angiotensin II- specific IgGs and resulted in the normalization of blood pressure [187]. In clinical trials, the AngQb vaccine was well tolerated and no serious adverse effects were observed. The immunization of patients with mild to moderate hypertension reduced blood pressure during the daytime and especially in the early morning [188].
301 APPENDIX II: VIRUS-BASED VACCINES
Alzheimer’s disease
Alzheimer’s disease is a neurodegenerative disorder characterized by a decline in cognitive ability accompanied by neuropathological features such as the loss of neurons in the hippocampus and neocortex and the accumulation of intracellular and extracellular protein deposits [189]. Extracellular protein deposits (amyloid plaques) contain the amyloid-β (Aβ) peptide, which is 42 amino acids in length [190, 191]. Previous reports have shown that immunization with the Aβ peptide reduced the deposition of amyloid plaques in transgenic mouse models [192]. Furthermore, passive immunization with Aβ antibodies had a similar effect [193]. However, a clinical trial (AN1792) using synthetic
Aβ peptides for immunization showed minimal efficacy and was stopped after meningoencephalitis was reported in 6% of the subjects [194]. This unanticipated effect was attributed to a T-cell-mediated autoimmune response caused by the adjuvant QS21
[194-196]. The safety of Aβ-derived immunotherapies could therefore be improved by triggering a predominantly Th2-based immune response by delivering only B-cell epitopes, which are found on the N-terminus of the Aβ peptide [197, 198]. VLPs based on HPV, bacteriophage Qβ, HBc, and BPV-1 have already been used to display Aβ peptides [86, 89, 90].
HPV-16 displaying Aβ peptides such as full-length Aβ (Aβ1-40), N-terminal Aβ
(Aβ1-9 and Aβ1-16), mid-domain Aβ (Aβ12-28), and C-terminal Aβ (Aβ17-40) have been tested as vaccines. HPV-Aβ1-40 elicited IgG responses without the use of Freund’s adjuvant in mice, whereas free Aβ1-40 required Freund’s adjuvant to achieve comparable
302 APPENDIX II: VIRUS-BASED VACCINES titers. HPV conjugated to peptides from the N-terminal domain of Aβ elicited higher antibody titers than HPV conjugated to peptides from either the mid or C-terminal domains, indicating that N-terminal Aβ peptides are the most immunogenic when presented on HPV particles. Importantly, these antibodies were predominantly of subtype
IgG1, indicating a Th2-biased immune response [86].
Aβ peptides have been directly conjugated to bacteriophage Qβ. The N-terminal modified peptide (Aβ1-9) with a C-terminal –GGC linker was conjugated to the bacteriophage using a bifunctional linker with both amine and sulfhydryl reactive arms
(SMPH). Mice immunized with Qβ-Aβ1-9 VLPs without adjuvant produced higher titers of antibodies than those immunized with HPV-Aβ1-9 and similar titers to those immunized with HPV-Aβ40. The inclusion of incomplete Freund’s adjuvant increased the
IgG titers even further [86].
Bacteriophage Qβ has also been conjugated with Aβ1-6, which is shorter than the typical T-cell epitope. Mice were immunized three times with Qβ-Aβ1-6 and did not activate Aβ-specific T cells. Additionally, mice immunized with Qβ-Aβ1-6 produced high antibody titers against the Aβ peptide and formed fewer plaques than control mice [199].
Qβ-Aβ1-6 was tested in a phase I trial (CAD106) and was deemed safe and tolerable in a double-blind, placebo-controlled, 52-week study. Importantly, no subjects recorded clinical or subclinical cases of meningoencephalitis, which halted the previous
Alzheimer’s disease immunotherapy clinical trial [88]. In a phase II trial, CAD106 was administered to 47 patients with Alzheimer’s disease (n = 11 for placebo). The patients received three subcutaneous or intramuscular doses of CAD106, followed by four
303 APPENDIX II: VIRUS-BASED VACCINES additional subcutaneous or intramuscular injections. Long-term treatment induced prolonged high titers of Aβ-specific antibodies suggesting that CAD106, which recently entered phase III trials, could be developed into an effective immunotherapy for
Alzheimer’s disease [87].
A C-terminally truncated version of the HBc protein (HBcΔ) was genetically modified to include two copies of Aβ1-15 in the MIR (Aβ-HBc) [89]. The MIR was chosen because epitopes inserted there tend to be highly antigenic and immunogenic compared to other insertion sites [200]. Preclinical studies in mice showed that anti-Aβ antibodies, predominantly IgG1 and IgG2b subtypes (indicating a Th2-biased immune response) were generated. Free Aβ peptide with adjuvant also elicited high antibody titers, but was dominated by the IgG2a subtype. Sera from immunized mice also prevented the formation of Aβ fibrils and reduced the toxicity of the Aβ peptide towards PC12 cells
[89].
In the final example based on BPV-1, the Aβ1-9 peptide was fused with the L1 protein and the chimeric Aβ-VLPs self-assembled to form a structure resembling the native virus particle. Preclinical vaccination studies in rabbits indicated that sera from the treated rabbits recognized Aβ1-9 and full length Aβ, and that Aβ fibril formation was inhibited in vitro. Transgenic APP/PS1 mice, which spontaneously form Aβ plaques, were also immunized with Aβ-VLP without adjuvant eliciting high titers of Aβ-specific antibodies. Higher levels of circulating Aβ peptide were detected in these mice compared to naïve controls, corresponding to lower levels of Aβ peptide in the brain [90].
304 APPENDIX II: VIRUS-BASED VACCINES
A2.5 Conclusions
Vaccines based on viruses could be developed for the prevention and/or treatment of diverse diseases, including infectious diseases, cancer, addiction, and chronic disorders.
One of the key advantages of viruses as a vaccine development platform is that they are naturally immunogenic, and are therefore ideal for the induction of immune responses even in the absence of an adjuvant. The success of prophylactic vaccines against HPV and HBV highlights the potential of this platform for the treatment of many other diseases. Many virus-based vaccines have shown promising results in non-human primates but a number of challenges remain to be overcome before such vaccines can be deployed in the clinic. The first barrier is safety: the natural immunogenicity of virus- based particles makes them ideal for the display of antigenic epitopes but increases the risk of toxicity. Bacteriophage Qβ vaccines for Alzheimer’s disease and nicotine addiction have recently completed phase I safety tests, but other platforms remain to be evaluated in clinical trials. Nevertheless, the phase I trials indicate that virus-based vaccines offer an alternative to other vaccine materials, and the promising results in primates indicate that this platform could be used to develop novel vaccines against a wide range of diseases.
A2.6 Works cited in this appendix
1. Clem AS. Fundamentals of vaccine immunology. J Glob Infect Dis 2011;3:73-8.
2. Pulendran B, Ahmed R. Immunological mechanisms of vaccination. Nat Immunol 2011;12:509-17.
3. Riedel S. Edward Jenner and the history of smallpox and vaccination. Proc (Bayl Univ Med Cent) 2005;18:21-5.
305 APPENDIX II: VIRUS-BASED VACCINES
4. Miller M, Barrett S, Henderson DA. Control and Eradication. In: Jamison DT, Breman JG, Measham AR, Alleyne G, Claeson M, Evans DB, et al., editors. Disease Control Priorities in Developing Countries. 2nd ed. Washington (DC)2006.
5. In: Knobler S, Lederberg J, Pray LA, editors. Considerations for Viral Disease Eradication: Lessons Learned and Future Strategies: Workshop Summary. Washington (DC)2002.
6. Hicks DJ, Fooks AR, Johnson N. Developments in rabies vaccines. Clin Exp Immunol 2012;169:199-204.
7. Geier MR, Geier DA, Zahalsky AC. A review of hepatitis B vaccination. Expert Opin Drug Saf 2003;2:113-22.
8. Lopez AL, Gonzales ML, Aldaba JG, Nair GB. Killed oral cholera vaccines: history, development and implementation challenges. Ther Adv Vaccines 2014;2:123-36.
9. Scorpio A, Blank TE, Day WA, Chabot DJ. Anthrax vaccines: Pasteur to the present. Cell Mol Life Sci 2006;63:2237-48.
10. Zhao L, Seth A, Wibowo N, Zhao CX, Mitter N, Yu C, et al. Nanoparticle vaccines. Vaccine 2014;32:327-37.
11. Gregory AE, Titball R, Williamson D. Vaccine delivery using nanoparticles. Front Cell Infect Microbiol 2013;3:13.
12. Lua LH, Connors NK, Sainsbury F, Chuan YP, Wibowo N, Middelberg AP. Bioengineering virus-like particles as vaccines. Biotechnol Bioeng 2014;111:425- 40.
13. Jennings GT, Bachmann MF. The coming of age of virus-like particle vaccines. Biol Chem 2008;389:521-36.
14. Peacey M, Wilson S, Baird MA, Ward VK. Versatile RHDV virus-like particles: incorporation of antigens by genetic modification and chemical conjugation. Biotechnol Bioeng 2007;98:968-77.
15. Zhang W, Wang L, Liu Y, Chen X, Liu Q, Jia J, et al. Immune responses to vaccines involving a combined antigen-nanoparticle mixture and nanoparticle- encapsulated antigen formulation. Biomaterials 2014;35:6086-97.
16. Heil F, Hemmi H, Hochrein H, Ampenberger F, Kirschning C, Akira S, et al. Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science 2004;303:1526-9.
306 APPENDIX II: VIRUS-BASED VACCINES
17. Jegerlehner A, Maurer P, Bessa J, Hinton HJ, Kopf M, Bachmann MF. TLR9 signaling in B cells determines class switch recombination to IgG2a. J Immunol 2007;178:2415-20.
18. Bessa J, Jegerlehner A, Hinton HJ, Pumpens P, Saudan P, Schneider P, et al. Alveolar macrophages and lung dendritic cells sense RNA and drive mucosal IgA responses. J Immunol 2009;183:3788-99.
19. Hou B, Saudan P, Ott G, Wheeler ML, Ji M, Kuzmich L, et al. Selective utilization of Toll-like receptor and MyD88 signaling in B cells for enhancement of the antiviral germinal center response. Immunity 2011;34:375-84.
20. Cubas R, Zhang S, Kwon S, Sevick-Muraca EM, Li M, Chen C, et al. Virus-like particle (VLP) lymphatic trafficking and immune response generation after immunization by different routes. J Immunother 2009;32:118-28.
21. Manolova V, Flace A, Bauer M, Schwarz K, Saudan P, Bachmann MF. Nanoparticles target distinct dendritic cell populations according to their size. Eur J Immunol 2008;38:1404-13.
22. Vogel FR. Improving vaccine performance with adjuvants. Clin Infect Dis 2000;30 Suppl 3:S266-70.
23. Goldman MaL, P.-H. Immunological safety of vaccines: facts, hypotheses, and allegation. In: Kaufmann SHE, editor. Novel Vaccination Strategies. Germany: Wiley-VCH; 2004. p. 595-611.
24. Schneemann A, Young MJ. Viral assembly using heterologous expression systems and cell extracts. Adv Protein Chem 2003;64:1-36.
25. Pokorski JK, Steinmetz NF. The art of engineering viral nanoparticles. Mol Pharm 2011;8:29-43.
26. Shukla S, Dickmeis C, Nagarajan AS, Fischer R, Commandeur U, Steinmetz NF. Molecular farming of fluorescent virus-based nanoparticles for optical imaging in plants, human cells and mouse models. Biomaterials Science 2014;2:784-97.
27. Marusic C, Rizza P, Lattanzi L, Mancini C, Spada M, Belardelli F, et al. Chimeric plant virus particles as immunogens for inducing murine and human immune responses against human immunodeficiency virus type 1. J Virol 2001;75:8434-9.
28. Tissot AC, Renhofa R, Schmitz N, Cielens I, Meijerink E, Ose V, et al. Versatile virus-like particle carrier for epitope based vaccines. PLoS One 2010;5:e9809.
29. Turpen TH, Reinl SJ, Charoenvit Y, Hoffman SL, Fallarme V, Grill LK. Malarial epitopes expressed on the surface of recombinant tobacco mosaic virus. Biotechnology (N Y) 1995;13:53-7.
307 APPENDIX II: VIRUS-BASED VACCINES
30. Denis J, Majeau N, Acosta-Ramirez E, Savard C, Bedard MC, Simard S, et al. Immunogenicity of papaya mosaic virus-like particles fused to a hepatitis C virus epitope: evidence for the critical function of multimerization. Virology 2007;363:59-68.
31. Tremblay MH, Majeau N, Gagne ME, Lecours K, Morin H, Duvignaud JB, et al. Effect of mutations K97A and E128A on RNA binding and self assembly of papaya mosaic potexvirus coat protein. FEBS J 2006;273:14-25.
32. Phelps JP, Dang N, Rasochova L. Inactivation and purification of cowpea mosaic virus-like particles displaying peptide antigens from Bacillus anthracis. J Virol Methods 2007;141:146-53.
33. O'Neil A, Prevelige PE, Basu G, Douglas T. Coconfinement of fluorescent proteins: spatially enforced communication of GFP and mCherry encapsulated within the P22 capsid. Biomacromolecules 2012;13:3902-7.
34. Patterson DP, Rynda-Apple A, Harmsen AL, Harmsen AG, Douglas T. Biomimetic antigenic nanoparticles elicit controlled protective immune response to influenza. ACS Nano 2013;7:3036-44.
35. Donnelly ML, Luke G, Mehrotra A, Li X, Hughes LE, Gani D, et al. Analysis of the aphthovirus 2A/2B polyprotein 'cleavage' mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal 'skip'. J Gen Virol 2001;82:1013-25.
36. Pastori C, Tudor D, Diomede L, Drillet AS, Jegerlehner A, Rohn TA, et al. Virus like particle based strategy to elicit HIV-protective antibodies to the alpha-helic regions of gp41. Virology 2012;431:1-11.
37. Chackerian B, Lowy DR, Schiller JT. Induction of autoantibodies to mouse CCR5 with recombinant papillomavirus particles. Proc Natl Acad Sci U S A 1999;96:2373-8.
38. Van Rompay KK, Hunter Z, Jayashankar K, Peabody J, Montefiori D, LaBranche CC, et al. A vaccine against CCR5 protects a subset of macaques upon intravaginal challenge with simian immunodeficiency virus SIVmac251. J Virol 2014;88:2011-24.
39. Adis International L. HIV gp120 vaccine - VaxGen: AIDSVAX, AIDSVAX B/B, AIDSVAX B/E, HIV gp120 vaccine - Genentech, HIV gp120 vaccine AIDSVAX - VaxGen, HIV vaccine AIDSVAX - VaxGen. Drugs R D 2003;4:249-53.
40. Kaleebu P, Njai HF, Wang L, Jones N, Ssewanyana I, Richardson P, et al. Immunogenicity of ALVAC-HIV vCP1521 in infants of HIV-1-infected women in Uganda (HPTN 027): the first pediatric HIV vaccine trial in Africa. J Acquir Immune Defic Syndr 2014;65:268-77.
308 APPENDIX II: VIRUS-BASED VACCINES
41. McFarland EJ, Johnson DC, Muresan P, Fenton T, Tomaras GD, McNamara J, et al. HIV-1 vaccine induced immune responses in newborns of HIV-1 infected mothers. AIDS 2006;20:1481-9.
42. Pitisuttithum P, Rerks-Ngarm S, Bussaratid V, Dhitavat J, Maekanantawat W, Pungpak S, et al. Safety and reactogenicity of canarypox ALVAC-HIV (vCP1521) and HIV-1 gp120 AIDSVAX B/E vaccination in an efficacy trial in Thailand. PLoS One 2011;6:e27837.
43. Rerks-Ngarm S, Pitisuttithum P, Nitayaphan S, Kaewkungwal J, Chiu J, Paris R, et al. Vaccination with ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand. N Engl J Med 2009;361:2209-20.
44. Dunkel A, Shen S, LaBranche CC, Montefiori D, McGettigan JP. A Bivalent, Chimeric Rabies Virus Expressing Simian Immunodeficiency Virus Envelope Induces Multifunctional Antibody Responses. AIDS Res Hum Retroviruses 2015.
45. Bomsel M, Tudor D, Drillet AS, Alfsen A, Ganor Y, Roger MG, et al. Immunization with HIV-1 gp41 subunit virosomes induces mucosal antibodies protecting nonhuman primates against vaginal SHIV challenges. Immunity 2011;34:269-80.
46. Martins KA, Steffens JT, van Tongeren SA, Wells JB, Bergeron AA, Dickson SP, et al. Toll-like receptor agonist augments virus-like particle-mediated protection from Ebola virus with transient immune activation. PLoS One 2014;9:e89735.
47. Ye L, Lin J, Sun Y, Bennouna S, Lo M, Wu Q, et al. Ebola virus-like particles produced in insect cells exhibit dendritic cell stimulating activity and induce neutralizing antibodies. Virology 2006;351:260-70.
48. Warfield KL, Swenson DL, Olinger GG, Kalina WV, Aman MJ, Bavari S. Ebola virus-like particle-based vaccine protects nonhuman primates against lethal Ebola virus challenge. J Infect Dis 2007;196 Suppl 2:S430-7.
49. Marzi A, Halfmann P, Hill-Batorski L, Feldmann F, Shupert WL, Neumann G, et al. Vaccines. An Ebola whole-virus vaccine is protective in nonhuman primates. Science 2015;348:439-42.
50. Henao-Restrepo AM, Longini IM, Egger M, Dean NE, Edmunds WJ, Camacho A, et al. Efficacy and effectiveness of an rVSV-vectored vaccine expressing Ebola surface glycoprotein: interim results from the Guinea ring vaccination cluster- randomised trial. Lancet 2015.
51. Blaney JE, Wirblich C, Papaneri AB, Johnson RF, Myers CJ, Juelich TL, et al. Inactivated or live-attenuated bivalent vaccines that confer protection against rabies and Ebola viruses. J Virol 2011;85:10605-16.
309 APPENDIX II: VIRUS-BASED VACCINES
52. Willet M, Kurup D, Papaneri A, Wirblich C, Hooper JW, Kwilas SA, et al. Preclinical Development of Inactivated Rabies Virus-Based Polyvalent Vaccine Against Rabies and Filoviruses. J Infect Dis 2015.
53. Perrone LA, Ahmad A, Veguilla V, Lu X, Smith G, Katz JM, et al. Intranasal vaccination with 1918 influenza virus-like particles protects mice and ferrets from lethal 1918 and H5N1 influenza virus challenge. J Virol 2009;83:5726-34.
54. Liu YV, Massare MJ, Pearce MB, Sun X, Belser JA, Maines TR, et al. Recombinant virus-like particles elicit protective immunity against avian influenza A(H7N9) virus infection in ferrets. Vaccine 2015;33:2152-8.
55. Lopez-Macias C, Ferat-Osorio E, Tenorio-Calvo A, Isibasi A, Talavera J, Arteaga-Ruiz O, et al. Safety and immunogenicity of a virus-like particle pandemic influenza A (H1N1) 2009 vaccine in a blinded, randomized, placebo- controlled trial of adults in Mexico. Vaccine 2011;29:7826-34.
56. Ward BJ, Landry N, Trepanier S, Mercier G, Dargis M, Couture M, et al. Human antibody response to N-glycans present on plant-made influenza virus-like particle (VLP) vaccines. Vaccine 2014;32:6098-106.
57. Ren Z, Ji X, Meng L, Wei Y, Wang T, Feng N, et al. H5N1 influenza virus-like particle vaccine protects mice from heterologous virus challenge better than whole inactivated virus. Virus Res 2015;200:9-18.
58. Chen S, Zheng D, Li C, Zhang W, Xu W, Liu X, et al. Protection against multiple subtypes of influenza viruses by virus-like particle vaccines based on a hemagglutinin conserved epitope. Biomed Res Int 2015;2015:901817.
59. Naskalska A, Szolajska E, Chaperot L, Angel J, Plumas J, Chroboczek J. Influenza recombinant vaccine: matrix protein M1 on the platform of the adenovirus dodecahedron. Vaccine 2009;27:7385-93.
60. Denis J, Acosta-Ramirez E, Zhao Y, Hamelin ME, Koukavica I, Baz M, et al. Development of a universal influenza A vaccine based on the M2e peptide fused to the papaya mosaic virus (PapMV) vaccine platform. Vaccine 2008;26:3395- 403.
61. Pascual E, Mata CP, Gomez-Blanco J, Moreno N, Barcena J, Blanco E, et al. Structural basis for the development of avian virus capsids that display influenza virus proteins and induce protective immunity. J Virol 2015;89:2563-74.
62. Babin C, Majeau N, Leclerc D. Engineering of papaya mosaic virus (PapMV) nanoparticles with a CTL epitope derived from influenza NP. J Nanobiotechnology 2013;11:10.
310 APPENDIX II: VIRUS-BASED VACCINES
63. Lico C, Mancini C, Italiani P, Betti C, Boraschi D, Benvenuto E, et al. Plant- produced potato virus X chimeric particles displaying an influenza virus-derived peptide activate specific CD8+ T cells in mice. Vaccine 2009;27:5069-76.
64. Richert LE, Servid AE, Harmsen AL, Rynda-Apple A, Han S, Wiley JA, et al. A virus-like particle vaccine platform elicits heightened and hastened local lung mucosal antibody production after a single dose. Vaccine 2012;30:3653-65.
65. Wiley JA, Richert LE, Swain SD, Harmsen A, Barnard DL, Randall TD, et al. Inducible bronchus-associated lymphoid tissue elicited by a protein cage nanoparticle enhances protection in mice against diverse respiratory viruses. PLoS One 2009;4:e7142.
66. Yin Z, Nguyen HG, Chowdhury S, Bentley P, Bruckman MA, Miermont A, et al. Tobacco mosaic virus as a new carrier for tumor associated carbohydrate antigens. Bioconjug Chem 2012;23:1694-703.
67. Yin Z, Comellas-Aragones M, Chowdhury S, Bentley P, Kaczanowska K, Benmohamed L, et al. Boosting immunity to small tumor-associated carbohydrates with bacteriophage qbeta capsids. ACS Chem Biol 2013;8:1253-62.
68. McCormick AA, Corbo TA, Wykoff-Clary S, Palmer KE, Pogue GP. Chemical conjugate TMV-peptide bivalent fusion vaccines improve cellular immunity and tumor protection. Bioconjug Chem 2006;17:1330-8.
69. Jobsri J, Allen A, Rajagopal D, Shipton M, Kanyuka K, Lomonossoff GP, et al. Plant virus particles carrying tumour antigen activate TLR7 and Induce high levels of protective antibody. PLoS One 2015;10:e0118096.
70. Pejawar-Gaddy S, Rajawat Y, Hilioti Z, Xue J, Gaddy DF, Finn OJ, et al. Generation of a tumor vaccine candidate based on conjugation of a MUC1 peptide to polyionic papillomavirus virus-like particles. Cancer Immunol Immunother 2010;59:1685-96.
71. Tumban E, Peabody J, Tyler M, Peabody DS, Chackerian B. VLPs displaying a single L2 epitope induce broadly cross-neutralizing antibodies against human papillomavirus. PLoS One 2012;7:e49751.
72. Tyler M, Tumban E, Peabody DS, Chackerian B. The use of hybrid virus-like particles to enhance the immunogenicity of a broadly protective HPV vaccine. Biotechnol Bioeng 2014;111:2398-406.
73. Huber B, Schellenbacher C, Jindra C, Fink D, Shafti-Keramat S, Kirnbauer R. A chimeric 18L1-45RG1 virus-like particle vaccine cross-protects against oncogenic alpha-7 human papillomavirus types. PLoS One 2015;10:e0120152.
74. Kaufmann AM, Nieland JD, Jochmus I, Baur S, Friese K, Gabelsberger J, et al. Vaccination trial with HPV16 L1E7 chimeric virus-like particles in women 311 APPENDIX II: VIRUS-BASED VACCINES
suffering from high grade cervical intraepithelial neoplasia (CIN 2/3). Int J Cancer 2007;121:2794-800.
75. Monroy-Garcia A, Gomez-Lim MA, Weiss-Steider B, Hernandez-Montes J, Huerta-Yepez S, Rangel-Santiago JF, et al. Immunization with an HPV-16 L1- based chimeric virus-like particle containing HPV-16 E6 and E7 epitopes elicits long-lasting prophylactic and therapeutic efficacy in an HPV-16 tumor mice model. Arch Virol 2014;159:291-305.
76. Ding FX, Wang F, Lu YM, Li K, Wang KH, He XW, et al. Multiepitope peptide- loaded virus-like particles as a vaccine against hepatitis B virus-related hepatocellular carcinoma. Hepatology 2009;49:1492-502.
77. Wiedermann U, Wiltschke C, Jasinska J, Kundi M, Zurbriggen R, Garner-Spitzer E, et al. A virosomal formulated Her-2/neu multi-peptide vaccine induces Her- 2/neu-specific immune responses in patients with metastatic breast cancer: a phase I study. Breast Cancer Res Treat 2010;119:673-83.
78. Patel JM, Kim MC, Vartabedian VF, Lee YN, He S, Song JM, et al. Protein transfer-mediated surface engineering to adjuvantate virus-like nanoparticles for enhanced anti-viral immune responses. Nanomedicine 2015;11:1097-107.
79. Tegerstedt K, Lindencrona JA, Curcio C, Andreasson K, Tullus C, Forni G, et al. A single vaccination with polyomavirus VP1/VP2Her2 virus-like particles prevents outgrowth of HER-2/neu-expressing tumors. Cancer Res 2005;65:5953-7.
80. Pouyanfard S, Bamdad T, Hashemi H, Bandehpour M, Kazemi B. Induction of protective anti-CTL epitope responses against HER-2-positive breast cancer based on multivalent T7 phage nanoparticles. PLoS One 2012;7:e49539.
81. Shukla S, Wen AM, Commandeur U, Steinmetz NF. Presentation of HER2 epitopes using a filamentous plant virus-based vaccination platform. Journal of Materials Chemistry B 2014;2:6249-58.
82. Montoya ID. Biologics (Vaccines, Antibodies, Enzymes) to Treat Drug Addictions. In: Nady el-Guebaly GC, Marc Galanter, editor. Textbook of Addiction Treatment: International Perspectives: Springer; 2015. p. 683-92.
83. De BP, Pagovich OE, Hicks MJ, Rosenberg JB, Moreno AY, Janda KD, et al. Disrupted adenovirus-based vaccines against small addictive molecules circumvent anti-adenovirus immunity. Hum Gene Ther 2013;24:58-66.
84. Hicks MJ, De BP, Rosenberg JB, Davidson JT, Moreno AY, Janda KD, et al. Cocaine analog coupled to disrupted adenovirus: a vaccine strategy to evoke high- titer immunity against addictive drugs. Mol Ther 2011;19:612-9.
85. Wee S, Hicks MJ, De BP, Rosenberg JB, Moreno AY, Kaminsky SM, et al. Novel cocaine vaccine linked to a disrupted adenovirus gene transfer vector 312 APPENDIX II: VIRUS-BASED VACCINES
blocks cocaine psychostimulant and reinforcing effects. Neuropsychopharmacology 2012;37:1083-91.
86. Chackerian B, Rangel M, Hunter Z, Peabody DS. Virus and virus-like particle- based immunogens for Alzheimer's disease induce antibody responses against amyloid-beta without concomitant T cell responses. Vaccine 2006;24:6321-31.
87. Farlow MR, Andreasen N, Riviere ME, Vostiar I, Vitaliti A, Sovago J, et al. Long-term treatment with active Abeta immunotherapy with CAD106 in mild Alzheimer's disease. Alzheimers Res Ther 2015;7:23.
88. Winblad B, Andreasen N, Minthon L, Floesser A, Imbert G, Dumortier T, et al. Safety, tolerability, and antibody response of active Abeta immunotherapy with CAD106 in patients with Alzheimer's disease: randomised, double-blind, placebo- controlled, first-in-human study. Lancet Neurol 2012;11:597-604.
89. Feng G, Wang W, Qian Y, Jin H. Anti-Abeta antibodies induced by Abeta-HBc virus-like particles prevent Abeta aggregation and protect PC12 cells against toxicity of Abeta1-40. J Neurosci Methods 2013;218:48-54.
90. Zamora E, Handisurya A, Shafti-Keramat S, Borchelt D, Rudow G, Conant K, et al. Papillomavirus-like particles are an effective platform for amyloid-beta immunization in rabbits and transgenic mice. J Immunol 2006;177:2662-70.
91. Maartens G, Celum C, Lewin SR. HIV infection: epidemiology, pathogenesis, treatment, and prevention. Lancet 2014;384:258-71.
92. Brechtl JR, Breitbart W, Galietta M, Krivo S, Rosenfeld B. The use of highly active antiretroviral therapy (HAART) in patients with advanced HIV infection: impact on medical, palliative care, and quality of life outcomes. J Pain Symptom Manage 2001;21:41-51.
93. Esparza J. A brief history of the global effort to develop a preventive HIV vaccine. Vaccine 2013;31:3502-18.
94. Dowdle W. The search for an AIDS vaccine. Public Health Rep 1986;101:232-3.
95. Buratti E, Tisminetzky SG, Scodeller ES, Baralle FE. Conformational display of two neutralizing epitopes of HIV-1 gp41 on the Flock House virus capsid protein. J Immunol Methods 1996;197:7-18.
96. Berkower I, Raymond M, Muller J, Spadaccini A, Aberdeen A. Assembly, structure, and antigenic properties of virus-like particles rich in HIV-1 envelope gp120. Virology 2004;321:75-86.
97. Sun S, Li W, Sun Y, Pan Y, Li J. A new RNA vaccine platform based on MS2 virus-like particles produced in Saccharomyces cerevisiae. Biochem Biophys Res Commun 2011;407:124-8.
313 APPENDIX II: VIRUS-BASED VACCINES
98. Yusibov V, Modelska A, Steplewski K, Agadjanyan M, Weiner D, Hooper DC, et al. Antigens produced in plants by infection with chimeric plant viruses immunize against rabies virus and HIV-1. Proc Natl Acad Sci U S A 1997;94:5784-8.
99. Sugiyama Y, Hamamoto H, Takemoto S, Watanabe Y, Okada Y. Systemic production of foreign peptides on the particle surface of tobacco mosaic virus. FEBS Lett 1995;359:247-50.
100. Lu Y, Xiao Y, Ding J, Dierich M, Chen YH. Immunogenicity of neutralizing epitopes on multiple-epitope vaccines against HIV-1. Int Arch Allergy Immunol 2000;121:80-4.
101. Arnold GF, Velasco PK, Holmes AK, Wrin T, Geisler SC, Phung P, et al. Broad neutralization of human immunodeficiency virus type 1 (HIV-1) elicited from human rhinoviruses that display the HIV-1 gp41 ELDKWA epitope. J Virol 2009;83:5087-100.
102. Alkhatib G, Combadiere C, Broder CC, Feng Y, Kennedy PE, Murphy PM, et al. CC CKR5: a RANTES, MIP-1alpha, MIP-1beta receptor as a fusion cofactor for macrophage-tropic HIV-1. Science 1996;272:1955-8.
103. Choe H, Farzan M, Sun Y, Sullivan N, Rollins B, Ponath PD, et al. The beta- chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates. Cell 1996;85:1135-48.
104. Deng H, Liu R, Ellmeier W, Choe S, Unutmaz D, Burkhart M, et al. Identification of a major co-receptor for primary isolates of HIV-1. Nature 1996;381:661-6.
105. Doranz BJ, Rucker J, Yi Y, Smyth RJ, Samson M, Peiper SC, et al. A dual-tropic primary HIV-1 isolate that uses fusin and the beta-chemokine receptors CKR-5, CKR-3, and CKR-2b as fusion cofactors. Cell 1996;85:1149-58.
106. Dragic T, Litwin V, Allaway GP, Martin SR, Huang Y, Nagashima KA, et al. HIV-1 entry into CD4+ cells is mediated by the chemokine receptor CC-CKR-5. Nature 1996;381:667-73.
107. Park DJ, Dudas G, Wohl S, Goba A, Whitmer SL, Andersen KG, et al. Ebola Virus Epidemiology, Transmission, and Evolution during Seven Months in Sierra Leone. Cell 2015;161:1516-26.
108. Goeijenbier M, van Kampen JJ, Reusken CB, Koopmans MP, van Gorp EC. Ebola virus disease: a review on epidemiology, symptoms, treatment and pathogenesis. Neth J Med 2014;72:442-8.
109. Baize S, Pannetier D, Oestereich L, Rieger T, Koivogui L, Magassouba N, et al. Emergence of Zaire Ebola virus disease in Guinea. N Engl J Med 2014;371:1418- 25.
314 APPENDIX II: VIRUS-BASED VACCINES
110. Matassov D, Marzi A, Latham T, Xu R, Ota-Setlik A, Feldmann F, et al. Vaccination With a Highly Attenuated Recombinant Vesicular Stomatitis Virus Vector Protects Against Challenge With a Lethal Dose of Ebola Virus. J Infect Dis 2015.
111. Jones SM, Stroher U, Fernando L, Qiu X, Alimonti J, Melito P, et al. Assessment of a vesicular stomatitis virus-based vaccine by use of the mouse model of Ebola virus hemorrhagic fever. J Infect Dis 2007;196 Suppl 2:S404-12.
112. Feldmann H, Jones SM, Daddario-DiCaprio KM, Geisbert JB, Stroher U, Grolla A, et al. Effective post-exposure treatment of Ebola infection. PLoS Pathog 2007;3:e2.
113. Geisbert TW, Feldmann H. Recombinant vesicular stomatitis virus-based vaccines against Ebola and Marburg virus infections. J Infect Dis 2011;204 Suppl 3:S1075-81.
114. de Wit E, Marzi A, Bushmaker T, Brining D, Scott D, Richt JA, et al. Safety of recombinant VSV-Ebola virus vaccine vector in pigs. Emerg Infect Dis 2015;21:702-4.
115. Papaneri AB, Wirblich C, Cooper K, Jahrling PB, Schnell MJ, Blaney JE. Further characterization of the immune response in mice to inactivated and live rabies vaccines expressing Ebola virus glycoprotein. Vaccine 2012;30:6136-41.
116. World Health Organization. Influenza (Seasonal) Fact Sheet. 2014.
117. Lambert LC, Fauci AS. Influenza vaccines for the future. N Engl J Med 2010;363:2036-44.
118. Krammer F. Emerging influenza viruses and the prospect of a universal influenza virus vaccine. Biotechnol J 2015;10:690-701.
119. Taubenberger JK, Morens DM. Influenza: the once and future pandemic. Public Health Rep 2010;125 Suppl 3:16-26.
120. World Health Organization Global Influenza Program Surveillance N. Evolution of H5N1 avian influenza viruses in Asia. Emerg Infect Dis 2005;11:1515-21.
121. Riedel S. Crossing the species barrier: the threat of an avian influenza pandemic. Proc (Bayl Univ Med Cent) 2006;19:16-20.
122. Fiore AE, Bridges CB, Cox NJ. Seasonal influenza vaccines. Curr Top Microbiol Immunol 2009;333:43-82.
123. Krammer F, Palese P. Influenza virus hemagglutinin stalk-based antibodies and vaccines. Curr Opin Virol 2013;3:521-30.
315 APPENDIX II: VIRUS-BASED VACCINES
124. de Jong JC, Beyer WE, Palache AM, Rimmelzwaan GF, Osterhaus AD. Mismatch between the 1997/1998 influenza vaccine and the major epidemic A(H3N2) virus strain as the cause of an inadequate vaccine-induced antibody response to this strain in the elderly. J Med Virol 2000;61:94-9.
125. Tricco AC, Chit A, Soobiah C, Hallett D, Meier G, Chen MH, et al. Comparing influenza vaccine efficacy against mismatched and matched strains: a systematic review and meta-analysis. BMC Med 2013;11:153.
126. Ito T, Gorman OT, Kawaoka Y, Bean WJ, Webster RG. Evolutionary analysis of the influenza A virus M gene with comparison of the M1 and M2 proteins. J Virol 1991;65:5491-8.
127. Epstein SL, Kong WP, Misplon JA, Lo CY, Tumpey TM, Xu L, et al. Protection against multiple influenza A subtypes by vaccination with highly conserved nucleoprotein. Vaccine 2005;23:5404-10.
128. Kawano M, Morikawa K, Suda T, Ohno N, Matsushita S, Akatsuka T, et al. Chimeric SV40 virus-like particles induce specific cytotoxicity and protective immunity against influenza A virus without the need of adjuvants. Virology 2014;448:159-67.
129. Moyron-Quiroz JE, Rangel-Moreno J, Hartson L, Kusser K, Tighe MP, Klonowski KD, et al. Persistence and responsiveness of immunologic memory in the absence of secondary lymphoid organs. Immunity 2006;25:643-54.
130. D'Aoust MA, Lavoie PO, Couture MM, Trepanier S, Guay JM, Dargis M, et al. Influenza virus-like particles produced by transient expression in Nicotiana benthamiana induce a protective immune response against a lethal viral challenge in mice. Plant Biotechnol J 2008;6:930-40.
131. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000;100:57-70.
132. Lizotte PH, Wen AM, Sheen MR, Fields J, Rojanasopondist P, Steinmetz NF, et al. In situ vaccination with cowpea mosaic virus nanoparticles suppresses metastatic cancer. Nat Nanotechnol 2016;11:295-303.
133. American Cancer Society. Global Cancer Facts & Figures, 3rd Edition. In: Society AC, editor. 3rd ed. Atlanta: American Cancer Society; 2015.
134. Castellsague X, Bosch FX. [Advances in cervical cancer prevention: HPV vaccines]. Farm Hosp 2007;31:261-3.
135. Bosch FX, de Sanjose S. The epidemiology of human papillomavirus infection and cervical cancer. Dis Markers 2007;23:213-27.
136. Clifford GM, Gallus S, Herrero R, Munoz N, Snijders PJ, Vaccarella S, et al. Worldwide distribution of human papillomavirus types in cytologically normal
316 APPENDIX II: VIRUS-BASED VACCINES
women in the International Agency for Research on Cancer HPV prevalence surveys: a pooled analysis. Lancet 2005;366:991-8.
137. Stanley M. Pathology and epidemiology of HPV infection in females. Gynecol Oncol 2010;117:S5-10.
138. Lehtinen M, Paavonen J, Wheeler CM, Jaisamrarn U, Garland SM, Castellsague X, et al. Overall efficacy of HPV-16/18 AS04-adjuvanted vaccine against grade 3 or greater cervical intraepithelial neoplasia: 4-year end-of-study analysis of the randomised, double-blind PATRICIA trial. Lancet Oncol 2012;13:89-99.
139. Munoz N, Kjaer SK, Sigurdsson K, Iversen OE, Hernandez-Avila M, Wheeler CM, et al. Impact of human papillomavirus (HPV)-6/11/16/18 vaccine on all HPV-associated genital diseases in young women. J Natl Cancer Inst 2010;102:325-39.
140. Brown DR, Kjaer SK, Sigurdsson K, Iversen OE, Hernandez-Avila M, Wheeler CM, et al. The impact of quadrivalent human papillomavirus (HPV; types 6, 11, 16, and 18) L1 virus-like particle vaccine on infection and disease due to oncogenic nonvaccine HPV types in generally HPV-naive women aged 16-26 years. J Infect Dis 2009;199:926-35.
141. Wheeler CM, Kjaer SK, Sigurdsson K, Iversen OE, Hernandez-Avila M, Perez G, et al. The impact of quadrivalent human papillomavirus (HPV; types 6, 11, 16, and 18) L1 virus-like particle vaccine on infection and disease due to oncogenic nonvaccine HPV types in sexually active women aged 16-26 years. J Infect Dis 2009;199:936-44.
142. Smith JF, Brownlow M, Brown M, Kowalski R, Esser MT, Ruiz W, et al. Antibodies from women immunized with Gardasil cross-neutralize HPV 45 pseudovirions. Hum Vaccin 2007;3:109-15.
143. Roden RB, Yutzy WHt, Fallon R, Inglis S, Lowy DR, Schiller JT. Minor capsid protein of human genital papillomaviruses contains subdominant, cross- neutralizing epitopes. Virology 2000;270:254-7.
144. Gambhira R, Karanam B, Jagu S, Roberts JN, Buck CB, Bossis I, et al. A protective and broadly cross-neutralizing epitope of human papillomavirus L2. J Virol 2007;81:13927-31.
145. Pastrana DV, Gambhira R, Buck CB, Pang YY, Thompson CD, Culp TD, et al. Cross-neutralization of cutaneous and mucosal Papillomavirus types with anti- sera to the amino terminus of L2. Virology 2005;337:365-72.
146. Schellenbacher C, Kwak K, Fink D, Shafti-Keramat S, Huber B, Jindra C, et al. Efficacy of RG1-VLP vaccination against infections with genital and cutaneous human papillomaviruses. J Invest Dermatol 2013;133:2706-13.
317 APPENDIX II: VIRUS-BASED VACCINES
147. Karanam B, Jagu S, Huh WK, Roden RB. Developing vaccines against minor capsid antigen L2 to prevent papillomavirus infection. Immunol Cell Biol 2009;87:287-99.
148. Tumban E, Peabody J, Peabody DS, Chackerian B. A pan-HPV vaccine based on bacteriophage PP7 VLPs displaying broadly cross-neutralizing epitopes from the HPV minor capsid protein, L2. PLoS One 2011;6:e23310.
149. zur Hausen H. Papillomaviruses causing cancer: evasion from host-cell control in early events in carcinogenesis. J Natl Cancer Inst 2000;92:690-8.
150. Muller M, Zhou J, Reed TD, Rittmuller C, Burger A, Gabelsberger J, et al. Chimeric papillomavirus-like particles. Virology 1997;234:93-111.
151. Schafer K, Muller M, Faath S, Henn A, Osen W, Zentgraf H, et al. Immune response to human papillomavirus 16 L1E7 chimeric virus-like particles: induction of cytotoxic T cells and specific tumor protection. Int J Cancer 1999;81:881-8.
152. Kaufmann AM, Nieland J, Schinz M, Nonn M, Gabelsberger J, Meissner H, et al. HPV16 L1E7 chimeric virus-like particles induce specific HLA-restricted T cells in humans after in vitro vaccination. Int J Cancer 2001;92:285-93.
153. Ohlschlager P, Osen W, Dell K, Faath S, Garcea RL, Jochmus I, et al. Human papillomavirus type 16 L1 capsomeres induce L1-specific cytotoxic T lymphocytes and tumor regression in C57BL/6 mice. J Virol 2003;77:4635-45.
154. London WT MK. Liver Cancer. In: Schottenfeld D FJJ, editor. Cancer Epidemiology and Prevention. 3rd ed. New York: Oxford University Press; 2006. p. 763-86.
155. Mittal S, El-Serag HB. Epidemiology of hepatocellular carcinoma: consider the population. J Clin Gastroenterol 2013;47 Suppl:S2-6.
156. El-Serag HB. Hepatocellular carcinoma. N Engl J Med 2011;365:1118-27.
157. McAleer WJ, Buynak EB, Maigetter RZ, Wampler DE, Miller WJ, Hilleman MR. Human hepatitis B vaccine from recombinant yeast. Nature 1984;307:178-80.
158. Andre FE, Safary A. Summary of clinical findings on Engerix-B, a genetically engineered yeast derived hepatitis B vaccine. Postgrad Med J 1987;63 Suppl 2:169-77.
159. Su F, Schneider RJ. Hepatitis B virus HBx protein sensitizes cells to apoptotic killing by tumor necrosis factor alpha. Proc Natl Acad Sci U S A 1997;94:8744-9.
318 APPENDIX II: VIRUS-BASED VACCINES
160. Sorlie T, Perou CM, Tibshirani R, Aas T, Geisler S, Johnsen H, et al. Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci U S A 2001;98:10869-74.
161. Ross JS, Slodkowska EA, Symmans WF, Pusztai L, Ravdin PM, Hortobagyi GN. The HER-2 receptor and breast cancer: ten years of targeted anti-HER-2 therapy and personalized medicine. Oncologist 2009;14:320-68.
162. Jhaveri K, Esteva FJ. Pertuzumab in the treatment of HER2+ breast cancer. J Natl Compr Canc Netw 2014;12:591-8.
163. Hortobagyi GN. Trastuzumab in the treatment of breast cancer. N Engl J Med 2005;353:1734-6.
164. Zurbriggen R. Immunostimulating reconstituted influenza virosomes. Vaccine 2003;21:921-4.
165. Bovier PA. Epaxal: a virosomal vaccine to prevent hepatitis A infection. Expert Rev Vaccines 2008;7:1141-50.
166. Thompson FM, Porter DW, Okitsu SL, Westerfeld N, Vogel D, Todryk S, et al. Evidence of blood stage efficacy with a virosomal malaria vaccine in a phase IIa clinical trial. PLoS One 2008;3:e1493.
167. Wagner S, Jasinska J, Breiteneder H, Kundi M, Pehamberger H, Scheiner O, et al. Delayed tumor onset and reduced tumor growth progression after immunization with a Her-2/neu multi-peptide vaccine and IL-12 in c-neu transgenic mice. Breast Cancer Res Treat 2007;106:29-38.
168. Chen XS, Stehle T, Harrison SC. Interaction of polyomavirus internal protein VP2 with the major capsid protein VP1 and implications for participation of VP2 in viral entry. EMBO J 1998;17:3233-40.
169. Carrera MR, Ashley JA, Parsons LH, Wirsching P, Koob GF, Janda KD. Suppression of psychoactive effects of cocaine by active immunization. Nature 1995;378:727-30.
170. Fox BS, Kantak KM, Edwards MA, Black KM, Bollinger BK, Botka AJ, et al. Efficacy of a therapeutic cocaine vaccine in rodent models. Nat Med 1996;2:1129-32.
171. Kinsey BM, Jackson DC, Orson FM. Anti-drug vaccines to treat substance abuse. Immunol Cell Biol 2009;87:309-14.
172. Maurer P, Jennings GT, Willers J, Rohner F, Lindman Y, Roubicek K, et al. A therapeutic vaccine for nicotine dependence: preclinical efficacy, and Phase I safety and immunogenicity. Eur J Immunol 2005;35:2031-40.
319 APPENDIX II: VIRUS-BASED VACCINES
173. Maoz A, Hicks MJ, Vallabhjosula S, Synan M, Kothari PJ, Dyke JP, et al. Adenovirus capsid-based anti-cocaine vaccine prevents cocaine from binding to the nonhuman primate CNS dopamine transporter. Neuropsychopharmacology 2013;38:2170-8.
174. Bachmann MF, Jennings GT. Therapeutic vaccines for chronic diseases: successes and technical challenges. Philos Trans R Soc Lond B Biol Sci 2011;366:2815-22.
175. Chackerian B, Lowy DR, Schiller JT. Conjugation of a self-antigen to papillomavirus-like particles allows for efficient induction of protective autoantibodies. J Clin Invest 2001;108:415-23.
176. Spohn G, Keller I, Beck M, Grest P, Jennings GT, Bachmann MF. Active immunization with IL-1 displayed on virus-like particles protects from autoimmune arthritis. Eur J Immunol 2008;38:877-87.
177. Spohn G, Bachmann MF. Targeting osteoporosis and rheumatoid arthritis by active vaccination against RANKL. Adv Exp Med Biol 2007;602:135-42.
178. Spohn G, Guler R, Johansen P, Keller I, Jacobs M, Beck M, et al. A virus-like particle-based vaccine selectively targeting soluble TNF-alpha protects from arthritis without inducing reactivation of latent tuberculosis. J Immunol 2007;178:7450-7.
179. Rohn TA, Jennings GT, Hernandez M, Grest P, Beck M, Zou Y, et al. Vaccination against IL-17 suppresses autoimmune arthritis and encephalomyelitis. Eur J Immunol 2006;36:2857-67.
180. Nakajima A, Seroogy CM, Sandora MR, Tarner IH, Costa GL, Taylor-Edwards C, et al. Antigen-specific T cell-mediated gene therapy in collagen-induced arthritis. J Clin Invest 2001;107:1293-301.
181. Spohn G, Schwarz K, Maurer P, Illges H, Rajasekaran N, Choi Y, et al. Protection against osteoporosis by active immunization with TRANCE/RANKL displayed on virus-like particles. J Immunol 2005;175:6211-8.
182. Sonderegger I, Rohn TA, Kurrer MO, Iezzi G, Zou Y, Kastelein RA, et al. Neutralization of IL-17 by active vaccination inhibits IL-23-dependent autoimmune myocarditis. Eur J Immunol 2006;36:2849-56.
183. Fulurija A, Lutz TA, Sladko K, Osto M, Wielinga PY, Bachmann MF, et al. Vaccination against GIP for the treatment of obesity. PLoS One 2008;3:e3163.
184. Chackerian B. Virus-like particles: flexible platforms for vaccine development. Expert Rev Vaccines 2007;6:381-90.
320 APPENDIX II: VIRUS-BASED VACCINES
185. Bachmann MF, Whitehead P. Active immunotherapy for chronic diseases. Vaccine 2013;31:1777-84.
186. Jennings GT, Bachmann MF. Immunodrugs: therapeutic VLP-based vaccines for chronic diseases. Annu Rev Pharmacol Toxicol 2009;49:303-26.
187. Ambuhl PM, Tissot AC, Fulurija A, Maurer P, Nussberger J, Sabat R, et al. A vaccine for hypertension based on virus-like particles: preclinical efficacy and phase I safety and immunogenicity. J Hypertens 2007;25:63-72.
188. Tissot AC, Maurer P, Nussberger J, Sabat R, Pfister T, Ignatenko S, et al. Effect of immunisation against angiotensin II with CYT006-AngQb on ambulatory blood pressure: a double-blind, randomised, placebo-controlled phase IIa study. Lancet 2008;371:821-7.
189. Liebscher S, Meyer-Luehmann M. A Peephole into the Brain: Neuropathological Features of Alzheimer's Disease Revealed by in vivo Two-Photon Imaging. Front Psychiatry 2012;3:26.
190. Hardy JA, Higgins GA. Alzheimer's disease: the amyloid cascade hypothesis. Science 1992;256:184-5.
191. Haass C, Selkoe DJ. Cellular processing of beta-amyloid precursor protein and the genesis of amyloid beta-peptide. Cell 1993;75:1039-42.
192. Schenk D, Barbour R, Dunn W, Gordon G, Grajeda H, Guido T, et al. Immunization with amyloid-beta attenuates Alzheimer-disease-like pathology in the PDAPP mouse. Nature 1999;400:173-7.
193. Bard F, Cannon C, Barbour R, Burke RL, Games D, Grajeda H, et al. Peripherally administered antibodies against amyloid beta-peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer disease. Nat Med 2000;6:916-9.
194. Orgogozo JM, Gilman S, Dartigues JF, Laurent B, Puel M, Kirby LC, et al. Subacute meningoencephalitis in a subset of patients with AD after Abeta42 immunization. Neurology 2003;61:46-54.
195. Nicoll JA, Wilkinson D, Holmes C, Steart P, Markham H, Weller RO. Neuropathology of human Alzheimer disease after immunization with amyloid- beta peptide: a case report. Nat Med 2003;9:448-52.
196. Ferrer I, Boada Rovira M, Sanchez Guerra ML, Rey MJ, Costa-Jussa F. Neuropathology and pathogenesis of encephalitis following amyloid-beta immunization in Alzheimer's disease. Brain Pathol 2004;14:11-20.
197. Bard F, Barbour R, Cannon C, Carretto R, Fox M, Games D, et al. Epitope and isotype specificities of antibodies to beta -amyloid peptide for protection against
321 APPENDIX II: VIRUS-BASED VACCINES
Alzheimer's disease-like neuropathology. Proc Natl Acad Sci U S A 2003;100:2023-8.
198. Seabrook TJ, Bloom JK, Iglesias M, Spooner ET, Walsh DM, Lemere CA. Species-specific immune response to immunization with human versus rodent A beta peptide. Neurobiol Aging 2004;25:1141-51.
199. Wiessner C, Wiederhold KH, Tissot AC, Frey P, Danner S, Jacobson LH, et al. The second-generation active Abeta immunotherapy CAD106 reduces amyloid accumulation in APP transgenic mice while minimizing potential side effects. J Neurosci 2011;31:9323-31.
200. Karpenko LI, Ivanisenko VA, Pika IA, Chikaev NA, Eroshkin AM, Veremeiko TA, et al. Insertion of foreign epitopes in HBcAg: how to make the chimeric particle assemble. Amino Acids 2000;18:329-37.
322 BIBLIOGRAPHY
Bibliography American Cancer Society. Global Cancer Facts & Figures, 3rd Edition. In: Society AC, editor. 3rd ed. Atlanta: American Cancer Society; 2015. A Guide to Radiation Therapy. In: Society AC, editor.2015. Abourbeh G, Shir A, Mishani E, Ogris M, Rödl W, Wagner E, et al. PolyIC GE11 polyplex inhibits EGFR-overexpressing tumors. IUBMB life 2012; 64:324-30. Abraham SA, Waterhouse DN, Mayer LD, Cullis PR, Madden TD, Bally MB. The liposomal formulation of doxorubicin. Methods Enzymol 2005; 391:71-97. Adis International L. HIV gp120 vaccine - VaxGen: AIDSVAX, AIDSVAX B/B, AIDSVAX B/E, HIV gp120 vaccine - Genentech, HIV gp120 vaccine AIDSVAX - VaxGen, HIV vaccine AIDSVAX - VaxGen. Drugs R D 2003; 4:249-53. Afonso LC, Scharton TM, Vieira LQ, Wysocka M, Trinchieri G, Scott P. The adjuvant effect of interleukin-12 in a vaccine against Leishmania major. Science 1994; 263:235-7. Aggarwal N, Santiago AM, Kessel D, Sloane BF. Photodynamic therapy as an effective therapeutic approach in MAME models of inflammatory breast cancer. Breast Cancer Res Treat 2015. Agostinis P, Berg K, Cengel KA, Foster TH, Girotti AW, Gollnick SO, et al. Photodynamic therapy of cancer: an update. CA Cancer J Clin 2011; 61:250-81. Agrawal A, Huang S, Wei Haw Lin A, Lee MH, Barton JK, Drezek RA, et al. Quantitative evaluation of optical coherence tomography signal enhancement with gold nanoshells. J Biomed Opt 2006; 11:041121. Agrawal A, Manchester M. Differential uptake of chemically modified cowpea mosaic virus nanoparticles in macrophage subpopulations present in inflammatory and tumor microenvironments. Biomacromolecules 2012; 13:3320-6. Aljabali AA, Sainsbury F, Lomonossoff GP, Evans DJ. Cowpea mosaic virus unmodified empty viruslike particles loaded with metal and metal oxide. Small 2010; 6:818- 21. Aljabali AA, Shukla S, Lomonossoff GP, Steinmetz NF, Evans DJ. CPMV-DOX delivers. Mol Pharm 2013; 10:3-10. Alkhatib G, Combadiere C, Broder CC, Feng Y, Kennedy PE, Murphy PM, et al. CC CKR5: a RANTES, MIP-1alpha, MIP-1beta receptor as a fusion cofactor for macrophage-tropic HIV-1. Science 1996; 272:1955-8. Allen TM, Brandeis E, Hansen CB, Kao GY, Zalipsky S. A new strategy for attachment of antibodies to sterically stabilized liposomes resulting in efficient targeting to cancer cells. Biochim Biophys Acta 1995; 1237:99-108. Allen TM, Cullis PR. Liposomal drug delivery systems: from concept to clinical applications. Adv Drug Deliv Rev 2013; 65:36-48. Allen TM, Hansen C, Martin F, Redemann C, Yau-Young A. Liposomes containing synthetic lipid derivatives of poly(ethylene glycol) show prolonged circulation half-lives in vivo. Biochim Biophys Acta 1991; 1066:29-36. Almeida JP, Lin AY, Langsner RJ, Eckels P, Foster AE, Drezek RA. In vivo immune cell distribution of gold nanoparticles in naive and tumor bearing mice. Small 2014; 10:812-9.
323 BIBLIOGRAPHY
Ambul PM, Tissot AC, Fulurija A, Maurer P, Nussberger J, Sabat R, et al. A vaccine for hypertension based on virus-like particles: preclinical efficacy and phase I safety and immunogenicity. J Hypertens 2007; 25:63-72. Andre FE, Safary A. Summary of clinical findings on Engerix-B, a genetically engineered yeast derived hepatitis B vaccine. Postgrad Med J 1987; 63 Suppl 2:169-77. Andtbacka RH, Kaufman HL, Collichio F, Amatruda T, Senzer N, Chesney J, et al. Talimogene Laherparepvec Improves Durable Response Rate in Patients With Advanced Melanoma. J Clin Oncol 2015; 33:2780-8. Anselmo AC, Mitragoti S. Nanoparticles in the Clinic. Bioengineering & Translational Medicine 2016. Antoni PM, Naik A, Albert I, Rubbiani R, Gupta S, Ruiz-Sanchez P, et al. (Metallo)porphyrins as potent phototoxic anti-cancer agents after irradiation with red light. Chemistry 2015; 21:1179-83. Arnida, Janat-Amsbury MM, Ray A, Peterson CM, Ghandehari H. Geometry and surface characteristics of gold nanoparticles influence their biodistribution and uptake by macrophages. Eur J Pharm Biopharm 2011; 77:417-23. Arnida, Janát-Amsbury MM, Ray A, Peterson CM, Ghandehari H. Geometry and surface characteristics of gold nanoparticles influence their biodistribution and uptake by macrophages. European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft für Pharmazeutische Verfahrenstechnik eV 2011; 77:417-23. Arnold GF, Velasco PK, Holmes AK, Wrin T, Geisler SC, Phung P, et al. Broad neutralization of human immunodeficiency virus type 1 (HIV-1) elicited from human rhinoviruses that display the HIV-1 gp41 ELDKWA epitope. J Virol 2009; 83:5087-100. Aryal M, Vykhodtseva N, Zhang YZ, McDannold N. Multiple sessions of liposomal doxorubicin delivery via focused ultrasound mediated blood-brain barrier disruption: a safety study. J Control Release 2015; 204:60-9. Azzarelli A, Gronchi A, Bertulli R, Tesoro JD, Baratti D, Pennacchioli E, et al. Low-dose chemotherapy with methotrexate and vinblastine for patients with advanced aggressive fibromatosis. Cancer 2001; 92:1259-64. Babin C, Majeau N, Leclerc D. Engineering of papaya mosaic virus (PapMV) nanoparticles with a CTL epitope derived from influenza NP. J Nanobiotechnology 2013; 11:10. Bachmann MF, Jennings GT. Therapeutic vaccines for chronic diseases: successes and technical challenges. Philos Trans R Soc Lond B Biol Sci 2011; 366:2815-22. Bachmann MF, Whitehead P. Active immunotherapy for chronic diseases. Vaccine 2013; 31:1777-84. Bagalkot V, Lee IH, Yu MK, Lee E, Park S, Lee JH, et al. A combined chemoimmunotherapy approach using a plasmid-doxorubicin complex. Mol Pharm 2009; 6:1019-28. Bainbridge JW, Mehat MS, Sundaram V, Robbie SJ, Barker SE, Ripamonti C, et al. Long-term effect of gene therapy on Leber's congenital amaurosis. N Engl J Med 2015; 372:1887-97.
324 BIBLIOGRAPHY
Baird JR, Byrne KT, Lizotte PH, Toraya-Brown S, Scarlett UK, Alexander MP, et al. Immune-mediated regression of established B16F10 melanoma by intratumoral injection of attenuated Toxoplasma gondii protects against rechallenge. J Immunol 2013; 190:469-78. Baiu DC, Brazel CS, Bao Y, Otto M. Interactions of iron oxide nanoparticles with the immune system: challenges and opportunities for their use in nano-oncology. Curr Pharm Des 2013; 19:6606-21. Baize S, Pannetier D, Oestereich L, Rieger T, Koivogui L, Magassouba N, et al. Emergence of Zaire Ebola virus disease in Guinea. N Engl J Med 2014; 371:1418- 25. Balch CM, Gershenwald JE, Soong SJ, Thompson JF, Atkins MB, Byrd DR, et al. Final version of 2009 AJCC melanoma staging and classification. J Clin Oncol 2009; 27:6199-206. Baldea I, Filip AG. Photodynamic therapy in melanoma--an update. J Physiol Pharmacol 2012; 63:109-18. Baldea I, Ion RM, Olteanu DE, Nenu I, Tudor D, Filip AG. Photodynamic therapy of melanoma using new, synthetic porphyrins and phthalocyanines as photosensitisers - a comparative study. Clujul Med 2015; 88:175-80. Balin-Gauthier D, Delord J-P, Rochaix P, Mallard V, Thomas F, Hennebelle I, et al. In vivo and in vitro antitumor activity of oxaliplatin in combination with cetuximab in human colorectal tumor cell lines expressing different level of EGFR. Cancer chemotherapy and pharmacology 2006; 57:709-18. Banerjee D, Liu AP, Voss NR, Schmid SL, Finn MG. Multivalent Display and Receptor- Mediated Endocytosis of Transferrin on Virus-Like Particles. Chembiochem 2010; 11:1273-9. Bard F, Barbour R, Cannon C, Carretto R, Fox M, Games D, et al. Epitope and isotype specificities of antibodies to beta -amyloid peptide for protection against Alzheimer's disease-like neuropathology. Proc Natl Acad Sci U S A 2003; 100:2023-8. Bard F, Cannon C, Barbour R, Burke RL, Games D, Grajeda H, et al. Peripherally administered antibodies against amyloid beta-peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer disease. Nat Med 2000; 6:916-9. Bardhan R, Lal S, Joshi A, Halas NJ. Theranostic nanoshells: from probe design to imaging and treatment of cancer. Acc Chem Res 2011; 44:936-46. Bardhan R, Mukherjee S, Mirin NA, Levit SD, Nordlander P, Halas NJ. Nanosphere-in- a-Nanoshell: A Simple Nanomatryushka. Journal of Physical Chemistry C 2010; 114:7378-83. Bareford LM, Swaan PW. Endocytic mechanisms for targeted drug delivery. Advanced drug delivery reviews 2007; 59:748-58. Barth M, Schietinger S, Schroder T, Aichele T, Benson O. Controlled coupling of NV defect centers to plasmonic and photonic nanostructures. Journal of Luminescence 2010; 130:1628-34. Barua S, Yoo JW, Kolhar P, Wakankar A, Gokarn YR, Mitragotri S. Particle shape enhances specificity of antibody-displaying nanoparticles. Proc Natl Acad Sci U S A 2013; 110:3270-5.
325 BIBLIOGRAPHY
Baselga J. The EGFR as a target for anticancer therapy—focus on cetuximab. European Journal of Cancer 2001; 37:16-22. Baskar R, Lee KA, Yeo R, Yeoh KW. Cancer and radiation therapy: current advances and future directions. Int J Med Sci 2012; 9:193-9. Basle E, Joubert N, Pucheault M. Protein chemical modification on endogenous amino acids. Chem Biol 2010; 17:213-27. Bazak R, Houri M, Achy SE, Hussein W, Refaat T. Passive targeting of nanoparticles to cancer: A comprehensive review of the literature. Mol Clin Oncol 2014; 2:904-8. Beer TM, Bernstein GT, Corman JM, Glode LM, Hall SJ, Poll WL, et al. Randomized trial of autologous cellular immunotherapy with sipuleucel-T in androgen- dependent prostate cancer. Clin Cancer Res 2011; 17:4558-67. Berkower I, Raymond M, Muller J, Spadaccini A, Aberdeen A. Assembly, structure, and antigenic properties of virus-like particles rich in HIV-1 envelope gp120. Virology 2004; 321:75-86. Bernardi RJ, Lowery AR, Thompson PA, Blaney SM, West JL. Immunonanoshells for targeted photothermal ablation in medulloblastoma and glioma: an in vitro evaluation using human cell lines. Journal of Neuro-Oncology 2008; 86:165-72. Bessa J, Jegerlehner A, Hinton HJ, Pumpens P, Saudan P, Schneider P, et al. Alveolar macrophages and lung dendritic cells sense RNA and drive mucosal IgA responses. J Immunol 2009; 183:3788-99. Bethge WA, Hegenbart U, Stuart MJ, Storer BE, Maris MB, Flowers ME, et al. Adoptive immunotherapy with donor lymphocyte infusions after allogeneic hematopoietic cell transplantation following nonmyeloablative conditioning. Blood 2004; 103:790-5. Biffi A, Montini E, Lorioli L, Cesani M, Fumagalli F, Plati T, et al. Lentiviral hematopoietic stem cell gene therapy benefits metachromatic leukodystrophy. Science 2013; 341:1233158. Blackadar CB. Historical review of the causes of cancer. World J Clin Oncol 2016; 7:54- 86. Blaney JE, Wirblich C, Papaneri AB, Johnson RF, Myers CJ, Juelich TL, et al. Inactivated or live-attenuated bivalent vaccines that confer protection against rabies and Ebola viruses. J Virol 2011; 85:10605-16. Bomsel M, Tudor D, Drillet AS, Alfsen A, Ganor Y, Roger MG, et al. Immunization with HIV-1 gp41 subunit virosomes induces mucosal antibodies protecting nonhuman primates against vaginal SHIV challenges. Immunity 2011; 34:269-80. Bosch FX, de Sanjose S. The epidemiology of human papillomavirus infection and cervical cancer. Dis Markers 2007; 23:213-27. Bottini M, Rosato N, Bottini N. PEG-Modified Carbon Nanotubes in Biomedicine: Current Status and Challenges Ahead. Biomacromolecules 2011; 12:3381-93. Bovier PA. Epaxal: a virosomal vaccine to prevent hepatitis A infection. Expert Rev Vaccines 2008; 7:1141-50. Brechtl JR, Breitbart W, Galietta M, Krivo S, Rosenfeld B. The use of highly active antiretroviral therapy (HAART) in patients with advanced HIV infection: impact on medical, palliative care, and quality of life outcomes. J Pain Symptom Manage 2001; 21:41-51.
326 BIBLIOGRAPHY
Brinson BE, Lassiter JB, Levin CS, Bardhan R, Mirin N, Halas NJ. Nanoshells Made Easy: Improving Au Layer Growth on Nanoparticle Surfaces. Langmuir 2008; 24:14166-71. Broekgaarden M, Weijer R, van Gulik TM, Hamblin MR, Heger M. Tumor cell survival pathways activated by photodynamic therapy: a molecular basis for pharmacological inhibition strategies. Cancer Metastasis Rev 2015; 34:643-90. Broersma S. J Chem Phys 1981; 74:6969-70. Brown DM, Johnston H, Gubbins E, Stone V. Serum enhanced cytokine responses of macrophages to silica and iron oxide particles and nanomaterials: a comparison of serum to lung lining fluid and albumin dispersions. J Appl Toxicol 2014. Brown DR, Kjaer SK, Sigurdsson K, Iversen OE, Hernandez-Avila M, Wheeler CM, et al. The impact of quadrivalent human papillomavirus (HPV; types 6, 11, 16, and 18) L1 virus-like particle vaccine on infection and disease due to oncogenic nonvaccine HPV types in generally HPV-naive women aged 16-26 years. J Infect Dis 2009; 199:926-35. Bruckman MA, Czapar AE, VanMeter A, Randolph LN, Steinmetz NF. Tobacco mosaic virus-based protein nanoparticles and nanorods for chemotherapy delivery targeting breast cancer. J Control Release 2016; 231:103-13. Bruckman MA, Hern S, Jiang K, Flask CA, Yu X, Steinmetz NF. Tobacco mosaic virus rods and spheres as supramolecular high-relaxivity MRI contrast agents. J Mater Chem B Mater Biol Med 2013; 1:1482-90. Bruckman Ma, Jiang K, Simpson EJ, Randolph LN, Luyt LG, Yu X, et al. Dual-modal magnetic resonance and fluorescence imaging of atherosclerotic plaques in vivo using VCAM-1 targeted tobacco mosaic virus. Nano letters 2014; 14:1551-8. Bruckman MA, Kaur G, Lee LA, Xie F, Sepulveda J, Breitenkamp R, et al. Surface modification of tobacco mosaic virus with "click" chemistry. Chembiochem 2008; 9:519-23. Bruckman MA, Randolph LN, Vanmeter A, Hern S, Shoffstall AJ, Taurog RE, et al. Biodistribution, pharmacokinetics, and blood compatibility of native and PEGylated tobacco mosaic virus nano-rods and -spheres in mice. Virology 2014; 449:163-73. Bruckman MA, Steinmetz NF. Chemical modification of the inner and outer surfaces of Tobacco Mosaic Virus (TMV). Methods Mol Biol 2014; 1108:173-85. Buratti E, Tisminetzky SG, Scodeller ES, Baralle FE. Conformational display of two neutralizing epitopes of HIV-1 gp41 on the Flock House virus capsid protein. J Immunol Methods 1996; 197:7-18. Cai S, Vijayan K, Cheng D, Lima EM, Discher DE. Micelles of different morphologies-- advantages of worm-like filomicelles of PEO-PCL in paclitaxel delivery. Pharm Res 2007; 24:2099-109. Cai S, Vijayan K, Cheng D, Lima EM, Discher DE. Micelles of Different Morphologies—Advantages of Worm-like Filomicelles of PEO-PCL in Paclitaxel Delivery. Pharmaceutical Research 2007; 24:2099-109. Caldorera-Moore M, Guimard N, Shi L, Roy K. Designer nanoparticles: incorporating size, shape and triggered release into nanoscale drug carriers. Expert opinion on drug delivery 2010; 7:479-95.
327 BIBLIOGRAPHY
Campbell RB, Fukumura D, Brown EB, Mazzola LM, Izumi Y, Jain RK, et al. Cationic charge determines the distribution of liposomes between the vascular and extravascular compartments of tumors. Cancer Research 2002; 62:6831-6. Cao J, Guenther RH, Sit TL, Opperman CH, Lommel SA, Willoughby JA. Loading and release mechanism of red clover necrotic mosaic virus derived plant viral nanoparticles for drug delivery of doxorubicin. Small 2014; 10:5126-36. Carelle N, Piotto E, Bellanger A, Germanaud J, Thuillier A, Khayat D. Changing patient perceptions of the side effects of cancer chemotherapy. Cancer 2002; 95:155-63. Carette N, Engelkamp H, Akpa E, Pierre SJ, Cameron NR, Christianen PC, et al. A virus- based biocatalyst. Nat Nanotechnol 2007; 2:226-9. Caron PC, Schwartz MA, Co MS, Queen C, Finn RD, Graham MC, et al. Murine and humanized constructs of monoclonal antibody M195 (anti-CD33) for the therapy of acute myelogenous leukemia. Cancer 1994; 73:1049-56. Carpenter BL, Feese E, Sadeghifar H, Argyropoulos DS, Ghiladi RA. Porphyrin- cellulose nanocrystals: a photobactericidal material that exhibits broad spectrum antimicrobial activity. Photochem Photobiol 2012; 88:527-36. Carpenter BL, Scholle F, Sadeghifar H, Francis AJ, Boltersdorf J, Weare WW, et al. Synthesis, Characterization, and Antimicrobial Efficacy of Photomicrobicidal Cellulose Paper. Biomacromolecules 2015; 16:2482-92. Carrera MR, Ashley JA, Parsons LH, Wirsching P, Koob GF, Janda KD. Suppression of psychoactive effects of cocaine by active immunization. Nature 1995; 378:727-30. Castano AP, Demidova TN, Hamblin MR. Mechanisms in photodynamic therapy: part one-photosensitizers, photochemistry and cellular localization. Photodiagnosis Photodyn Ther 2004; 1:279-93. Castellsague X, Bosch FX. [Advances in cervical cancer prevention: HPV vaccines]. Farm Hosp 2007; 31:261-3. Cawley LJMC, Brien PO, Hudson LG. Overexpression of the Epidermal Growth Factor Receptor Contributes to Enhanced Ligand-Mediated Motility in Keratinocyte Cell Lines. Endocrinology 2014; 138:121-7. Cerovska N, Hoffmeisterova H, Moravec T, Plchova H, Folwarczna J, Synkova H, et al. Transient expression of Human papillomavirus type 16 L2 epitope fused to N- and C-terminus of coat protein of Potato virus X in plants. J Biosci 2012; 37:125- 33. Chabner BA, Roberts TG, Jr. Timeline: Chemotherapy and the war on cancer. Nat Rev Cancer 2005; 5:65-72. Chackerian B. Virus-like particles: flexible platforms for vaccine development. Expert Rev Vaccines 2007; 6:381-90. Chackerian B, Lowy DR, Schiller JT. Induction of autoantibodies to mouse CCR5 with recombinant papillomavirus particles. Proc Natl Acad Sci U S A 1999; 96:2373-8. Chackerian B, Lowy DR, Schiller JT. Conjugation of a self-antigen to papillomavirus- like particles allows for efficient induction of protective autoantibodies. J Clin Invest 2001; 108:415-23. Chackerian B, Rangel M, Hunter Z, Peabody DS. Virus and virus-like particle-based immunogens for Alzheimer's disease induce antibody responses against amyloid- beta without concomitant T cell responses. Vaccine 2006; 24:6321-31.
328 BIBLIOGRAPHY
Champion JA, Mitragotri S. Role of target geometry in phagocytosis. Proc Natl Acad Sci U S A 2006; 103:4930-4. Chang BM, Lin HH, Su LJ, Lin WD, Lin RJ, Tzeng YK, et al. Highly Fluorescent Nanodiamonds Protein-Functionalized for Cell Labeling and Targeting. Advanced Functional Materials 2013; 23:5737-45. Chariou PL, Lee KL, Pokorski JK, Saidel GM, Steinmetz NF. Diffusion and Uptake of Tobacco Mosaic Virus as Therapeutic Carrier in Tumor Tissue: Effect of Nanoparticle Aspect Ratio. J Phys Chem B 2016. Chatterjee K, Zhang J, Honbo N, Karliner JS. Doxorubicin cardiomyopathy. Cardiology 2010; 115:155-62. Chatterji A, Ochoa WF, Paine M, Ratna BR, Johnson JE, Lin T. New addresses on an addressable virus nanoblock; uniquely reactive Lys residues on cowpea mosaic virus. Chemistry & biology 2004; 11:855-63. Chauhan VP, Popovic Z, Chen O, Cui J, Fukumura D, Bawendi MG, et al. Fluorescent nanorods and nanospheres for real-time in vivo probing of nanoparticle shape- dependent tumor penetration. Angew Chem Int Ed Engl 2011; 50:11417-20. Chauhan VP, Popović Z, Chen O, Cui J, Fukumura D, Bawendi MG, et al. Fluorescent nanorods and nanospheres for real-time in vivo probing of nanoparticle shape- dependent tumor penetration. Angewandte Chemie (International ed in English) 2011; 50:11417-20. Cheever MA, Higano CS. PROVENGE (Sipuleucel-T) in prostate cancer: the first FDA- approved therapeutic cancer vaccine. Clin Cancer Res 2011; 17:3520-6. Chen DS, Mellman I. Oncology meets immunology: the cancer-immunity cycle. Immunity 2013; 39:1-10. Chen GX, Liu Y, Song M, Wu BT, Wu E, Zeng HP. Photoluminescence Enhancement Dependent on the Orientations of Single NV Centers in Nanodiamonds on a Gold Film. Ieee Journal of Selected Topics in Quantum Electronics 2013; 19. Chen NT, Wu CY, Chung CY, Hwu Y, Cheng SH, Mou CY, et al. Probing the dynamics of doxorubicin-DNA intercalation during the initial activation of apoptosis by fluorescence lifetime imaging microscopy (FLIM). PLoS One 2012; 7:e44947. Chen S, Zheng D, Li C, Zhang W, Xu W, Liu X, et al. Protection against multiple subtypes of influenza viruses by virus-like particle vaccines based on a hemagglutinin conserved epitope. Biomed Res Int 2015; 2015:901817. Chen XS, Stehle T, Harrison SC. Interaction of polyomavirus internal protein VP2 with the major capsid protein VP1 and implications for participation of VP2 in viral entry. EMBO J 1998; 17:3233-40. Chen YS, Frey W, Kim S, Kruizinga P, Homan K, Emelianov S. Silica-coated gold nanorods as photoacoustic signal nanoamplifiers. Nano Lett 2011; 11:348-54. Chen ZG. Small-molecule delivery by nanoparticles for anticancer therapy. Trends Mol Med 2010; 16:594-602. Cheng LC, Chen HM, Lai TC, Chan YC, Liu RS, Sung JC, et al. Targeting polymeric fluorescent nanodiamond-gold/silver multi-functional nanoparticles as a light- transforming hyperthermia reagent for cancer cells. Nanoscale 2013; 5:3931-40. Cheng Y, Cheng H, Jiang C, Qiu X, Wang K, Huan W, et al. Perfluorocarbon nanoparticles enhance reactive oxygen levels and tumour growth inhibition in photodynamic therapy. Nat Commun 2015; 6:8785.
329 BIBLIOGRAPHY
Cheung-Ong K, Giaever G, Nislow C. DNA-damaging agents in cancer chemotherapy: serendipity and chemical biology. Chem Biol 2013; 20:648-59. Chi YZ, Chen GX, Jelezko F, Wu E, Zeng HP. Enhanced Photoluminescence of Single- Photon Emitters in Nanodiamonds on a Gold Film. Ieee Photonics Technology Letters 2011; 23:374-6. Chiodoni C, Stoppacciaro A, Sangaletti S, Gri G, Cappetti B, Koezuka Y, et al. Different requirements for alpha-galactosylceramide and recombinant IL-12 antitumor activity in the treatment of C-26 colon carcinoma hepatic metastases. Eur J Immunol 2001; 31:3101-10. Chitale R. Merck hopes to extend gardasil vaccine to men. J Natl Cancer Inst 2009; 101:222-3. Cho K, Wang X, Nie S, Chen ZG, Shin DM. Therapeutic nanoparticles for drug delivery in cancer. Clinical cancer research : an official journal of the American Association for Cancer Research 2008; 14:1310-6. Choe H, Farzan M, Sun Y, Sullivan N, Rollins B, Ponath PD, et al. The beta-chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates. Cell 1996; 85:1135-48. Chow EK, Zhang XQ, Chen M, Lam R, Robinson E, Huang H, et al. Nanodiamond therapeutic delivery agents mediate enhanced chemoresistant tumor treatment. Sci Transl Med 2011; 3:73ra21. Chow EKH, Ho D. Cancer Nanomedicine: From Drug Delivery to Imaging. Science Translational Medicine 2013; 5. Christian DA, Cai S, Garbuzenko OB, Harada T, Allison L, Minko T, et al. Flexible filaments for in vivo imaging and delivery: persistent circulation of filomicelles opens the dosage window for sustained tumor shrinkage. Molecular pharmaceutics 2009; 6:1343-52. Christian DA, Cai S, Garbuzenko OB, Harada T, Zajac AL, Minko T, et al. Flexible filaments for in vivo imaging and delivery: persistent circulation of filomicelles opens the dosage window for sustained tumor shrinkage. Mol Pharm 2009; 6:1343-52. Chu JH, Yu S, Hayward SW, Chan FL. Development of a three-dimensional culture model of prostatic epithelial cells and its use for the study of epithelial- mesenchymal transition and inhibition of PI3K pathway in prostate cancer. Prostate 2009; 69:428-42. Claes A, Idema AJ, Wesseling P. Diffuse glioma growth: a guerilla war. Acta Neuropathol 2007; 114:443-58. Clem AS. Fundamentals of vaccine immunology. J Glob Infect Dis 2011; 3:73-8. Clifford GM, Gallus S, Herrero R, Munoz N, Snijders PJ, Vaccarella S, et al. Worldwide distribution of human papillomavirus types in cytologically normal women in the International Agency for Research on Cancer HPV prevalence surveys: a pooled analysis. Lancet 2005; 366:991-8. Cohen BA, Bergkvist M. Targeted in vitro photodynamic therapy via aptamer-labeled, porphyrin-loaded virus capsids. J Photochem Photobiol B 2013; 121:67-74.
330 BIBLIOGRAPHY
Colombat P, Salles G, Brousse N, Eftekhari P, Soubeyran P, Delwail V, et al. Rituximab (anti-CD20 monoclonal antibody) as single first-line therapy for patients with follicular lymphoma with a low tumor burden: clinical and molecular evaluation. Blood 2001; 97:101-6. Colombo MP, Trinchieri G. Interleukin-12 in anti-tumor immunity and immunotherapy. Cytokine Growth Factor Rev 2002; 13:155-68. Couvreur P, Kante B, Grislain L, Roland M, Speiser P. Toxicity of polyalkylcyanoacrylate nanoparticles II: Doxorubicin-loaded nanoparticles. J Pharm Sci 1982; 71:790-2. Coxon TP, Fallows TW, Gough JE, Webb SJ. A versatile approach towards multivalent saccharide displays on magnetic nanoparticles and phospholipid vesicles. Org Biomol Chem 2015; 13:10751-61. Crank J. The mathematics of diffusion. Oxford: Oxford University Press; 2004. Croyle MA, Chirmule N, Zhang Y, Wilson JM. "Stealth" adenoviruses blunt cell- mediated and humoral immune responses against the virus and allow for significant gene expression upon readministration in the lung. Journal of Virology 2001; 75:4792-801. Cubas R, Zhang S, Kwon S, Sevick-Muraca EM, Li M, Chen C, et al. Virus-like particle (VLP) lymphatic trafficking and immune response generation after immunization by different routes. J Immunother 2009; 32:118-28. Czapar AE, Zheng YR, Riddell IA, Shukla S, Awuah SG, Lippard SJ, et al. Tobacco mosaic virus delivery of phenanthriplatin for cancer therapy. ACS Nano 2016. D'Aoust MA, Lavoie PO, Couture MM, Trepanier S, Guay JM, Dargis M, et al. Influenza virus-like particles produced by transient expression in Nicotiana benthamiana induce a protective immune response against a lethal viral challenge in mice. Plant Biotechnol J 2008; 6:930-40. Dai X, Yue Z, Eccleston ME, Swartling J, Slater NK, Kaminski CF. Fluorescence intensity and lifetime imaging of free and micellar-encapsulated doxorubicin in living cells. Nanomedicine 2008; 4:49-56. Danhier F, Feron O, Preat V. To exploit the tumor microenvironment: Passive and active tumor targeting of nanocarriers for anti-cancer drug delivery. J Control Release 2010; 148:135-46. Danhier F, Feron O, Préat V. To exploit the tumor microenvironment: Passive and active tumor targeting of nanocarriers for anti-cancer drug delivery. Journal of controlled release : official journal of the Controlled Release Society 2010; 148:135-46. Daniel MC, Astruc D. Gold nanoparticles: assembly, supramolecular chemistry, quantum-size-related properties, and applications toward biology, catalysis, and nanotechnology. Chem Rev 2004; 104:293-346. Daniels TR, Bernabeu E, Rodriguez JA, Patel S, Kozman M, Chiappetta DA, et al. The transferrin receptor and the targeted delivery of therapeutic agents against cancer. Biochimica Et Biophysica Acta-General Subjects 2012; 1820:291-317. Daniels TR, Delgado T, Helguera G, Penichet ML. The transferrin receptor part II: Targeted delivery of therapeutic agents into cancer cells. Clinical Immunology 2006; 121:159-76.
331 BIBLIOGRAPHY
Daum N, Tscheka C, Neumeyer A, Schneider M. Novel approaches for drug delivery systems in nanomedicine: effects of particle design and shape. Wiley Interdisciplinary Reviews-Nanomedicine and Nanobiotechnology 2012; 4:52-65. Davis ME, Chen ZG, Shin DM. Nanoparticle therapeutics: an emerging treatment modality for cancer. Nat Rev Drug Discov 2008; 7:771-82. De BP, Pagovich OE, Hicks MJ, Rosenberg JB, Moreno AY, Janda KD, et al. Disrupted adenovirus-based vaccines against small addictive molecules circumvent anti- adenovirus immunity. Hum Gene Ther 2013; 24:58-66. De Geest B, Snoeys J, Van Linthout S, Lievens J, Collen D. Elimination of innate immune responses and liver inflammation by PEGylation of adenoviral vectors and methylprednisolone. Hum Gene Ther 2005; 16:1439-51. de Gennes PG. Polymers at an interface: a simplified view. Adv Colloid Interface Sci 1987; 27:189-209. de Jong JC, Beyer WE, Palache AM, Rimmelzwaan GF, Osterhaus AD. Mismatch between the 1997/1998 influenza vaccine and the major epidemic A(H3N2) virus strain as the cause of an inadequate vaccine-induced antibody response to this strain in the elderly. J Med Virol 2000; 61:94-9. de Wit E, Marzi A, Bushmaker T, Brining D, Scott D, Richt JA, et al. Safety of recombinant VSV-Ebola virus vaccine vector in pigs. Emerg Infect Dis 2015; 21:702-4. Decuzzi P, Ferrari M. The adhesive strength of non-spherical particles mediated by specific interactions. Biomaterials 2006; 27:5307-14. Decuzzi P, Godin B, Tanaka T, Lee S-Y, Chiappini C, Liu X, et al. Size and shape effects in the biodistribution of intravascularly injected particles. Journal of controlled release : official journal of the Controlled Release Society 2010; 141:320-7. Decuzzi P, Godin B, Tanaka T, Lee SY, Chiappini C, Liu X, et al. Size and shape effects in the biodistribution of intravascularly injected particles. J Control Release 2010; 141:320-7. Decuzzi P, Pasqualini R, Arap W, Ferrari M. Intravascular delivery of particulate systems: does geometry really matter? Pharmaceutical Research 2009; 26:235-43. Degennes PG. Polymers at an Interface - a Simplified View. Advances in Colloid and Interface Science 1987; 27:189-209. Dell'Orco D, Lundqvist M, Cedervall T, Linse S. Delivery success rate of engineered nanoparticles in the presence of the protein corona: a systems-level screening. Nanomedicine 2012; 8:1271-81. Dellian M, Yuan F, Trubetskoy VS, Torchilin VP, Jain RK. Vascular permeability in a human tumour xenograft: molecular charge dependence. British journal of cancer 2000; 82:1513-8. Demir M, Stowell MHB. A chemoselective biomolecular template for assembling diverse nanotubular materials. Nanotechnology 2002; 13:541-4. Deng H, Liu R, Ellmeier W, Choe S, Unutmaz D, Burkhart M, et al. Identification of a major co-receptor for primary isolates of HIV-1. Nature 1996; 381:661-6. Denis J, Acosta-Ramirez E, Zhao Y, Hamelin ME, Koukavica I, Baz M, et al. Development of a universal influenza A vaccine based on the M2e peptide fused to the papaya mosaic virus (PapMV) vaccine platform. Vaccine 2008; 26:3395- 403.
332 BIBLIOGRAPHY
Denis J, Majeau N, Acosta-Ramirez E, Savard C, Bedard MC, Simard S, et al. Immunogenicity of papaya mosaic virus-like particles fused to a hepatitis C virus epitope: evidence for the critical function of multimerization. Virology 2007; 363:59-68. Desai N. Challenges in development of nanoparticle-based therapeutics. AAPS J 2012; 14:282-95. Ding FX, Wang F, Lu YM, Li K, Wang KH, He XW, et al. Multiepitope peptide-loaded virus-like particles as a vaccine against hepatitis B virus-related hepatocellular carcinoma. Hepatology 2009; 49:1492-502. Dolmans DE, Fukumura D, Jain RK. Photodynamic therapy for cancer. Nat Rev Cancer 2003; 3:380-7. Donnelly ML, Luke G, Mehrotra A, Li X, Hughes LE, Gani D, et al. Analysis of the aphthovirus 2A/2B polyprotein 'cleavage' mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal 'skip'. J Gen Virol 2001; 82:1013-25. Doranz BJ, Rucker J, Yi Y, Smyth RJ, Samson M, Peiper SC, et al. A dual-tropic primary HIV-1 isolate that uses fusin and the beta-chemokine receptors CKR-5, CKR-3, and CKR-2b as fusion cofactors. Cell 1996; 85:1149-58. Doshi N, Prabhakarpandian B, Rea-Ramsey A, Pant K, Sundaram S, Mitragotri S. Flow and adhesion of drug carriers in blood vessels depend on their shape: a study using model synthetic microvascular networks. Journal of controlled release : official journal of the Controlled Release Society 2010; 146:196-200. Dougherty TJ, Gomer CJ, Henderson BW, Jori G, Kessel D, Korbelik M, et al. Photodynamic therapy. J Natl Cancer Inst 1998; 90:889-905. Dowdle W. The search for an AIDS vaccine. Public Health Rep 1986; 101:232-3. Doxorubicin Hydrochloride. National Cancer Institute; 2014. Dragic T, Litwin V, Allaway GP, Martin SR, Huang Y, Nagashima KA, et al. HIV-1 entry into CD4+ cells is mediated by the chemokine receptor CC-CKR-5. Nature 1996; 381:667-73. Drake CG. Combination immunotherapy approaches. Ann Oncol 2012; 23 Suppl 8:viii41-6. Dufau I, Frongia C, Sicard F, Dedieu L, Cordelier P, Ausseil F, et al. Multicellular tumor spheroid model to evaluate spatio-temporal dynamics effect of chemotherapeutics: application to the gemcitabine/CHK1 inhibitor combination in pancreatic cancer. BMC cancer 2012; 12:15. Dufes C, Muller JM, Couet W, Olivier JC, Uchegbu IF, Schatzlein AG. Anticancer drug delivery with transferrin targeted polymeric chitosan vesicles. Pharm Res 2004; 21:101-7. Dunkel A, Shen S, LaBranche CC, Montefiori D, McGettigan JP. A Bivalent, Chimeric Rabies Virus Expressing Simian Immunodeficiency Virus Envelope Induces Multifunctional Antibody Responses. AIDS Res Hum Retroviruses 2015. El-Serag HB. Hepatocellular carcinoma. N Engl J Med 2011; 365:1118-27. Elliott JA, Winn WC, Jr. Treatment of alveolar macrophages with cytochalasin D inhibits uptake and subsequent growth of Legionella pneumophila. Infect Immun 1986; 51:31-6.
333 BIBLIOGRAPHY
Epstein SL, Kong WP, Misplon JA, Lo CY, Tumpey TM, Xu L, et al. Protection against multiple influenza A subtypes by vaccination with highly conserved nucleoprotein. Vaccine 2005; 23:5404-10. Esparza J. A brief history of the global effort to develop a preventive HIV vaccine. Vaccine 2013; 31:3502-18. Fang J, Nakamura H, Maeda H. The EPR effect: Unique features of tumor blood vessels for drug delivery, factors involved, and limitations and augmentation of the effect. Adv Drug Deliv Rev 2011; 63:136-51. Farkas ME, Aanei IL, Behrens CR, Tong GJ, Murphy ST, Neil JPO, et al. PET Imaging and Biodistribution of Chemically Modified Bacteriophage MS2. Molecular pharmaceutics 2013; 10:69-76. Farkas ME, Aanei IL, Behrens CR, Tong GJ, Murphy ST, O'Neil JP, et al. PET Imaging and biodistribution of chemically modified bacteriophage MS2. Mol Pharm 2013; 10:69-76. Farlow MR, Andreasen N, Riviere ME, Vostiar I, Vitaliti A, Sovago J, et al. Long-term treatment with active Abeta immunotherapy with CAD106 in mild Alzheimer's disease. Alzheimers Res Ther 2015; 7:23. Farokhzad OC, Langer R. Impact of Nanotechnology on Drug Delivery. Acs Nano 2009; 3:16-20. Feese E. Development of Novel Photosensitizers for Photodynamic Inactivation of Bacteria. [Raleigh, North Carolina]: North Carolina State University; 2012. Feese E, Sadeghifar H, Gracz HS, Argyropoulos DS, Ghiladi RA. Photobactericidal porphyrin-cellulose nanocrystals: synthesis, characterization, and antimicrobial properties. Biomacromolecules 2011; 12:3528-39. Feldmann H, Jones SM, Daddario-DiCaprio KM, Geisbert JB, Stroher U, Grolla A, et al. Effective post-exposure treatment of Ebola infection. PLoS Pathog 2007; 3:e2. Feng G, Wang W, Qian Y, Jin H. Anti-Abeta antibodies induced by Abeta-HBc virus- like particles prevent Abeta aggregation and protect PC12 cells against toxicity of Abeta1-40. J Neurosci Methods 2013; 218:48-54. Ferrari M. Cancer nanotechnology: Opportunities and challenges. Nature Reviews Cancer 2005; 5:161-71. Ferrer I, Boada Rovira M, Sanchez Guerra ML, Rey MJ, Costa-Jussa F. Neuropathology and pathogenesis of encephalitis following amyloid-beta immunization in Alzheimer's disease. Brain Pathol 2004; 14:11-20. Fiore AE, Bridges CB, Cox NJ. Seasonal influenza vaccines. Curr Top Microbiol Immunol 2009; 333:43-82. Firme CP, 3rd, Bandaru PR. Toxicity issues in the application of carbon nanotubes to biological systems. Nanomedicine : nanotechnology, biology, and medicine 2010; 6:245-56. Fletcher JI, Haber M, Henderson MJ, Norris MD. ABC transporters in cancer: more than just drug efflux pumps. Nature reviews Cancer 2010; 10:147-56. Foster GD, Taylor S. Plant Virology Protocols: From Virus Isolation to Transgenic Resistance. Humana Press; 1998. Fox BS, Kantak KM, Edwards MA, Black KM, Bollinger BK, Botka AJ, et al. Efficacy of a therapeutic cocaine vaccine in rodent models. Nat Med 1996; 2:1129-32.
334 BIBLIOGRAPHY
Fulurija A, Lutz TA, Sladko K, Osto M, Wielinga PY, Bachmann MF, et al. Vaccination against GIP for the treatment of obesity. PLoS One 2008; 3:e3163. Galizia G, Lieto E, Vita F, Orditura M, Castellano P, Troiani T, et al. Cetuximab, a chimeric human mouse anti-epidermal growth factor receptor monoclonal antibody, in the treatment of human colorectal cancer. Oncogene 2007; 26:3654- 60. Gambhira R, Karanam B, Jagu S, Roberts JN, Buck CB, Bossis I, et al. A protective and broadly cross-neutralizing epitope of human papillomavirus L2. J Virol 2007; 81:13927-31. Gameiro SR, Jammeh ML, Wattenberg MM, Tsang KY, Ferrone S, Hodge JW. Radiation-induced immunogenic modulation of tumor enhances antigen processing and calreticulin exposure, resulting in enhanced T-cell killing. Oncotarget 2014; 5:403-16. Gandra N, Abbineni G, Qu X, Huai Y, Wang L, Mao C. Bacteriophage bionanowire as a carrier for both cancer-targeting peptides and photosensitizers and its use in selective cancer cell killing by photodynamic therapy. Small 2013; 9:215-21. Ganson NJ, Povsic TJ, Sullenger BA, Alexander JH, Zelenkofske SL, Sailstad JM, et al. Pre-existing anti-polyethylene glycol antibody linked to first-exposure allergic reactions to pegnivacogin, a PEGylated RNA aptamer. J Allergy Clin Immunol 2016; 137:1610-3 e7. Geier MR, Geier DA, Zahalsky AC. A review of hepatitis B vaccination. Expert Opin Drug Saf 2003; 2:113-22. Geisbert TW, Feldmann H. Recombinant vesicular stomatitis virus-based vaccines against Ebola and Marburg virus infections. J Infect Dis 2011; 204 Suppl 3:S1075-81. Gelderblom H, Verweij J, Nooter K, Sparreboom A. Cremophor EL: the drawbacks and advantages of vehicle selection for drug formulation. Eur J Cancer 2001; 37:1590-8. Geng Y, Dalhaimer P, Cai S, Tsai R, Tewari M, Minko T, et al. Shape effects of filaments versus spherical particles in flow and drug delivery. Nat Nanotechnol 2007; 2:249-55. Gentile F, Chiappini C, Fine D, Bhavane RC, Peluccio MS, Cheng MM, et al. The effect of shape on the margination dynamics of non-neutrally buoyant particles in two- dimensional shear flows. J Biomech 2008; 41:2312-8. Gentile F, Chiappini C, Fine D, Bhavane RC, Peluccio MS, Cheng MM-C, et al. The effect of shape on the margination dynamics of non-neutrally buoyant particles in two-dimensional shear flows. Journal of biomechanics 2008; 41:2312-8. Gobin AM, Lee MH, Halas NJ, James WD, Drezek RA, West JL. Near-infrared resonant nanoshells for combined optical imaging and photothermal cancer therapy. Nano Lett 2007; 7:1929-34. Godugu C, Patel AR, Desai U, Andey T, Sams A, Singh M. AlgiMatrix based 3D cell culture system as an in-vitro tumor model for anticancer studies. PLoS One 2013; 8:e53708. Goeijenbier M, van Kampen JJ, Reusken CB, Koopmans MP, van Gorp EC. Ebola virus disease: a review on epidemiology, symptoms, treatment and pathogenesis. Neth J Med 2014; 72:442-8.
335 BIBLIOGRAPHY
Goldman MaL, P.-H. Immunological safety of vaccines: facts, hypotheses, and allegation. In: Kaufmann SHE, editor. Novel Vaccination Strategies. Germany: Wiley-VCH; 2004. p. 595-611. Gradishar WJ, Tjulandin S, Davidson N, Shaw H, Desai N, Bhar P, et al. Phase III trial of nanoparticle albumin-bound paclitaxel compared with polyethylated castor oil- based paclitaxel in women with breast cancer. J Clin Oncol 2005; 23:7794-803. Graf C, Vossen DLJ, Imhof A, van Blaaderen A. A general method to coat colloidal particles with silica. Langmuir 2003; 19:6693-700. Green MR, Manikhas GM, Orlov S, Afanasyev B, Makhson AM, Bhar P, et al. Abraxane, a novel Cremophor-free, albumin-bound particle form of paclitaxel for the treatment of advanced non-small-cell lung cancer. Ann Oncol 2006; 17:1263-8. Gregory AE, Titball R, Williamson D. Vaccine delivery using nanoparticles. Front Cell Infect Microbiol 2013; 3:13. Gulley JL, Madan RA, Tsang KY, Jochems C, Marte JL, Farsaci B, et al. Immune impact induced by PROSTVAC (PSA-TRICOM), a therapeutic vaccine for prostate cancer. Cancer Immunol Res 2014; 2:133-41. Gunawan C, Lim M, Marquis CP, Amal R. Nanoparticle-protein corona complexes govern the biological fates and functions of nanoparticles. Journal of Materials Chemistry B 2014; 2:2060-83. Haass C, Selkoe DJ. Cellular processing of beta-amyloid precursor protein and the genesis of amyloid beta-peptide. Cell 1993; 75:1039-42. Hackel PO, Zwick E, Prenzel N, Ullrich A. Epidermal growth factor receptors: critical mediators of multiple receptor pathways. Current Opinion in Cell Biology 1999; 11:184-9. Haddad R, Blumenfeld A, Siegal A, Kaplan O, Cohen M, Skornick Y, et al. In vitro and in vivo effects of photodynamic therapy on murine malignant melanoma. Ann Surg Oncol 1998; 5:241-7. Hamilton RF, Jr., Wu Z, Mitra S, Shaw PK, Holian A. Effect of MWCNT size, carboxylation, and purification on in vitro and in vivo toxicity, inflammation and lung pathology. Part Fibre Toxicol 2013; 10:57. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000; 100:57-70. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011; 144:646- 74. Hardy JA, Higgins GA. Alzheimer's disease: the amyloid cascade hypothesis. Science 1992; 256:184-5. Harris JM, Chess RB. Effect of pegylation on pharmaceuticals. Nat Rev Drug Discov 2003; 2:214-21. He Z, Schulz A, Wan X, Seitz J, Bludau H, Alakhova DY, et al. Poly(2-oxazoline) based micelles with high capacity for 3rd generation taxoids: preparation, in vitro and in vivo evaluation. J Control Release 2015; 208:67-75. Heil F, Hemmi H, Hochrein H, Ampenberger F, Kirschning C, Akira S, et al. Species- specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science 2004; 303:1526-9.
336 BIBLIOGRAPHY
Henao-Restrepo AM, Longini IM, Egger M, Dean NE, Edmunds WJ, Camacho A, et al. Efficacy and effectiveness of an rVSV-vectored vaccine expressing Ebola surface glycoprotein: interim results from the Guinea ring vaccination cluster-randomised trial. Lancet 2015. Henry AI, Bingham JM, Ringe E, Marks LD, Schatz GC, Van Duyne RP. Correlated Structure and Optical Property Studies of Plasmonic Nanoparticles. Journal of Physical Chemistry C 2011; 115:9291-305. Hicks DJ, Fooks AR, Johnson N. Developments in rabies vaccines. Clin Exp Immunol 2012; 169:199-204. Hicks MJ, De BP, Rosenberg JB, Davidson JT, Moreno AY, Janda KD, et al. Cocaine analog coupled to disrupted adenovirus: a vaccine strategy to evoke high-titer immunity against addictive drugs. Mol Ther 2011; 19:612-9. Hillaireau H, Couvreur P. Nanocarriers' entry into the cell: relevance to drug delivery. Cellular and molecular life sciences : CMLS 2009; 66:2873-96. Ho RL, Maccubbin D, Zaleskis G, Krawczyk C, Wing K, Mihich E, et al. Development of a safe and effective adriamycin plus interleukin 2 therapy against both adriamycin-sensitive and -resistant lymphomas. Oncol Res 1993; 5:373-81. Ho RL, Maccubbin DL, Ujhazy P, Zaleskis G, Eppolito C, Mihich E, et al. Immunological responses critical to the therapeutic effects of adriamycin plus interleukin 2 in C57BL/6 mice bearing syngeneic EL4 lymphoma. Oncol Res 1993; 5:363-72. Hodge JW, Garnett CT, Farsaci B, Palena C, Tsang KY, Ferrone S, et al. Chemotherapy- induced immunogenic modulation of tumor cells enhances killing by cytotoxic T lymphocytes and is distinct from immunogenic cell death. Int J Cancer 2013; 133:624-36. Hodi FS, O'Day SJ, McDermott DF, Weber RW, Sosman JA, Haanen JB, et al. Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med 2010; 363:711-23. Holmes FO. A Comparison of the Experimental Host Ranges of Tobacco-Etch and Tobacco-Mosaic Viruses. Phytopathology 1946; 36:643-59. Hong V, Presolski SI, Ma C, Finn MG. Analysis and Optimization of Copper-Catalyzed Azide-Alkyne Cycloaddition for Bioconjugation. Angewandte Chemie- International Edition 2009; 48:9879-83. Hortobagyi GN. Trastuzumab in the treatment of breast cancer. N Engl J Med 2005; 353:1734-6. Hou B, Saudan P, Ott G, Wheeler ML, Ji M, Kuzmich L, et al. Selective utilization of Toll-like receptor and MyD88 signaling in B cells for enhancement of the antiviral germinal center response. Immunity 2011; 34:375-84. Hovlid ML, Steinmetz NF, Laufer B, Lau JL, Kuzelka J, Wang Q, et al. Guiding plant virus particles to integrin-displaying cells. Nanoscale 2012; 4:3698-705. Howarth M, Liu WH, Puthenveetil S, Zheng Y, Marshall LF, Schmidt MM, et al. Monovalent, reduced-size quantum dots for imaging receptors on living cells. Nature Methods 2008; 5:397-9. Huang RK, Steinmetz NF, Fu CY, Manchester M, Johnson JE. Transferrin-mediated targeting of bacteriophage HK97 nanoparticles into tumor cells. Nanomedicine (Lond) 2011; 6:55-68.
337 BIBLIOGRAPHY
Huang X, El-Sayed IH, Qian W, El-Sayed MA. Cancer cell imaging and photothermal therapy in the near-infrared region by using gold nanorods. J Am Chem Soc 2006; 128:2115-20. Huang X, Stein BD, Cheng H, Malyutin A, Tsvetkova IB, Baxter DV, et al. Magnetic virus-like nanoparticles in N. benthamiana plants: a new paradigm for environmental and agronomic biotechnological research. ACS Nano 2011; 5:4037-45. Huang Z. A review of progress in clinical photodynamic therapy. Technol Cancer Res Treat 2005; 4:283-93. Huber B, Schellenbacher C, Jindra C, Fink D, Shafti-Keramat S, Kirnbauer R. A chimeric 18L1-45RG1 virus-like particle vaccine cross-protects against oncogenic alpha-7 human papillomavirus types. PLoS One 2015; 10:e0120152. Hughes B. Antibody-drug conjugates for cancer: poised to deliver? Nat Rev Drug Discov 2010; 9:665-7. Hui YY, Cheng CL, Chang HC. Nanodiamonds for optical bioimaging. Journal of Physics D-Applied Physics 2010; 43. Hui YY, Lu YC, Su LJ, Fang CY, Hsu JH, Chang HC. Tip-enhanced sub-diffraction fluorescence imaging of nitrogen-vacancy centers in nanodiamonds. Applied Physics Letters 2013; 102. Huisman MJ, Linthorst HJ, Bol JF, Cornelissen JC. The complete nucleotide sequence of potato virus X and its homologies at the amino acid level with various plus- stranded RNA viruses. J Gen Virol 1988; 69 ( Pt 8):1789-98. Iinuma H, Maruyama K, Okinaga K, Sasaki K, Sekine T, Ishida S, et al. Intracellular targeting therapy of cisplatin-encapsulated transferrin-polyethylene glycol liposome on peritoneal dissemination of gastric cancer. International Journal of Cancer 2002; 99:130-7. Ikeda H, Chamoto K, Tsuji T, Suzuki Y, Wakita D, Takeshima T, et al. The critical role of type-1 innate and acquired immunity in tumor immunotherapy. Cancer Sci 2004; 95:697-703. Ismaili H, Workentin MS. Covalent diamond-gold nanojewel hybrids via photochemically generated carbenes. Chemical Communications 2011; 47:7788- 90. Ito T, Gorman OT, Kawaoka Y, Bean WJ, Webster RG. Evolutionary analysis of the influenza A virus M gene with comparison of the M1 and M2 proteins. J Virol 1991; 65:5491-8. Jain RK. Transport of molecules, particles, and cells in solid tumors. Annual review of biomedical engineering 1999; 1:241-63. Jain RK, Stylianopoulos T. Delivering nanomedicine to solid tumors. Nat Rev Clin Oncol 2010; 7:653-64. Jegerlehner A, Maurer P, Bessa J, Hinton HJ, Kopf M, Bachmann MF. TLR9 signaling in B cells determines class switch recombination to IgG2a. J Immunol 2007; 178:2415-20. Jennings GT, Bachmann MF. The coming of age of virus-like particle vaccines. Biol Chem 2008; 389:521-36. Jennings GT, Bachmann MF. Immunodrugs: therapeutic VLP-based vaccines for chronic diseases. Annu Rev Pharmacol Toxicol 2009; 49:303-26.
338 BIBLIOGRAPHY
Jhaveri K, Esteva FJ. Pertuzumab in the treatment of HER2+ breast cancer. J Natl Compr Canc Netw 2014; 12:591-8. Jobsri J, Allen A, Rajagopal D, Shipton M, Kanyuka K, Lomonossoff GP, et al. Plant virus particles carrying tumour antigen activate TLR7 and Induce high levels of protective antibody. PLoS One 2015; 10:e0118096. Johansson A, Hamzah J, Payne CJ, Ganss R. Tumor-targeted TNFalpha stabilizes tumor vessels and enhances active immunotherapy. Proc Natl Acad Sci U S A 2012; 109:7841-6. Johnston SR, Dowsett M. Aromatase inhibitors for breast cancer: lessons from the laboratory. Nat Rev Cancer 2003; 3:821-31. Jokerst JV, Lobovkina T, Zare RN, Gambhir SS. Nanoparticle PEGylation for imaging and therapy. Nanomedicine 2011; 6:715-28. Jones MR, Osberg KD, Macfarlane RJ, Langille MR, Mirkin CA. Templated techniques for the synthesis and assembly of plasmonic nanostructures. Chem Rev 2011; 111:3736-827. Jones SM, Stroher U, Fernando L, Qiu X, Alimonti J, Melito P, et al. Assessment of a vesicular stomatitis virus-based vaccine by use of the mouse model of Ebola virus hemorrhagic fever. J Infect Dis 2007; 196 Suppl 2:S404-12. Kafri T, Morgan D, Krahl T, Sarvetnick N, Sherman L, Verma I. Cellular immune response to adenoviral vector infected cells does not require de novo viral gene expression: implications for gene therapy. Proc Natl Acad Sci U S A 1998; 95:11377-82. Kah JC, Chow TH, Ng BK, Razul SG, Olivo M, Sheppard CJ. Concentration dependence of gold nanoshells on the enhancement of optical coherence tomography images: a quantitative study. Appl Opt 2009; 48:D96-D108. Kaiser CR, Flenniken ML, Gillitzer E, Harmsen AL, Harmsen AG, Jutila MA, et al. Biodistribution studies of protein cage nanoparticles demonstrate broad tissue distribution and rapid clearance in vivo. Int J Nanomedicine 2007; 2:715-33. Kaleebu P, Njai HF, Wang L, Jones N, Ssewanyana I, Richardson P, et al. Immunogenicity of ALVAC-HIV vCP1521 in infants of HIV-1-infected women in Uganda (HPTN 027): the first pediatric HIV vaccine trial in Africa. J Acquir Immune Defic Syndr 2014; 65:268-77. Kalos M, Levine BL, Porter DL, Katz S, Grupp SA, Bagg A, et al. T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia. Sci Transl Med 2011; 3:95ra73. Kaltsas GA, Mukherjee JJ, Plowman PN, Monson JP, Grossman AB, Besser GM. The role of cytotoxic chemotherapy in the management of aggressive and malignant pituitary tumors. J Clin Endocrinol Metab 1998; 83:4233-8. Kamba SA, Ismail M, Hussein-Al-Ali SH, Ibrahim TA, Zakaria ZA. In vitro delivery and controlled release of Doxorubicin for targeting osteosarcoma bone cancer. Molecules 2013; 18:10580-98. Kaneno R, Shurin GV, Kaneno FM, Naiditch H, Luo J, Shurin MR. Chemotherapeutic agents in low noncytotoxic concentrations increase immunogenicity of human colon cancer cells. Cell Oncol (Dordr) 2011; 34:97-106.
339 BIBLIOGRAPHY
Kang KA, Wang JT, Jasinski JB, Achilefu S. Fluorescence Manipulation by Gold Nanoparticles: From Complete Quenching to Extensive Enhancement. Journal of Nanobiotechnology 2011; 9. Karanam B, Jagu S, Huh WK, Roden RB. Developing vaccines against minor capsid antigen L2 to prevent papillomavirus infection. Immunol Cell Biol 2009; 87:287- 99. Karpenko LI, Ivanisenko VA, Pika IA, Chikaev NA, Eroshkin AM, Veremeiko TA, et al. Insertion of foreign epitopes in HBcAg: how to make the chimeric particle assemble. Amino Acids 2000; 18:329-37. Karukstis KK, Thompson EH, Whiles JA, Rosenfeld RJ. Deciphering the fluorescence signature of daunomycin and doxorubicin. Biophys Chem 1998; 73:249-63. Kattan J, Droz JP, Couvreur P, Marino JP, Boutan-Laroze A, Rougier P, et al. Phase I clinical trial and pharmacokinetic evaluation of doxorubicin carried by polyisohexylcyanoacrylate nanoparticles. Invest New Drugs 1992; 10:191-9. Kaufmann AM, Nieland J, Schinz M, Nonn M, Gabelsberger J, Meissner H, et al. HPV16 L1E7 chimeric virus-like particles induce specific HLA-restricted T cells in humans after in vitro vaccination. Int J Cancer 2001; 92:285-93. Kaufmann AM, Nieland JD, Jochmus I, Baur S, Friese K, Gabelsberger J, et al. Vaccination trial with HPV16 L1E7 chimeric virus-like particles in women suffering from high grade cervical intraepithelial neoplasia (CIN 2/3). Int J Cancer 2007; 121:2794-800. Kawai H, Minamiya Y, Kitamura M, Matsuzaki I, Hashimoto M, Suzuki H, et al. Direct measurement of doxorubicin concentration in the intact, living single cancer cell during hyperthermia. Cancer 1997; 79:214-9. Kawamotos T, Mendelsohnsq J, Le A, Sato GH, Lazarlt CS, Gill GN. Relation of Epidermal Growth Factor Receptor Concentration to Growth of Human Epidermoid Carcinoma A431 Cells. Journal of Biological Chemistry 1984; 259:7761-6. Kawano M, Morikawa K, Suda T, Ohno N, Matsushita S, Akatsuka T, et al. Chimeric SV40 virus-like particles induce specific cytotoxicity and protective immunity against influenza A virus without the need of adjuvants. Virology 2014; 448:159- 67. Kelf T.A S, V.K.A, Sun, J, Kim, E.J, Goldys, E.M, Zvyagin, A.V. Non-specific cellular uptake of surface-functionalized quantum dots. Nanotechnlogy 2010; 21:1-14. Kelly TA, Ng KW, Wang CC, Ateshian GA, Hung CT. Spatial and temporal development of chondrocyte-seeded agarose constructs in free-swelling and dynamically loaded cultures. J Biomech 2006; 39:1489-97. Kendall A, McDonald M, Bian W, Bowles T, Baumgarten SC, Shi J, et al. Structure of flexible filamentous plant viruses. J Virol 2008; 82:9546-54. Kepp O, Galluzzi L, Martins I, Schlemmer F, Adjemian S, Michaud M, et al. Molecular determinants of immunogenic cell death elicited by anticancer chemotherapy. Cancer Metastasis Rev 2011; 30:61-9. Khan HA, Abdelhalim MA, Alhomida AS, Al Ayed MS. Transient increase in IL-1beta, IL-6 and TNF-alpha gene expression in rat liver exposed to gold nanoparticles. Genet Mol Res 2013; 12:5851-7.
340 BIBLIOGRAPHY
Khlebtsov NG, Dykman LA. Optical properties and biomedical applications of plasmonic nanoparticles. Journal of Quantitative Spectroscopy & Radiative Transfer 2010; 111:1-35. Kickhoefer VA, Han M, Raval-fernandes S, Poderycki MJ, Moniz RJ, Vaccari D, et al. Targeting Vault Nanoparticles to Specific Cell Surface Receptors. ACS Nano 2009; 3:27-36. Kim JB, Stein R, O'Hare MJ. Three-dimensional in vitro tissue culture models of breast cancer-- a review. Breast Cancer Res Treat 2004; 85:281-91. Kinsey BM, Jackson DC, Orson FM. Anti-drug vaccines to treat substance abuse. Immunol Cell Biol 2009; 87:309-14. Kitai Y, Fukuda H, Enomoto T, Asakawa Y, Suzuki T, Inouye S, et al. Cell selective targeting of a simian virus 40 virus-like particle conjugated to epidermal growth factor. Journal of biotechnology 2011; 155:251-6. Klibanov AL, Maruyama K, Torchilin VP, Huang L. Amphipathic polyethyleneglycols effectively prolong the circulation time of liposomes. FEBS Lett 1990; 268:235-7. Knobler S, Lederberg J, Pray LA, editors. Considerations for Viral Disease Eradication: Lessons Learned and Future Strategies: Workshop Summary. Washington (DC)2002. Kochenderfer JN, Dudley ME, Feldman SA, Wilson WH, Spaner DE, Maric I, et al. B- cell depletion and remissions of malignancy along with cytokine-associated toxicity in a clinical trial of anti-CD19 chimeric-antigen-receptor-transduced T cells. Blood 2012; 119:2709-20. Kochenderfer JN, Dudley ME, Kassim SH, Somerville RP, Carpenter RO, Stetler- Stevenson M, et al. Chemotherapy-refractory diffuse large B-cell lymphoma and indolent B-cell malignancies can be effectively treated with autologous T cells expressing an anti-CD19 chimeric antigen receptor. J Clin Oncol 2015; 33:540-9. Kostarelos K. Carbon nanotubes: Fibrillar pharmacology. Nat Mater 2010; 9:793-5. Kraft JC, Freeling JP, Wang Z, Ho RJ. Emerging research and clinical development trends of liposome and lipid nanoparticle drug delivery systems. J Pharm Sci 2014; 103:29-52. Krammer F. Emerging influenza viruses and the prospect of a universal influenza virus vaccine. Biotechnol J 2015; 10:690-701. Krammer F, Palese P. Influenza virus hemagglutinin stalk-based antibodies and vaccines. Curr Opin Virol 2013; 3:521-30. Labarbera DV, Reid BG, Yoo BH. The multicellular tumor spheroid model for high- throughput cancer drug discovery. Expert opinion on drug discovery 2012; 7:819- 30. Lam HY. Tamoxifen is a calmodulin antagonist in the activation of cAMP phosphodiesterase. Biochem Biophys Res Commun 1984; 118:27-32. Lambert LC, Fauci AS. Influenza vaccines for the future. N Engl J Med 2010; 363:2036- 44. Lammers T, Kiessling F, Hennink WE, Storm G. Drug targeting to tumors: Principles, pitfalls and (pre-) clinical progress. Journal of Controlled Release 2012; 161:175- 87.
341 BIBLIOGRAPHY
Lang SH, Sharrard RM, Stark M, Villette JM, Maitland NJ. Prostate epithelial cell lines form spheroids with evidence of glandular differentiation in three-dimensional Matrigel cultures. Br J Cancer 2001; 85:590-9. Larson N, Ghandehari H. Polymeric conjugates for drug delivery. Chem Mater 2012; 24:840-53. Lebel ME, Chartrand K, Tarrab E, Savard P, Leclerc D, Lamarre A. Potentiating Cancer Immunotherapy Using Papaya Mosaic Virus-Derived Nanoparticles. Nano Lett 2016; 16:1826-32. Leberman R. The isolation of plant viruses by means of "simple" coacervates. Virology 1966; 30:341-7. Lee DW, Kochenderfer JN, Stetler-Stevenson M, Cui YK, Delbrook C, Feldman SA, et al. T cells expressing CD19 chimeric antigen receptors for acute lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial. Lancet 2015; 385:517-28. Lee GK, Maheshri N, Kaspar B, Schaffer DV. PEG conjugation moderately protects adeno-associated viral vectors against antibody neutralization. Biotechnology and Bioengineering 2005; 92:24-34. Lee GY, Kim J-H, Oh GT, Lee B-H, Kwon IC, Kim I-S. Molecular targeting of atherosclerotic plaques by a stabilin-2-specific peptide ligand. Journal of controlled release : official journal of the Controlled Release Society 2011; 155:211-7. Lee KL, Hubbard LC, Hern S, Yildiz I, Gratzl M, Steinmetz NF. Shape matters: the diffusion rates of TMV rods and CPMV icosahedrons in a spheroid model of extracellular matrix are distinct. Biomater Sci 2013; 1. Lee KL, Twyman RM, Fiering S, Steinmetz NF. Virus-based nanoparticles as platform technologies for modern vaccines. Wiley Interdiscip Rev Nanomed Nanobiotechnol 2016. Lee KL, Uhde-Holzem K, Fischer R, Commandeur U, Steinmetz NF. Genetic engineering and chemical conjugation of potato virus X. Methods Mol Biol 2014; 1108:3-21. Lee KL, Uhde-Holzem K, Fisher R, Commendeur U, Steinmetz N. Genetic engineering and chemical conjugation of potato virus X. Methods Mol Biol2014. p. 3-21. Lee RJ, Low PS. Delivery of liposomes into cultured KB cells via folate receptor- mediated endocytosis. J Biol Chem 1994; 269:3198-204. Lee S-Y, Ferrari M, Decuzzi P. Shaping nano-/micro-particles for enhanced vascular interaction in laminar flows. Nanotechnology 2009; 20:495101. Lee SY, Ferrari M, Decuzzi P. Shaping nano-/micro-particles for enhanced vascular interaction in laminar flows. Nanotechnology 2009; 20:495101. Leffers N, Lambeck AJ, Gooden MJ, Hoogeboom BN, Wolf R, Hamming IE, et al. Immunization with a P53 synthetic long peptide vaccine induces P53-specific immune responses in ovarian cancer patients, a phase II trial. Int J Cancer 2009; 125:2104-13. Lehtinen M, Paavonen J, Wheeler CM, Jaisamrarn U, Garland SM, Castellsague X, et al. Overall efficacy of HPV-16/18 AS04-adjuvanted vaccine against grade 3 or greater cervical intraepithelial neoplasia: 4-year end-of-study analysis of the randomised, double-blind PATRICIA trial. Lancet Oncol 2012; 13:89-99.
342 BIBLIOGRAPHY
Li Y, Huang Y, Wang Z, Carniato F, Xie Y, Patterson JP, et al. Polycatechol Nanoparticle MRI Contrast Agents. Small 2016; 12:668-77. Li Z, Zhao R, Wu X, Sun Y, Yao M, Li J, et al. Identification and characterization of a novel peptide ligand of epidermal growth factor receptor for targeted delivery of therapeutics. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2005; 19:1978-85. Liang Y, Jeong J, DeVolder RJ, Cha C, Wang F, Tong YW, et al. A cell-instructive hydrogel to regulate malignancy of 3D tumor spheroids with matrix rigidity. Biomaterials 2011; 32:9308-15. Lico C, Mancini C, Italiani P, Betti C, Boraschi D, Benvenuto E, et al. Plant-produced potato virus X chimeric particles displaying an influenza virus-derived peptide activate specific CD8+ T cells in mice. Vaccine 2009; 27:5069-76. Liebscher S, Meyer-Luehmann M. A Peephole into the Brain: Neuropathological Features of Alzheimer's Disease Revealed by in vivo Two-Photon Imaging. Front Psychiatry 2012; 3:26. Lim TS, Fu CC, Lee KC, Lee HY, Chen K, Cheng WF, et al. Fluorescence enhancement and lifetime modification of single nanodiamonds near a nanocrystalline silver surface. Phys Chem Chem Phys 2009; 11:1508-14. Lin S, Wang X, Ji Z, Chang CH, Dong Y, Meng H, et al. Aspect Ratio Plays a Role in the Hazard Potential of CeO Nanoparticles in Mouse Lung and Zebrafish Gastrointestinal Tract. Acs Nano 2014. Lin S, Wang X, Ji Z, Chang CH, Dong Y, Meng H, et al. Aspect ratio plays a role in the hazard potential of CeO2 nanoparticles in mouse lung and zebrafish gastrointestinal tract. ACS Nano 2014; 8:4450-64. Lipson EJ, Drake CG. Ipilimumab: an anti-CTLA-4 antibody for metastatic melanoma. Clin Cancer Res 2011; 17:6958-62. Liu B, Fang M, Schmidt M, Lu Y, Mendelsohn J, Fan Z. Induction of apoptosis and activation of the caspase cascade by anti-EGF receptor monoclonal antibodies in DiFi human colon cancer cells do not involve the c-jun N-terminal kinase activity. British Journal of Cancer 2000; 82:1991-9. Liu R, Vaishnav RA, Roberts AM, Friedland RP. Humans have antibodies against a plant virus: evidence from tobacco mosaic virus. PLoS One 2013; 8:e60621. Liu T, Yuan X, Jia T, Liu C, Ni Z, Qin Z, et al. Polymeric prodrug of bufalin for increasing solubility and stability: Synthesis and anticancer study in vitro and in vivo. Int J Pharm 2016; 506:382-93. Liu TC, Galanis E, Kirn D. Clinical trial results with oncolytic virotherapy: a century of promise, a decade of progress. Nat Clin Pract Oncol 2007; 4:101-17. Liu Y, Peterson DA, Kimura H, Schubert D. Mechanism of cellular 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction. J Neurochem 1997; 69:581-93. Liu YL, Sun KW. Plasmon-enhanced photoluminescence from bioconjugated gold nanoparticle and nanodiamond assembly. Applied Physics Letters 2011; 98. Liu YV, Massare MJ, Pearce MB, Sun X, Belser JA, Maines TR, et al. Recombinant virus-like particles elicit protective immunity against avian influenza A(H7N9) virus infection in ferrets. Vaccine 2015; 33:2152-8.
343 BIBLIOGRAPHY
Liu Z, Davis C, Cai W, He L, Chen X, Dai H. Circulation and long-term fate of functionalized, biocompatible single-walled carbon nanotubes in mice probed by Raman spectroscopy. Proc Natl Acad Sci U S A 2008; 105:1410-5. Lizotte PH, Wen AM, Sheen MR, Fields J, Rojanasopondist P, Steinmetz NF, et al. In situ vaccination with cowpea mosaic virus nanoparticles suppresses metastatic cancer. Nat Nanotechnol 2016; 11:295-303. Lockney DM, Guenther RN, Loo L, Overton W, Antonelli R, Clark J, et al. The Red clover necrotic mosaic virus capsid as a multifunctional cell targeting plant viral nanoparticle. Bioconjug Chem 2011; 22:67-73. London WT MK. Liver Cancer. In: Schottenfeld D FJJ, editor. Cancer Epidemiology and Prevention. 3rd ed. New York: Oxford University Press; 2006. p. 763-86. Longmire M, Choyke PL, Kobayashi H. Clearance properties of nano-sized particles and molecules as imaging agents: considerations and caveats. Nanomedicine (Lond) 2008; 3:703-17. Lopez AL, Gonzales ML, Aldaba JG, Nair GB. Killed oral cholera vaccines: history, development and implementation challenges. Ther Adv Vaccines 2014; 2:123-36. Lopez-Macias C, Ferat-Osorio E, Tenorio-Calvo A, Isibasi A, Talavera J, Arteaga-Ruiz O, et al. Safety and immunogenicity of a virus-like particle pandemic influenza A (H1N1) 2009 vaccine in a blinded, randomized, placebo-controlled trial of adults in Mexico. Vaccine 2011; 29:7826-34. Lu W, Xiong C, Zhang G, Huang Q, Zhang R, Zhang JZ, et al. Targeted photothermal ablation of murine melanomas with melanocyte-stimulating hormone analog- conjugated hollow gold nanospheres. Clin Cancer Res 2009; 15:876-86. Lu WD, Zhang L, Wu CL, Liu ZG, Lei GY, Liu J, et al. Development of an acellular tumor extracellular matrix as a three-dimensional scaffold for tumor engineering. PLoS One 2014; 9:e103672. Lu Y, Xiao Y, Ding J, Dierich M, Chen YH. Immunogenicity of neutralizing epitopes on multiple-epitope vaccines against HIV-1. Int Arch Allergy Immunol 2000; 121:80- 4. Lua LH, Connors NK, Sainsbury F, Chuan YP, Wibowo N, Middelberg AP. Bioengineering virus-like particles as vaccines. Biotechnol Bioeng 2014; 111:425- 40. Lucky SS, Soo KC, Zhang Y. Nanoparticles in photodynamic therapy. Chem Rev 2015; 115:1990-2042. Lundin J, Kimby E, Bjorkholm M, Broliden PA, Celsing F, Hjalmar V, et al. Phase II trial of subcutaneous anti-CD52 monoclonal antibody alemtuzumab (Campath- 1H) as first-line treatment for patients with B-cell chronic lymphocytic leukemia (B-CLL). Blood 2002; 100:768-73. Lundqvist M. Nanoparticles: Tracking protein corona over time. Nat Nanotechnol 2013; 8:701-2. Lunov O, Syrovets T, Loos C, Nienhaus GU, Mailander V, Landfester K, et al. Amino- functionalized polystyrene nanoparticles activate the NLRP3 inflammasome in human macrophages. ACS Nano 2011; 5:9648-57. Luxenhofer R, Han Y, Schulz A, Tong J, He Z, Kabanov AV, et al. Poly(2-oxazoline)s as polymer therapeutics. Macromol Rapid Commun 2012; 33:1613-31.
344 BIBLIOGRAPHY
Maartens G, Celum C, Lewin SR. HIV infection: epidemiology, pathogenesis, treatment, and prevention. Lancet 2014; 384:258-71. Mac Keon S, Ruiz MS, Gazzaniga S, Wainstok R. Dendritic cell-based vaccination in cancer: therapeutic implications emerging from murine models. Front Immunol 2015; 6:243. Maeda H. The enhanced permeability and retention (EPR) effect in tumor vasculature: the key role of tumor-selective macromolecular drug targeting. Advances in Enzyme Regulation 2001; 41:189-207. Manchester M, Singh P. Virus-based nanoparticles (VNPs): platform technologies for diagnostic imaging. Adv Drug Deliv Rev 2006; 58:1505-22. Manolova V, Flace A, Bauer M, Schwarz K, Saudan P, Bachmann MF. Nanoparticles target distinct dendritic cell populations according to their size. Eur J Immunol 2008; 38:1404-13. Maoz A, Hicks MJ, Vallabhjosula S, Synan M, Kothari PJ, Dyke JP, et al. Adenovirus capsid-based anti-cocaine vaccine prevents cocaine from binding to the nonhuman primate CNS dopamine transporter. Neuropsychopharmacology 2013; 38:2170-8. Marinho-Soriano E. Agar polysaccharides from Gracilaria species (Rhodophyta, Gracilariaceae). J Biotechnol 2001; 89:81-4. Martins I, Tesniere A, Kepp O, Michaud M, Schlemmer F, Senovilla L, et al. Chemotherapy induces ATP release from tumor cells. Cell Cycle 2009; 8:3723-8. Martins KA, Steffens JT, van Tongeren SA, Wells JB, Bergeron AA, Dickson SP, et al. Toll-like receptor agonist augments virus-like particle-mediated protection from Ebola virus with transient immune activation. PLoS One 2014; 9:e89735. Marusic C, Rizza P, Lattanzi L, Mancini C, Spada M, Belardelli F, et al. Chimeric plant virus particles as immunogens for inducing murine and human immune responses against human immunodeficiency virus type 1. J Virol 2001; 75:8434-9. Maruyama K, Yuda T, Okamoto A, Kojima S, Suginaka A, Iwatsuru M. Prolonged circulation time in vivo of large unilamellar liposomes composed of distearoyl phosphatidylcholine and cholesterol containing amphipathic poly(ethylene glycol). Biochim Biophys Acta 1992; 1128:44-9. Marzi A, Halfmann P, Hill-Batorski L, Feldmann F, Shupert WL, Neumann G, et al. Vaccines. An Ebola whole-virus vaccine is protective in nonhuman primates. Science 2015; 348:439-42. Matar P, Rojo F, Cassia R, Moreno-Bueno G, Di Cosimo S, Tabernero J, et al. Combined epidermal growth factor receptor targeting with the tyrosine kinase inhibitor gefitinib (ZD1839) and the monoclonal antibody cetuximab (IMC-C225): superiority over single-agent receptor targeting. Clin Cancer Res 2004; 10:6487- 501. Matassov D, Marzi A, Latham T, Xu R, Ota-Setlik A, Feldmann F, et al. Vaccination With a Highly Attenuated Recombinant Vesicular Stomatitis Virus Vector Protects Against Challenge With a Lethal Dose of Ebola Virus. J Infect Dis 2015. Matsumura Y, Maeda H. A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs. Cancer Res 1986; 46:6387-92.
345 BIBLIOGRAPHY
Maurer P, Jennings GT, Willers J, Rohner F, Lindman Y, Roubicek K, et al. A therapeutic vaccine for nicotine dependence: preclinical efficacy, and Phase I safety and immunogenicity. Eur J Immunol 2005; 35:2031-40. McAleer WJ, Buynak EB, Maigetter RZ, Wampler DE, Miller WJ, Hilleman MR. Human hepatitis B vaccine from recombinant yeast. Nature 1984; 307:178-80. McCormick AA, Corbo TA, Wykoff-Clary S, Palmer KE, Pogue GP. Chemical conjugate TMV-peptide bivalent fusion vaccines improve cellular immunity and tumor protection. Bioconjug Chem 2006; 17:1330-8. McFarland EJ, Johnson DC, Muresan P, Fenton T, Tomaras GD, McNamara J, et al. HIV-1 vaccine induced immune responses in newborns of HIV-1 infected mothers. AIDS 2006; 20:1481-9. Mei BC, Susumu K, Medintz IL, Delehanty JB, Mountziaris TJ, Mattoussi H. Modular poly(ethylene glycol) ligands for biocompatible semiconductor and gold nanocrystals with extended pH and ionic stability. Journal of Materials Chemistry 2008; 18:4949-58. Mellman I, Coukos G, Dranoff G. Cancer immunotherapy comes of age. Nature 2011; 480:480-9. Menard C, Martin F, Apetoh L, Bouyer F, Ghiringhelli F. Cancer chemotherapy: not only a direct cytotoxic effect, but also an adjuvant for antitumor immunity. Cancer Immunol Immunother 2008; 57:1579-87. Meng J, Yang M, Jia F, Xu Z, Kong H, Xu H. Immune responses of BALB/c mice to subcutaneously injected multi-walled carbon nanotubes. Nanotoxicology 2011; 5:583-91. Meunier E, Coste A, Olagnier D, Authier H, Lefevre L, Dardenne C, et al. Double-walled carbon nanotubes trigger IL-1beta release in human monocytes through Nlrp3 inflammasome activation. Nanomedicine 2012; 8:987-95. Michel R, Pasche S, Textor M, Castner DG. Influence of PEG architecture on protein adsorption and conformation. Langmuir 2005; 21:12327-32. Michielsen AJ, Hogan AE, Marry J, Tosetto M, Cox F, Hyland JM, et al. Tumour tissue microenvironment can inhibit dendritic cell maturation in colorectal cancer. PLoS One 2011; 6:e27944. Mickler FM, Mo L, Ruthardt N, Ogris M, Wagner E. Tuning Nanoparticle Uptake: Live- Cell Imaging Reveals Two Distinct Endocytosis Mechanisms Mediated by Natural and Arti fi cial EGFR Targeting Ligand. 2012. Miele E, Spinelli GP, Miele E, Tomao F, Tomao S. Albumin-bound formulation of paclitaxel (Abraxane ABI-007) in the treatment of breast cancer. Int J Nanomedicine 2009; 4:99-105. Mihlon Ft, Ray CE, Jr., Messersmith W. Chemotherapy agents: a primer for the interventional radiologist. Semin Intervent Radiol 2010; 27:384-90. Miller M, Barrett S, Henderson DA. Control and Eradication. In: Jamison DT, Breman JG, Measham AR, Alleyne G, Claeson M, Evans DB, et al., editors. Disease Control Priorities in Developing Countries. 2nd ed. Washington (DC)2006. Missirlis D, Kawamura R, Tirelli N, Hubbell JA. Doxorubicin encapsulation and diffusional release from stable, polymeric, hydrogel nanoparticles. Eur J Pharm Sci 2006; 29:120-9.
346 BIBLIOGRAPHY
Mitchison TJ. The proliferation rate paradox in antimitotic chemotherapy. Mol Biol Cell 2012; 23:1-6. Mittal S, El-Serag HB. Epidemiology of hepatocellular carcinoma: consider the population. J Clin Gastroenterol 2013; 47 Suppl:S2-6. Miyoshi S, Flexman JA, Cross DJ, Maravilla KR, Kim Y, Anzai Y, et al. Transfection of neuroprogenitor cells with iron nanoparticles for magnetic resonance imaging tracking: cell viability, differentiation, and intracellular localization. Mol Imaging Biol 2005; 7:286-95. Moan J, Berg K. The photodegradation of porphyrins in cells can be used to estimate the lifetime of singlet oxygen. Photochem Photobiol 1991; 53:549-53. Mohammad AK, Reineke JJ. Quantitative Detection of PLGA Nanoparticle Degradation in Tissues following Intravenous Administration. Molecular Pharmaceutics 2013; 10:2183-9. Mohan N, Chen CS, Hsieh HH, Wu YC, Chang HC. In Vivo Imaging and Toxicity Assessments of Fluorescent Nanodiamonds in Caenorhabditis elegans. Nano Letters 2010; 10:3692-9. Mohan P, Rapoport N. Doxorubicin as a molecular nanotheranostic agent: effect of doxorubicin encapsulation in micelles or nanoemulsions on the ultrasound- mediated intracellular delivery and nuclear trafficking. Mol Pharm 2010; 7:1959- 73. Moitra K, Lou H, Dean M. Multidrug efflux pumps and cancer stem cells: insights into multidrug resistance and therapeutic development. Clinical pharmacology and therapeutics 2011; 89:491-502. Mok H, Palmer DJ, Ng P, Barry MA. Evaluation of polyethylene glycol modification of first-generation and helper-dependent adenoviral vectors to reduce innate immune responses. Mol Ther 2005; 11:66-79. Molenaar TJ, Michon I, de Haas SA, van Berkel TJ, Kuiper J, Biessen EA. Uptake and processing of modified bacteriophage M13 in mice: implications for phage display. Virology 2002; 293:182-91. Monjazeb AM, Zamora AE, Grossenbacher SK, Mirsoian A, Sckisel GD, Murphy WJ. Immunoediting and antigen loss: overcoming the achilles heel of immunotherapy with antigen non-specific therapies. Front Oncol 2013; 3:197. Monroy-Garcia A, Gomez-Lim MA, Weiss-Steider B, Hernandez-Montes J, Huerta- Yepez S, Rangel-Santiago JF, et al. Immunization with an HPV-16 L1-based chimeric virus-like particle containing HPV-16 E6 and E7 epitopes elicits long- lasting prophylactic and therapeutic efficacy in an HPV-16 tumor mice model. Arch Virol 2014; 159:291-305. Montet X, Funovics M, Montet-Abou K, Weissleder R, Josephson L. Multivalent effects of RGD peptides obtained by nanoparticle display. J Med Chem 2006; 49:6087-93. Montoya ID. Biologics (Vaccines, Antibodies, Enzymes) to Treat Drug Addictions. In: Nady el-Guebaly GC, Marc Galanter, editor. Textbook of Addiction Treatment: International Perspectives: Springer; 2015. p. 683-92. Moore AE. The destructive effect of the virus of Russian Far East encephalitis on the transplantable mouse sarcoma 180. Cancer 1949; 2:525-34.
347 BIBLIOGRAPHY
Morgenfeld M, Segretin ME, Wirth S, Lentz E, Zelada A, Mentaberry A, et al. Potato virus X coat protein fusion to human papillomavirus 16 E7 oncoprotein enhance antigen stability and accumulation in tobacco chloroplast. Mol Biotechnol 2009; 43:243-9. Morice P, Brehier-Ollive D, Rey A, Atallah D, Lhomme C, Pautier P, et al. Results of interval debulking surgery in advanced stage ovarian cancer: an exposed-non- exposed study. Ann Oncol 2003; 14:74-7. Morice P, Dubernard G, Rey A, Atallah D, Pautier P, Pomel C, et al. Results of interval debulking surgery compared with primary debulking surgery in advanced stage ovarian cancer. J Am Coll Surg 2003; 197:955-63. Moriuchi S, Wolfe D, Tamura M, Yoshimine T, Miura F, Cohen JB, et al. Double suicide gene therapy using a replication defective herpes simplex virus vector reveals reciprocal interference in a malignant glioma model. Gene Ther 2002; 9:584-91. Morstyn G, Campbell L, Souza LM, Alton NK, Keech J, Green M, et al. Effect of granulocyte colony stimulating factor on neutropenia induced by cytotoxic chemotherapy. Lancet 1988; 1:667-72. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983; 65:55-63. Moyron-Quiroz JE, Rangel-Moreno J, Hartson L, Kusser K, Tighe MP, Klonowski KD, et al. Persistence and responsiveness of immunologic memory in the absence of secondary lymphoid organs. Immunity 2006; 25:643-54. Muller M, Zhou J, Reed TD, Rittmuller C, Burger A, Gabelsberger J, et al. Chimeric papillomavirus-like particles. Virology 1997; 234:93-111. Munoz N, Kjaer SK, Sigurdsson K, Iversen OE, Hernandez-Avila M, Wheeler CM, et al. Impact of human papillomavirus (HPV)-6/11/16/18 vaccine on all HPV- associated genital diseases in young women. J Natl Cancer Inst 2010; 102:325-39. Murahari MS, Yergeri MC. Identification and usage of fluorescent probes as nanoparticle contrast agents in detecting cancer. Curr Pharm Des 2013; 19:4622-40. Nagata Y, Lan KH, Zhou X, Tan M, Esteva FJ, Sahin AA, et al. PTEN activation contributes to tumor inhibition by trastuzumab, and loss of PTEN predicts trastuzumab resistance in patients. Cancer Cell 2004; 6:117-27. Nakajima A, Seroogy CM, Sandora MR, Tarner IH, Costa GL, Taylor-Edwards C, et al. Antigen-specific T cell-mediated gene therapy in collagen-induced arthritis. J Clin Invest 2001; 107:1293-301. Nakano K, Bando Y, Tozuka Y, Takeuchi H. Cellular interaction of PEGylated PLGA nanospheres with macrophage J774 cells using flow cytometry. Asian Journal of Pharmaceutical Sciences 2007; 2:220-6. Naldini L. Gene therapy returns to centre stage. Nature 2015; 526:351-60. Narayanan J, Xiong J-Y, Liu X-Y. Determination of agarose gel pore size: Absorbance measurements vis a vis other techniques. Journal of Physics 2006; 28:83-6. Naskalska A, Szolajska E, Chaperot L, Angel J, Plumas J, Chroboczek J. Influenza recombinant vaccine: matrix protein M1 on the platform of the adenovirus dodecahedron. Vaccine 2009; 27:7385-93. Nayerossadat N, Maedeh T, Ali PA. Viral and nonviral delivery systems for gene delivery. Adv Biomed Res 2012; 1:27.
348 BIBLIOGRAPHY
Ng KW, Wang CC, Mauck RL, Kelly TA, Chahine NO, Costa KD, et al. A layered agarose approach to fabricate depth-dependent inhomogeneity in chondrocyte- seeded constructs. Journal of orthopaedic research : official publication of the Orthopaedic Research Society 2005; 23:134-41. Ni F, Jiang L, Yang R, Chen Z, Qi X, Wang J. Effects of PEG length and iron oxide nanoparticles size on reduced protein adsorption and non-specific uptake by macrophage cells. J Nanosci Nanotechnol 2012; 12:2094-100. Nicholson RI, Gee JMW, Harper ME. EGFR and cancer prognosis. European Journal of Cancer 2001; 37:9-15. Nicoll JA, Wilkinson D, Holmes C, Steart P, Markham H, Weller RO. Neuropathology of human Alzheimer disease after immunization with amyloid-beta peptide: a case report. Nat Med 2003; 9:448-52. Niedre M, Patterson MS, Wilson BC. Direct near-infrared luminescence detection of singlet oxygen generated by photodynamic therapy in cells in vitro and tissues in vivo. Photochem Photobiol 2002; 75:382-91. Nwogu C, Pera P, Bshara W, Attwood K, Pandey R. Photodynamic therapy of human lung cancer xenografts in mice. J Surg Res 2016; 200:8-12. O'Neil A, Prevelige PE, Basu G, Douglas T. Coconfinement of fluorescent proteins: spatially enforced communication of GFP and mCherry encapsulated within the P22 capsid. Biomacromolecules 2012; 13:3902-7. O'riordan CR, Lachapelle A, Delgado C, Parkes V, Wadsworth SC, Smith AE, et al. PEGylation of adenovirus with retention of infectivity and protection from neutralizing antibody in vitro and in vivo. Hum Gene Ther 1999; 10:1349-58. Obata T, Mori S, Suzuki Y, Kashiwagi T, Tokunaga E, Shibata N, et al. Photodynamic Therapy Using Novel Zinc Phthalocyanine Derivatives and a Diode Laser for Superficial Tumors in Experimental Animals. Journal of Cancer Therapy 2015; 6:53-61. Obeid M, Tesniere A, Ghiringhelli F, Fimia GM, Apetoh L, Perfettini JL, et al. Calreticulin exposure dictates the immunogenicity of cancer cell death. Nat Med 2007; 13:54-61. Ohlschlager P, Osen W, Dell K, Faath S, Garcea RL, Jochmus I, et al. Human papillomavirus type 16 L1 capsomeres induce L1-specific cytotoxic T lymphocytes and tumor regression in C57BL/6 mice. J Virol 2003; 77:4635-45. Oldenburg SJ, Averitt RD, Westcott SL, Halas NJ. Nanoengineering of optical resonances. Chemical Physics Letters 1998; 288:243-7. Oltra NS, Swift J, Mahmud A, Rajagopal K, Loverde SM, Discher DE. Filomicelles in nanomedicine - from flexible, fragmentable, and ligand-targetable drug carrier designs to combination therapy for brain tumors. Journal of Materials Chemistry B 2013; 1:5177-85. Orgogozo JM, Gilman S, Dartigues JF, Laurent B, Puel M, Kirby LC, et al. Subacute meningoencephalitis in a subset of patients with AD after Abeta42 immunization. Neurology 2003; 61:46-54. Ott PA, Hodi FS. Talimogene Laherparepvec for the Treatment of Advanced Melanoma. Clin Cancer Res 2016. Owens DE, 3rd, Peppas NA. Opsonization, biodistribution, and pharmacokinetics of polymeric nanoparticles. Int J Pharm 2006; 307:93-102.
349 BIBLIOGRAPHY
Pal SK, Childs BH, Pegram M. Triple negative breast cancer: unmet medical needs. Breast Cancer Res Treat 2011; 125:627-36. Papaneri AB, Wirblich C, Cooper K, Jahrling PB, Schnell MJ, Blaney JE. Further characterization of the immune response in mice to inactivated and live rabies vaccines expressing Ebola virus glycoprotein. Vaccine 2012; 30:6136-41. Paraskevakou G, Allen C, Nakamura T, Zollman P, James CD, Peng KW, et al. Epidermal Growth Factor Receptor ( EGFR )– Retargeted Measles Virus Strains Effectively Target EGFR- or EGFRvIII Expressing Gliomas. 2007; 15:677-86. Pardoll DM. The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer 2012; 12:252-64. Park DJ, Dudas G, Wohl S, Goba A, Whitmer SL, Andersen KG, et al. Ebola Virus Epidemiology, Transmission, and Evolution during Seven Months in Sierra Leone. Cell 2015; 161:1516-26. Park EJ, Umh HN, Kim SW, Cho MH, Kim JH, Kim Y. ERK pathway is activated in bare-FeNPs-induced autophagy. Archives of Toxicology 2014; 88:323-36. Park JW. Liposome-based drug delivery in breast cancer treatment. Breast Cancer Res 2002; 4:95-9. Pascual E, Mata CP, Gomez-Blanco J, Moreno N, Barcena J, Blanco E, et al. Structural basis for the development of avian virus capsids that display influenza virus proteins and induce protective immunity. J Virol 2015; 89:2563-74. Pastori C, Tudor D, Diomede L, Drillet AS, Jegerlehner A, Rohn TA, et al. Virus like particle based strategy to elicit HIV-protective antibodies to the alpha-helic regions of gp41. Virology 2012; 431:1-11. Pastrana DV, Gambhira R, Buck CB, Pang YY, Thompson CD, Culp TD, et al. Cross- neutralization of cutaneous and mucosal Papillomavirus types with anti-sera to the amino terminus of L2. Virology 2005; 337:365-72. Patel JM, Kim MC, Vartabedian VF, Lee YN, He S, Song JM, et al. Protein transfer- mediated surface engineering to adjuvantate virus-like nanoparticles for enhanced anti-viral immune responses. Nanomedicine 2015; 11:1097-107. Patterson DP, Rynda-Apple A, Harmsen AL, Harmsen AG, Douglas T. Biomimetic antigenic nanoparticles elicit controlled protective immune response to influenza. ACS Nano 2013; 7:3036-44. Pavani C, Uchoa AF, Oliveira CS, Iamamoto Y, Baptista MS. Effect of zinc insertion and hydrophobicity on the membrane interactions and PDT activity of porphyrin photosensitizers. Photochem Photobiol Sci 2009; 8:233-40. Peacey M, Wilson S, Baird MA, Ward VK. Versatile RHDV virus-like particles: incorporation of antigens by genetic modification and chemical conjugation. Biotechnol Bioeng 2007; 98:968-77. Peiris PM, Toy R, Doolittle E, Pansky J, Abramowski A, Tam M, et al. Imaging metastasis using an integrin-targeting chain-shaped nanoparticle. ACS Nano 2012; 6:8783-95. Pejawar-Gaddy S, Rajawat Y, Hilioti Z, Xue J, Gaddy DF, Finn OJ, et al. Generation of a tumor vaccine candidate based on conjugation of a MUC1 peptide to polyionic papillomavirus virus-like particles. Cancer Immunol Immunother 2010; 59:1685- 96.
350 BIBLIOGRAPHY
Perez EA. Microtubule inhibitors: Differentiating tubulin-inhibiting agents based on mechanisms of action, clinical activity, and resistance. Mol Cancer Ther 2009; 8:2086-95. Perrone LA, Ahmad A, Veguilla V, Lu X, Smith G, Katz JM, et al. Intranasal vaccination with 1918 influenza virus-like particles protects mice and ferrets from lethal 1918 and H5N1 influenza virus challenge. J Virol 2009; 83:5726-34. Perry JL, Reuter KG, Kai MP, Herlihy KP, Jones SW, Luft JC, et al. PEGylated PRINT Nanoparticles: The Impact of PEG Density on Protein Binding, Macrophage Association, Biodistribution, and Pharmacokinetics. Nano Lett 2012; 12:5304-10. Petros RA, DeSimone JM. Strategies in the design of nanoparticles for therapeutic applications. Nat Rev Drug Discov 2010; 9:615-27. Phelps JP, Dang N, Rasochova L. Inactivation and purification of cowpea mosaic virus- like particles displaying peptide antigens from Bacillus anthracis. J Virol Methods 2007; 141:146-53. Phumyen A, Jantasorn S, Jumnainsong A, Leelayuwat C. Doxorubicin-conjugated bacteriophages carrying anti-MHC class I chain-related A for targeted cancer therapy in vitro. Onco Targets Ther 2014; 7:2183-95. Pitek AS, Jameson SA, Veliz FA, Shukla S, Steinmetz NF. Serum albumin 'camouflage' of plant virus based nanoparticles prevents their antibody recognition and enhances pharmacokinetics. Biomaterials 2016; 89:89-97. Pitisuttithum P, Rerks-Ngarm S, Bussaratid V, Dhitavat J, Maekanantawat W, Pungpak S, et al. Safety and reactogenicity of canarypox ALVAC-HIV (vCP1521) and HIV-1 gp120 AIDSVAX B/E vaccination in an efficacy trial in Thailand. PLoS One 2011; 6:e27837. Plchova H, Moravec T, Hoffmeisterova H, Folwarczna J, Cerovska N. Expression of Human papillomavirus 16 E7ggg oncoprotein on N- and C-terminus of Potato virus X coat protein in bacterial and plant cells. Protein Expr Purif 2011; 77:146- 52. Pokorski JK, Breitenkamp K, Liepold LO, Qazi S, Finn MG. Functional virus-based polymer-protein nanoparticles by atom transfer radical polymerization. J Am Chem Soc 2011; 133:9242-5. Pokorski JK, Hovlid ML, Finn MG. Cell targeting with hybrid Qbeta virus-like particles displaying epidermal growth factor. Chembiochem 2011; 12:2441-7. Pokorski JK, Hovlid ML, Finn MG. Cell targeting with hybrid Qβ virus-like particles displaying epidermal growth factor. Chembiochem : a European journal of chemical biology 2011; 12:2441-7. Pokorski JK, Steinmetz NF. The art of engineering viral nanoparticles. Mol Pharm 2011; 8:29-43. Portney NG, Ozkan M. Nano-oncology: drug delivery, imaging, and sensing. Analytical and bioanalytical chemistry 2006; 384:620-30. Porumb H. The solution spectroscopy of drugs and the drug-nucleic acid interactions. Prog Biophys Mol Biol 1978; 34:175-95. Pouyanfard S, Bamdad T, Hashemi H, Bandehpour M, Kazemi B. Induction of protective anti-CTL epitope responses against HER-2-positive breast cancer based on multivalent T7 phage nanoparticles. PLoS One 2012; 7:e49539.
351 BIBLIOGRAPHY
Prabhakar U, Maeda H, Jain RK, Sevick-Muraca EM, Zamboni W, Farokhzad OC, et al. Challenges and key considerations of the enhanced permeability and retention effect for nanomedicine drug delivery in oncology. Cancer Res 2013; 73:2412-7. Prasuhn DE, Jr., Singh P, Strable E, Brown S, Manchester M, Finn MG. Plasma clearance of bacteriophage Qbeta particles as a function of surface charge. J Am Chem Soc 2008; 130:1328-34. Presolski SI, Hong VP, Finn MG. Copper-Catalyzed Azide-Alkyne Click Chemistry for Bioconjugation. Curr Protoc Chem Biol 2011; 3:153-62. Press D. Cyclic RGD peptide-modified liposomal drug delivery system : enhanced cellular uptake in vitro and improved pharmacokinetics in rats. International Journal of Nanomedicine 2012; 7:3803-11. Presta LG, Chen H, O'Connor SJ, Chisholm V, Meng YG, Krummen L, et al. Humanization of an anti-vascular endothelial growth factor monoclonal antibody for the therapy of solid tumors and other disorders. Cancer Res 1997; 57:4593-9. Prieto PA, Yang JC, Sherry RM, Hughes MS, Kammula US, White DE, et al. CTLA-4 blockade with ipilimumab: long-term follow-up of 177 patients with metastatic melanoma. Clin Cancer Res 2012; 18:2039-47. Prokop A, Davidson JM. Nanovehicular intracellular delivery systems. Journal of Pharmaceutical Sciences 2008; 97:3518-90. Pulendran B, Ahmed R. Immunological mechanisms of vaccination. Nat Immunol 2011; 12:509-17. Qazi S, Liepold LO, Abedin MJ, Johnson B, Prevelige P, Frank JA, et al. P22 viral capsids as nanocomposite high-relaxivity MRI contrast agents. Mol Pharm 2013; 10:11-7. Quan Q, Xie J, Gao H, Yang M, Zhang F, Liu G, et al. HSA coated iron oxide nanoparticles as drug delivery vehicles for cancer therapy. Mol Pharm 2011; 8:1669-76. Rae JM, Scheys JO, Clark KM, Chadwick RB, Kiefer MC, Lippman ME. EGFR and EGFRvIII expression in primary breast cancer and cell lines. Breast cancer research and treatment 2004; 87:87-95. Raff JP, Rajdev L, Malik U, Novik Y, Manalo JM, Negassa A, et al. Phase II Study of Weekly Docetaxel Alone or in Combination with Trastuzumab in Patients with Metastatic Breast Cancer. Clinical Breast Cancer 2004; 4:420-7. Raja KS, Wang Q, Gonzalez MJ, Manchester M, Johnson JE, Finn MG. Hybrid virus- polymer materials. 1. Synthesis and properties of PEG-decorated cowpea mosaic virus. Biomacromolecules 2003; 4:472-6. Rangasamy S, Ju H, Um S, Oh DC, Song JM. Mitochondria and DNA Targeting of 5,10,15,20-Tetrakis(7-sulfonatobenzo[b]thiophene) Porphyrin-Induced Photodynamic Therapy via Intrinsic and Extrinsic Apoptotic Cell Death. J Med Chem 2015; 58:6864-74. Rasch MR, Sokolov KV, Korgel BA. Limitations on the Optical Tunability of Small Diameter Gold Nanoshells. Langmuir 2009; 25:11777-85. Redaniel MT, Martin RM, Gillatt D, Wade J, Jeffreys M. Time from diagnosis to surgery and prostate cancer survival: a retrospective cohort study. BMC Cancer 2013; 13:559.
352 BIBLIOGRAPHY
Rehor I, Cigler P. Precise estimation of HPHT nanodiamond size distribution based on transmission electron microscopy image analysis. Diamond and Related Materials 2014; 46:21-4. Rehor I, Mackova H, Filippov SK, Kucka J, Proks V, Slegerova J, et al. Fluorescent Nanodiamonds with Bioorthogonally Reactive Protein-Resistant Polymeric Coatings. Chempluschem 2014; 79:21-4. Rehor I, Slegerova J, Kucka J, Proks V, Petrakova V, Adam MP, et al. Fluorescent Nanodiamonds Embedded in Biocompatible Translucent Shells. Small 2014; 10:1106-15. Ren Y, Wong SM, Lim LY. Folic acid-conjugated protein cages of a plant virus: a novel delivery platform for doxorubicin. Bioconjug Chem 2007; 18:836-43. Ren Z, Ji X, Meng L, Wei Y, Wang T, Feng N, et al. H5N1 influenza virus-like particle vaccine protects mice from heterologous virus challenge better than whole inactivated virus. Virus Res 2015; 200:9-18. Rerks-Ngarm S, Pitisuttithum P, Nitayaphan S, Kaewkungwal J, Chiu J, Paris R, et al. Vaccination with ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand. N Engl J Med 2009; 361:2209-20. Restifo NP, Dudley ME, Rosenberg SA. Adoptive immunotherapy for cancer: harnessing the T cell response. Nat Rev Immunol 2012; 12:269-81. Reynolds JG, Geretti E, Hendriks BS, Lee H, Leonard SC, Klinz SG, et al. HER2- targeted liposomal doxorubicin displays enhanced anti-tumorigenic effects without associated cardiotoxicity. Toxicol Appl Pharmacol 2012; 262:1-10. Rhee JK, Baksh M, Nycholat C, Paulson JC, Kitagishi H, Finn MG. Glycan-targeted virus-like nanoparticles for photodynamic therapy. Biomacromolecules 2012; 13:2333-8. Richert LE, Servid AE, Harmsen AL, Rynda-Apple A, Han S, Wiley JA, et al. A virus- like particle vaccine platform elicits heightened and hastened local lung mucosal antibody production after a single dose. Vaccine 2012; 30:3653-65. Riedel S. Edward Jenner and the history of smallpox and vaccination. Proc (Bayl Univ Med Cent) 2005; 18:21-5. Riedel S. Crossing the species barrier: the threat of an avian influenza pandemic. Proc (Bayl Univ Med Cent) 2006; 19:16-20. Roberts MJ, Bentley MD, Harris JM. Chemistry for peptide and protein PEGylation. Adv Drug Deliv Rev 2002; 54:459-76. Robertson CA, Abrahamse H, Evans D. The in vitro PDT efficacy of a novel metallophthalocyanine (MPc) derivative and established 5-ALA photosensitizing dyes against human metastatic melanoma cells. Lasers Surg Med 2010; 42:926-36. Rocha-lima CM, Soares HP, Raez LE, Singal R. EGFR Targeting of Solid Tumors. Journal of the Moffitt Cancer Center 2007; 14:295-304. Roden RB, Yutzy WHt, Fallon R, Inglis S, Lowy DR, Schiller JT. Minor capsid protein of human genital papillomaviruses contains subdominant, cross-neutralizing epitopes. Virology 2000; 270:254-7. Rodriguez PL, Harada T, Christian DA, Pantano DA, Tsai RK, Discher DE. Minimal "Self" peptides that inhibit phagocytic clearance and enhance delivery of nanoparticles. Science 2013; 339:971-5.
353 BIBLIOGRAPHY
Rohn TA, Jennings GT, Hernandez M, Grest P, Beck M, Zou Y, et al. Vaccination against IL-17 suppresses autoimmune arthritis and encephalomyelitis. Eur J Immunol 2006; 36:2857-67. Rosenberg SA. IL-2: the first effective immunotherapy for human cancer. J Immunol 2014; 192:5451-8. Ross JS, Slodkowska EA, Symmans WF, Pusztai L, Ravdin PM, Hortobagyi GN. The HER-2 receptor and breast cancer: ten years of targeted anti-HER-2 therapy and personalized medicine. Oncologist 2009; 14:320-68. Roy A, Singh MS, Upadhyay P, Bhaskar S. Nanoparticle mediated co-delivery of paclitaxel and a TLR-4 agonist results in tumor regression and enhanced immune response in the tumor microenvironment of a mouse model. Int J Pharm 2013; 445:171-80. Ruggiero A, Villa CH, Bander E, Rey DA, Bergkvist M, Batt CA, et al. Paradoxical glomerular filtration of carbon nanotubes. Proc Natl Acad Sci U S A 2010; 107:12369-74. Ryan MD, Drew J. Foot-and-mouth disease virus 2A oligopeptide mediated cleavage of an artificial polyprotein. EMBO J 1994; 13:928-33. Sabel MS, Diehl KM, Chang AE. Priniciples of surgical therapy in oncology. Oncology. New York: Springer; 2006. p. 58-72. Sanda MG, Smith DC, Charles LG, Hwang C, Pienta KJ, Schlom J, et al. Recombinant vaccinia-PSA (PROSTVAC) can induce a prostate-specific immune response in androgen-modulated human prostate cancer. Urology 1999; 53:260-6. Sanson C, Schatz C, Le Meins JF, Soum A, Thevenot J, Garanger E, et al. A simple method to achieve high doxorubicin loading in biodegradable polymersomes. J Control Release 2010; 147:428-35. Santini MT, Rainaldi G. Three-dimensional spheroid model in tumor biology. Pathobiology : journal of immunopathology, molecular and cellular biology 1999; 67:148-57. Schafer K, Muller M, Faath S, Henn A, Osen W, Zentgraf H, et al. Immune response to human papillomavirus 16 L1E7 chimeric virus-like particles: induction of cytotoxic T cells and specific tumor protection. Int J Cancer 1999; 81:881-8. Schellenbacher C, Kwak K, Fink D, Shafti-Keramat S, Huber B, Jindra C, et al. Efficacy of RG1-VLP vaccination against infections with genital and cutaneous human papillomaviruses. J Invest Dermatol 2013; 133:2706-13. Schenk D, Barbour R, Dunn W, Gordon G, Grajeda H, Guido T, et al. Immunization with amyloid-beta attenuates Alzheimer-disease-like pathology in the PDAPP mouse. Nature 1999; 400:173-7. Schietinger S, Barth M, Aichele T, Benson O. Plasmon-enhanced single photon emission from a nanoassembled metal-diamond hybrid structure at room temperature. Nano Lett 2009; 9:1694-8. Schlick TL, Ding Z, Kovacs EW, Francis MB. Dual-surface modification of the tobacco mosaic virus. Journal of the American Chemical Society 2005; 127:3718-23. Schmitt-Sody M, Strieth S, Krasnici S, Sauer B, Schulze B, Teifel M, et al. Neovascular targeting therapy: paclitaxel encapsulated in cationic liposomes improves antitumoral efficacy. Clinical cancer research : an official journal of the American Association for Cancer Research 2003; 9:2335-41.
354 BIBLIOGRAPHY
Schneemann A, Young MJ. Viral assembly using heterologous expression systems and cell extracts. Adv Protein Chem 2003; 64:1-36. Schroeder A, Heller DA, Winslow MM, Dahlman JE, Pratt GW, Langer R, et al. Treating metastatic cancer with nanotechnology. Nat Rev Cancer 2012; 12:39-50. Schwartzentruber DJ, Lawson DH, Richards JM, Conry RM, Miller DM, Treisman J, et al. gp100 peptide vaccine and interleukin-2 in patients with advanced melanoma. N Engl J Med 2011; 364:2119-27. Scorpio A, Blank TE, Day WA, Chabot DJ. Anthrax vaccines: Pasteur to the present. Cell Mol Life Sci 2006; 63:2237-48. Scott AM, Wolchok JD, Old LJ. Antibody therapy of cancer. Nat Rev Cancer 2012; 12:278-87. Seabrook TJ, Bloom JK, Iglesias M, Spooner ET, Walsh DM, Lemere CA. Species- specific immune response to immunization with human versus rodent A beta peptide. Neurobiol Aging 2004; 25:1141-51. Segeren CM, Sonneveld P, van der Holt B, Baars JW, Biesma DH, Cornellissen JJ, et al. Vincristine, doxorubicin and dexamethasone (VAD) administered as rapid intravenous infusion for first-line treatment in untreated multiple myeloma. Br J Haematol 1999; 105:127-30. Semple SC, Harasym TO, Clow KA, Ansell SM, Klimuk SK, Hope MJ. Immunogenicity and rapid blood clearance of liposomes containing polyethylene glycol-lipid conjugates and nucleic Acid. J Pharmacol Exp Ther 2005; 312:1020-6. Sen Gupta S, Kuzelka J, Singh P, Lewis WG, Manchester M, Finn MG. Accelerated bioorthogonal conjugation: a practical method for the ligation of diverse functional molecules to a polyvalent virus scaffold. Bioconjug Chem 2005; 16:1572-9. Shao J, DeHaven J, Lamm D, Weissman DN, Malanga CJ, Rojanasakul Y, et al. A cell- based drug delivery system for lung targeting: II. Therapeutic activities on B16- F10 melanoma in mouse lungs. Drug Deliv 2001; 8:71-6. Sharma G, Valenta DT, Altman Y, Harvey S, Xie H, Mitragotri S, et al. Polymer particle shape independently influences binding and internalization by macrophages. J Control Release 2010; 147:408-12. Shen F, Chu S, Bence AK, Bailey B, Xue X, Erickson PA, et al. Quantitation of doxorubicin uptake, efflux, and modulation of multidrug resistance (MDR) in MDR human cancer cells. J Pharmacol Exp Ther 2008; 324:95-102. Sheng Y, Yuan Y, Liu CS, Tao XY, Shan XQ, Xu F. In vitro macrophage uptake and in vivo biodistribution of PLA-PEG nanoparticles loaded with hemoglobin as blood substitutes: effect of PEG content. Journal of Materials Science-Materials in Medicine 2009; 20:1881-91. Sheth. Multielectrode platform for measuring oxygenation status in multicellular tumor spheroids. PhD Thesis, CWRU 2011. Shimizu T, Ishida T, Kiwada H. Transport of PEGylated liposomes from the splenic marginal zone to the follicle in the induction phase of the accelerated blood clearance phenomenon. Immunobiology 2013; 218:725-32. Shin CS, Kwak B, Han B, Park K. Development of an in vitro 3D tumor model to study therapeutic efficiency of an anticancer drug. Mol Pharm 2013; 10:2167-75.
355 BIBLIOGRAPHY
Shirakawa T. Clinical trial design for adenoviral gene therapy products. Drug News Perspect 2009; 22:140-5. Shukla S, Ablack A, Wen A, Lee K, Lewis J, Steinmetz NF. Increased tumor homing and tissue penetration of the filamentous plant viral nanoparticle Potato virus X. Molecular pharmaceutics 2012. Shukla S, Ablack AL, Wen AM, Lee KL, Lewis JD, Steinmetz NF. Increased tumor homing and tissue penetration of the filamentous plant viral nanoparticle Potato virus X. Mol Pharm 2013; 10:33-42. Shukla S, Dickmeis C, Nagarajan AS, Fischer R, Commandeur U, Steinmetz NF. Molecular farming of fluorescent virus-based nanoparticles for optical imaging in plants, human cells and mouse models. Biomaterials Science 2014; 2:784 - 97. Shukla S, DiFranco NA, Wen AM, Commandeur U, Steinmetz NF. To Target or Not to Target: Active vs. Passive Tumor Homing of Filamentous Nanoparticles Based on Potato virus X. Cellular and Molecular Bioengineering 2015; 8:433-44. Shukla S, Dorand RD, Myers JT, Woods SE, Gulati NM, Stewart PL, et al. Multiple Administrations of Viral Nanoparticles Alter in Vivo Behavior-Insights from Intravital Microscopy. Acs Biomaterials-Science & Engineering 2016; 2:829-37. Shukla S, Eber FJ, Nagarajan AS, DiFranco NA, Schmidt N, Wen AM, et al. The Impact of Aspect Ratio on the Biodistribution and Tumor Homing of Rigid Soft-Matter Nanorods. Adv Healthc Mater 2015; 4:874-82. Shukla S, Steinmetz NF. Virus-based nanomaterials as positron emission tomography and magnetic resonance contrast agents: from technology development to translational medicine. Wiley Interdiscip Rev Nanomed Nanobiotechnol 2015; 7:708-21. Shukla S, Wen AM, Ayat NR, Commandeur U, Gopalkrishnan R, Broome AM, et al. Biodistribution and clearance of a filamentous plant virus in healthy and tumor- bearing mice. Nanomedicine (Lond) 2014; 9:221-35. Shukla S, Wen AM, Commandeur U, Steinmetz NF. Presentation of HER2 epitopes using a filamentous plant virus-based vaccination platform. Journal of Materials Chemistry B 2014; 2:6249. Silberman AW. Surgical debulking of tumors. Surg Gynecol Obstet 1982; 155:577-85. Simone EA, Dziubla TD, Discher DE, Muzykantov VR. Filamentous polymer nanocarriers of tunable stiffness that encapsulate the therapeutic enzyme catalase. Biomacromolecules 2009; 10:1324-30. Singh P, Prasuhn D, Yeh RM, Destito G, Rae CS, Osborn K, et al. Bio-distribution, toxicity and pathology of cowpea mosaic virus nanoparticles in vivo. J Control Release 2007; 120:41-50. Singh R, Pantarotto D, Lacerda L, Pastorin G, Klumpp C, Prato M, et al. Tissue biodistribution and blood clearance rates of intravenously administered carbon nanotube radiotracers. Proc Natl Acad Sci U S A 2006; 103:3357-62. Slegerova J, Hajek M, Rehor I, Sedlak F, Stursa J, Hruby M, et al. Designing the nanobiointerface of fluorescent nanodiamonds: highly selective targeting of glioma cancer cells. Nanoscale 2015; 7:415-20. Slegerova J, Rehor I, Havlik J, Raabova H, Muchova E, Cigler P. Nanodiamonds as Intracellular Probes for Imaging in Biology and Medicine. Intracellular Delivery Ii: Fundamentals and Applications 2014; 7:363-401.
356 BIBLIOGRAPHY
Small EJ, Schellhammer PF, Higano CS, Redfern CH, Nemunaitis JJ, Valone FH, et al. Placebo-controlled phase III trial of immunologic therapy with sipuleucel-T (APC8015) in patients with metastatic, asymptomatic hormone refractory prostate cancer. J Clin Oncol 2006; 24:3089-94. Smith JF, Brownlow M, Brown M, Kowalski R, Esser MT, Ruiz W, et al. Antibodies from women immunized with Gardasil cross-neutralize HPV 45 pseudovirions. Hum Vaccin 2007; 3:109-15. Smyth MJ, Taniguchi M, Street SE. The anti-tumor activity of IL-12: mechanisms of innate immunity that are model and dose dependent. J Immunol 2000; 165:2665- 70. Sonderegger I, Rohn TA, Kurrer MO, Iezzi G, Zou Y, Kastelein RA, et al. Neutralization of IL-17 by active vaccination inhibits IL-23-dependent autoimmune myocarditis. Eur J Immunol 2006; 36:2849-56. Song S, Liu D, Peng J, Deng H, Guo Y, Xu LX, et al. Novel peptide ligand directs liposomes toward EGF-R high-expressing cancer cells in vitro and in vivo. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2009; 23:1396-404. Song S, Liu D, Peng J, Sun Y, Li Z, Gu J-R, et al. Peptide ligand-mediated liposome distribution and targeting to EGFR expressing tumor in vivo. International journal of pharmaceutics 2008; 363:155-61. Sorlie T, Perou CM, Tibshirani R, Aas T, Geisler S, Johnsen H, et al. Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci U S A 2001; 98:10869-74. Sosnovik DE, Nahrendorf M, Weissleder R. Magnetic nanoparticles for MR imaging: agents, techniques and cardiovascular applications. Basic Res Cardiol 2008; 103:122-30. Spohn G, Bachmann MF. Targeting osteoporosis and rheumatoid arthritis by active vaccination against RANKL. Adv Exp Med Biol 2007; 602:135-42. Spohn G, Guler R, Johansen P, Keller I, Jacobs M, Beck M, et al. A virus-like particle- based vaccine selectively targeting soluble TNF-alpha protects from arthritis without inducing reactivation of latent tuberculosis. J Immunol 2007; 178:7450-7. Spohn G, Keller I, Beck M, Grest P, Jennings GT, Bachmann MF. Active immunization with IL-1 displayed on virus-like particles protects from autoimmune arthritis. Eur J Immunol 2008; 38:877-87. Spohn G, Schwarz K, Maurer P, Illges H, Rajasekaran N, Choi Y, et al. Protection against osteoporosis by active immunization with TRANCE/RANKL displayed on virus-like particles. J Immunol 2005; 175:6211-8. Stanley M. Pathology and epidemiology of HPV infection in females. Gynecol Oncol 2010; 117:S5-10. Steinmetz NF. Viral nanoparticles as platforms for next-generation therapeutics and imaging devices. Nanomedicine 2010; 6:634-41. Steinmetz NF, Ablack A, Hickey JL, Ablack J, Manocha B, Mymryk JS, et al. Intravital Imaging of Human Prostate Cancer using Viral Nanoparticles Targeted to Gastrin-Releasing Peptide Receptors. Small 2011; in press.
357 BIBLIOGRAPHY
Steinmetz NF, Ablack AL, Hickey JL, Ablack J, Manocha B, Mymryk JS, et al. Intravital imaging of human prostate cancer using viral nanoparticles targeted to gastrin- releasing Peptide receptors. Small 2011; 7:1664-72. Steinmetz NF, Cho CF, Ablack A, Lewis JD, Manchester M. Cowpea mosaic virus nanoparticles target surface vimentin on cancer cells. Nanomedicine (Lond) 2011; 6:351-64. Steinmetz NF, Manchester M. PEGylated Viral Nanoparticles for Biomedicine: The Impact of PEG Chain Length on VNP Cell Interactions In Vitro and Ex Vivo. Biomacromolecules 2009; 10:784-92. Steinmetz NF, Mertens ME, Taurog RE, Johnson JE, Commandeur U, Fischer R, et al. Potato virus X as a novel platform for potential biomedical applications. Nano Lett 2010; 10:305-12. Stephanopoulos N, Tong GJ, Hsiao SC, Francis MB. Dual-surface modified virus capsids for targeted delivery of photodynamic agents to cancer cells. ACS Nano 2010; 4:6014-20. Stern JM, Stanfield J, Kabbani W, Hsieh JT, Cadeddu JA. Selective prostate cancer thermal ablation with laser activated gold nanoshells. J Urol 2008; 179:748-53. Stoll S, Delon J, Brotz TM, Germain RN. Dynamic imaging of T cell-dendritic cell interactions in lymph nodes. Science 2002; 296:1873-6. Stylianopoulos T, Diop-Frimpong B, Munn LL, Jain RK. Diffusion anisotropy in collagen gels and tumors: the effect of fiber network orientation. Biophys J 2010; 99:3119-28. Stylianopoulos T, Poh MZ, Insin N, Bawendi MG, Fukumura D, Munn LL, et al. Diffusion of particles in the extracellular matrix: the effect of repulsive electrostatic interactions. Biophys J 2010; 99:1342-9. Su F, Schneider RJ. Hepatitis B virus HBx protein sensitizes cells to apoptotic killing by tumor necrosis factor alpha. Proc Natl Acad Sci U S A 1997; 94:8744-9. Suci PA, Varpness Z, Gillitzer E, Douglas T, Young M. Targeting and photodynamic killing of a microbial pathogen using protein cage architectures functionalized with a photosensitizer. Langmuir 2007; 23:12280-6. Sugiyama Y, Hamamoto H, Takemoto S, Watanabe Y, Okada Y. Systemic production of foreign peptides on the particle surface of tobacco mosaic virus. FEBS Lett 1995; 359:247-50. Sun S, Li W, Sun Y, Pan Y, Li J. A new RNA vaccine platform based on MS2 virus-like particles produced in Saccharomyces cerevisiae. Biochem Biophys Res Commun 2011; 407:124-8. Susumu K, Mei BC, Mattoussi H. Multifunctional ligands based on dihydrolipoic acid and polyethylene glycol to promote biocompatibility of quantum dots. Nature Protocols 2009; 4:424-36. Szurko A, Kramer-Marek G, Widel M, Ratuszna A, Habdas J, Kus P. Photodynamic effects of two water soluble porphyrins evaluated on human malignant melanoma cells in vitro. Acta Biochim Pol 2003; 50:1165-74. Tachikawa S, El-Zaria ME, Inomata R, Sato S, Nakamura H. Synthesis of protoporphyrin-lipids and biological evaluation of micelles and liposomes. Bioorg Med Chem 2014; 22:4745-51.
358 BIBLIOGRAPHY
Tan J, Shah S, Thomas A, Ou-Yang HD, Liu Y. The influence of size, shape and vessel geometry on nanoparticle distribution. Microfluidics and nanofluidics 2013; 14:77-87. Tang H, Chen X, Rui M, Sun W, Chen J, Peng J, et al. Effects of Surface Displayed Targeting Ligand GE11 on Liposome Distribution and Extravasation in Tumor. Molecular pharmaceutics 2014; 11:3242-50. Taubenberger JK, Morens DM. Influenza: the once and future pandemic. Public Health Rep 2010; 125 Suppl 3:16-26. Tegerstedt K, Lindencrona JA, Curcio C, Andreasson K, Tullus C, Forni G, et al. A single vaccination with polyomavirus VP1/VP2Her2 virus-like particles prevents outgrowth of HER-2/neu-expressing tumors. Cancer Res 2005; 65:5953-7. Teng IT, Chang YJ, Wang LS, Lu HY, Wu LC, Yang CM, et al. Phospholipid- functionalized mesoporous silica nanocarriers for selective photodynamic therapy of cancer. Biomaterials 2013; 34:7462-70. Tenzer S, Docter D, Kuharev J, Musyanovych A, Fetz V, Hecht R, et al. Rapid formation of plasma protein corona critically affects nanoparticle pathophysiology. Nat Nanotechnol 2013; 8:772-81. Teow Y, Valiyaveettil S. Active targeting of cancer cells using folic acid-conjugated platinum nanoparticles. Nanoscale 2010; 2:2607-13. Testori A, Rutkowski P, Marsden J, Bastholt L, Chiarion-Sileni V, Hauschild A, et al. Surgery and radiotherapy in the treatment of cutaneous melanoma. Ann Oncol 2009; 20 Suppl 6:vi22-9. Thomas CE, Ehrhardt A, Kay MA. Progress and problems with the use of viral vectors for gene therapy. Nat Rev Genet 2003; 4:346-58. Thomas CE, Schiedner G, Kochanek S, Castro MG, Lowenstein PR. Preexisting antiadenoviral immunity is not a barrier to efficient and stable transduction of the brain, mediated by novel high-capacity adenovirus vectors. Hum Gene Ther 2001; 12:839-46. Thompson FM, Porter DW, Okitsu SL, Westerfeld N, Vogel D, Todryk S, et al. Evidence of blood stage efficacy with a virosomal malaria vaccine in a phase IIa clinical trial. PLoS One 2008; 3:e1493. Thorn CF, Oshiro C, Marsh S, Hernandez-Boussard T, McLeod H, Klein TE, et al. Doxorubicin pathways: pharmacodynamics and adverse effects. Pharmacogenet Genomics 2011; 21:440-6. Tirado MM, Martinez CL, Garcia de la Torre J. J Chem Phys 1984; 81:2047-52. Tissot AC, Maurer P, Nussberger J, Sabat R, Pfister T, Ignatenko S, et al. Effect of immunisation against angiotensin II with CYT006-AngQb on ambulatory blood pressure: a double-blind, randomised, placebo-controlled phase IIa study. Lancet 2008; 371:821-7. Tissot AC, Renhofa R, Schmitz N, Cielens I, Meijerink E, Ose V, et al. Versatile virus- like particle carrier for epitope based vaccines. PLoS One 2010; 5:e9809. Toy R, Hayden E, Shoup C, Baskaran H, Karathanasis E. The effects of particle size, density and shape on margination of nanoparticles in microcirculation. Nanotechnology 2011; 22:115101.
359 BIBLIOGRAPHY
Toy R, Peiris PM, Ghaghada KB, Karathanasis E. Shaping cancer nanomedicine: the effect of particle shape on the in vivo journey of nanoparticles. Nanomedicine (Lond) 2014; 9:121-34. Tremblay MH, Majeau N, Gagne ME, Lecours K, Morin H, Duvignaud JB, et al. Effect of mutations K97A and E128A on RNA binding and self assembly of papaya mosaic potexvirus coat protein. FEBS J 2006; 273:14-25. Tricco AC, Chit A, Soobiah C, Hallett D, Meier G, Chen MH, et al. Comparing influenza vaccine efficacy against mismatched and matched strains: a systematic review and meta-analysis. BMC Med 2013; 11:153. Tugues S, Burkhard SH, Ohs I, Vrohlings M, Nussbaum K, Vom Berg J, et al. New insights into IL-12-mediated tumor suppression. Cell Death Differ 2015; 22:237- 46. Tumban E, Peabody J, Peabody DS, Chackerian B. A pan-HPV vaccine based on bacteriophage PP7 VLPs displaying broadly cross-neutralizing epitopes from the HPV minor capsid protein, L2. PLoS One 2011; 6:e23310. Tumban E, Peabody J, Tyler M, Peabody DS, Chackerian B. VLPs displaying a single L2 epitope induce broadly cross-neutralizing antibodies against human papillomavirus. PLoS One 2012; 7:e49751. Turpen TH, Reinl SJ, Charoenvit Y, Hoffman SL, Fallarme V, Grill LK. Malarial epitopes expressed on the surface of recombinant tobacco mosaic virus. Biotechnology (N Y) 1995; 13:53-7. Tyler M, Tumban E, Peabody DS, Chackerian B. The use of hybrid virus-like particles to enhance the immunogenicity of a broadly protective HPV vaccine. Biotechnol Bioeng 2014; 111:2398-406. Uhde-Holzem K, Schlosser V, Viazov S, Fischer R, Commandeur U. Immunogenic properties of chimeric potato virus X particles displaying the hepatitis C virus hypervariable region I peptide R9. J Virol Methods 2010; 166:12-20. Vacha R, Martinez-Veracoechea FJ, Frenkel D. Receptor-mediated endocytosis of nanoparticles of various shapes. Nano letters 2011; 11:5391-5. Vaijayanthimala V, Cheng PY, Yeh SH, Liu KK, Hsiao CH, Chao JI, et al. The long-term stability and biocompatibility of fluorescent nanodiamond as an in vivo contrast agent. Biomaterials 2012; 33:7794-802. Vaine CA, Patel MK, Zhu J, Lee E, Finberg RW, Hayward RC, et al. Tuning innate immune activation by surface texturing of polymer microparticles: the role of shape in inflammasome activation. J Immunol 2013; 190:3525-32. Van Rompay KK, Hunter Z, Jayashankar K, Peabody J, Montefiori D, LaBranche CC, et al. A vaccine against CCR5 protects a subset of macaques upon intravaginal challenge with simian immunodeficiency virus SIVmac251. J Virol 2014; 88:2011-24. Venter PA, Dirksen A, Thomas D, Manchester M, Dawson PE, Schneemann A. Multivalent display of proteins on viral nanoparticles using molecular recognition and chemical ligation strategies. Biomacromolecules 2011; 12:2293-301. Verhoef JJ, Carpenter JF, Anchordoquy TJ, Schellekens H. Potential induction of anti- PEG antibodies and complement activation toward PEGylated therapeutics. Drug Discov Today 2014; 19:1945-52.
360 BIBLIOGRAPHY
Veronese FM, Pasut G. PEGylation, successful approach to drug delivery. Drug Discov Today 2005; 10:1451-8. Vogel FR. Improving vaccine performance with adjuvants. Clin Infect Dis 2000; 30 Suppl 3:S266-70. Von Hoff DD, Ramanathan RK, Borad MJ, Laheru DA, Smith LS, Wood TE, et al. Gemcitabine plus nab-paclitaxel is an active regimen in patients with advanced pancreatic cancer: a phase I/II trial. J Clin Oncol 2011; 29:4548-54. Wagner S, Jasinska J, Breiteneder H, Kundi M, Pehamberger H, Scheiner O, et al. Delayed tumor onset and reduced tumor growth progression after immunization with a Her-2/neu multi-peptide vaccine and IL-12 in c-neu transgenic mice. Breast Cancer Res Treat 2007; 106:29-38. Wang J, Saffold S, Cao X, Krauss J, Chen W. Eliciting T cell immunity against poorly immunogenic tumors by immunization with dendritic cell-tumor fusion vaccines. J Immunol 1998; 161:5516-24. Wang K, Wang K, Li W, Huang T, Li R, Wang D, et al. Characterizing breast cancer xenograft epidermal growth factor receptor expression by using near-infrared optical imaging. Acta radiologica (Stockholm, Sweden : 1987) 2009; 50:1095-103. Wang X, Ishida T, Kiwada H. Anti-PEG IgM elicited by injection of liposomes is involved in the enhanced blood clearance of a subsequent dose of PEGylated liposomes. J Control Release 2007; 119:236-44. Wang Z, Li Y, Huang Y, Thompson MP, LeGuyader CL, Sahu S, et al. Enzyme- regulated topology of a cyclic peptide brush polymer for tuning assembly. Chem Commun (Camb) 2015; 51:17108-11. Ward BJ, Landry N, Trepanier S, Mercier G, Dargis M, Couture M, et al. Human antibody response to N-glycans present on plant-made influenza virus-like particle (VLP) vaccines. Vaccine 2014; 32:6098-106. Warfield KL, Swenson DL, Olinger GG, Kalina WV, Aman MJ, Bavari S. Ebola virus- like particle-based vaccine protects nonhuman primates against lethal Ebola virus challenge. J Infect Dis 2007; 196 Suppl 2:S430-7. Washington-Hughes CL, Cheng Y, Duan X, Cai L, Lee LA, Wang Q. In vivo virus-based macrofluorogenic probes target azide-labeled surface glycans in MCF-7 breast cancer cells. Mol Pharm 2013; 10:43-50. Wattendorf U, Merkle HP. PEGylation as a tool for the biomedical engineering of surface modified microparticles. J Pharm Sci 2008. Webb JA, Bardhan R. Emerging advances in nanomedicine with engineered gold nanostructures. Nanoscale 2014; 6:2502-30. Wee S, Hicks MJ, De BP, Rosenberg JB, Moreno AY, Kaminsky SM, et al. Novel cocaine vaccine linked to a disrupted adenovirus gene transfer vector blocks cocaine psychostimulant and reinforcing effects. Neuropsychopharmacology 2012; 37:1083-91. Weinstein JN, Magin RL, Yatvin MB, Zaharko DS. Liposomes and local hyperthermia: selective delivery of methotrexate to heated tumors. Science 1979; 204:188-91. Wen AM, Infusino M, De Luca A, Kernan DL, Czapar AE, Strangi G, et al. Interface of physics and biology: engineering virus-based nanoparticles for biophotonics. Bioconjug Chem 2015; 26:51-62.
361 BIBLIOGRAPHY
Wen AM, Ryan MJ, Yang AC, Breitenkamp K, Pokorski JK, Steinmetz NF. Photodynamic activity of viral nanoparticles conjugated with C60. Chem Commun (Camb) 2012; 48:9044-6. Weng MF, Chang BJ, Chiang SY, Wang NS, Niu H. Cellular uptake and phototoxicity of surface-modified fluorescent nanodiamonds. Diamond and Related Materials 2012; 22:96-104. Wheeler CM, Kjaer SK, Sigurdsson K, Iversen OE, Hernandez-Avila M, Perez G, et al. The impact of quadrivalent human papillomavirus (HPV; types 6, 11, 16, and 18) L1 virus-like particle vaccine on infection and disease due to oncogenic nonvaccine HPV types in sexually active women aged 16-26 years. J Infect Dis 2009; 199:936-44. World Health Organization. Influenza (Seasonal) Fact Sheet. 2014. Wiedermann U, Wiltschke C, Jasinska J, Kundi M, Zurbriggen R, Garner-Spitzer E, et al. A virosomal formulated Her-2/neu multi-peptide vaccine induces Her-2/neu- specific immune responses in patients with metastatic breast cancer: a phase I study. Breast Cancer Res Treat 2010; 119:673-83. Wiessner C, Wiederhold KH, Tissot AC, Frey P, Danner S, Jacobson LH, et al. The second-generation active Abeta immunotherapy CAD106 reduces amyloid accumulation in APP transgenic mice while minimizing potential side effects. J Neurosci 2011; 31:9323-31. Wiley HS. Anomalous Binding of Epidermal Growth Factor to A431 Cells Is Due to the Effect of High Receptor Densities and a Saturable Endocytic System. 1988; 107:801-10. Wiley JA, Richert LE, Swain SD, Harmsen A, Barnard DL, Randall TD, et al. Inducible bronchus-associated lymphoid tissue elicited by a protein cage nanoparticle enhances protection in mice against diverse respiratory viruses. PLoS One 2009; 4:e7142. Willet M, Kurup D, Papaneri A, Wirblich C, Hooper JW, Kwilas SA, et al. Preclinical Development of Inactivated Rabies Virus-Based Polyvalent Vaccine Against Rabies and Filoviruses. J Infect Dis 2015. Winblad B, Andreasen N, Minthon L, Floesser A, Imbert G, Dumortier T, et al. Safety, tolerability, and antibody response of active Abeta immunotherapy with CAD106 in patients with Alzheimer's disease: randomised, double-blind, placebo- controlled, first-in-human study. Lancet Neurol 2012; 11:597-604. Wolfram J, Yang Y, Shen J, Moten A, Chen C, Shen H, et al. The nano-plasma interface: Implications of the protein corona. Colloids Surf B Biointerfaces 2014. Woodburn KW, Fan Q, Kessel D, Luo Y, Young SW. Photodynamic therapy of B16F10 murine melanoma with lutetium texaphyrin. J Invest Dermatol 1998; 110:746-51. World Health Organization Global Influenza Program Surveillance N. Evolution of H5N1 avian influenza viruses in Asia. Emerg Infect Dis 2005; 11:1515-21. Wortmann A, Vohringer S, Engler T, Corjon S, Schirmbeck R, Reimann J, et al. Fully detargeted polyethylene glycol-coated adenovirus vectors are potent genetic vaccines and escape from pre-existing anti-adenovirus antibodies. Mol Ther 2008; 16:154-62.
362 BIBLIOGRAPHY
Wu G, Mikhailovsky A, Khant HA, Fu C, Chiu W, Zasadzinski JA. Remotely triggered liposome release by near-infrared light absorption via hollow gold nanoshells. J Am Chem Soc 2008; 130:8175-7. Wu M, Shi J, Fan D, Zhou Q, Wang F, Niu Z, et al. Biobehavior in normal and tumor- bearing mice of tobacco mosaic virus. Biomacromolecules 2013; 14:4032-7. Wu W, Hsiao SC, Carrico ZM, Francis MB. Genome-free viral capsids as multivalent carriers for taxol delivery. Angew Chem Int Ed Engl 2009; 48:9493-7. Wu Y, Feng S, Zan X, Lin Y, Wang Q. Aligned Electroactive TMV Nanofibers as Enabling Scaffold for Neural Tissue Engineering. Biomacromolecules 2015; 16:3466-72. Wyffels L, Verbrugghen T, Monnery BD, Glassner M, Stroobants S, Hoogenboom R, et al. muPET imaging of the pharmacokinetic behavior of medium and high molar mass Zr-labeled poly(2-ethyl-2-oxazoline) in comparison to poly(ethylene glycol). J Control Release 2016; 235:63-71. Xie J, Xu C, Kohler N, Hou Y, Sun S. Controlled PEGylation of monodisperse Fe3O4 nanoparticles for reduced non-specific uptake by macrophage cells. Advanced Materials 2007; 19:3163-+. Xu J, Wong DH, Byrne JD, Chen K, Bowerman C, DeSimone JM. Future of the particle replication in nonwetting templates (PRINT) technology. Angew Chem Int Ed Engl 2013; 52:6580-9. Xue X, Liang XJ. Overcoming drug efflux-based multidrug resistance in cancer with nanotechnology. Chin J Cancer 2012; 31:100-9. Yamada KM, Cukierman E. Modeling tissue morphogenesis and cancer in 3D. Cell 2007; 130:601-10. Yang Q, Jones SW, Parker CL, Zamboni WC, Bear JE, Lai SK. Evading Immune Cell Uptake and Clearance Requires PEG Grafting at Densities Substantially Exceeding the Minimum for Brush Conformation. Mol Pharm 2014. Yatvin MB, Kreutz W, Horwitz BA, Shinitzky M. pH-sensitive liposomes: possible clinical implications. Science 1980; 210:1253-5. Yazdi AS, Guarda G, Riteau N, Drexler SK, Tardivel A, Couillin I, et al. Nanoparticles activate the NLR pyrin domain containing 3 (Nlrp3) inflammasome and cause pulmonary inflammation through release of IL-1alpha and IL-1beta. Proc Natl Acad Sci U S A 2010; 107:19449-54. Ye L, Lin J, Sun Y, Bennouna S, Lo M, Wu Q, et al. Ebola virus-like particles produced in insect cells exhibit dendritic cell stimulating activity and induce neutralizing antibodies. Virology 2006; 351:260-70. Yhee JY, Lee SJ, Lee S, Song S, Min HS, Kang SW, et al. Tumor-targeting transferrin nanoparticles for systemic polymerized siRNA delivery in tumor-bearing mice. Bioconjug Chem 2013; 24:1850-60. Yi HM, Nisar S, Lee SY, Powers MA, Bentley WE, Payne GF, et al. Patterned assembly of genetically modified viral nanotemplates via nucleic acid hybridization. Nano Letters 2005; 5:1931-6. Yildiz I, Lee KL, Chen K, Shukla S, Steinmetz NF. Infusion of imaging and therapeutic molecules into the plant virus-based carrier cowpea mosaic virus: cargo-loading and delivery. J Control Release 2013; 172:568-78.
363 BIBLIOGRAPHY
Yildiz I, Tsvetkova I, Wen AM, Shukla S, Masarapu MH, Dragnea B, et al. Engineering of Brome mosaic virus for biomedical applications. Rsc Advances 2012; 2:3670-7. Yin Z, Comellas-Aragones M, Chowdhury S, Bentley P, Kaczanowska K, Benmohamed L, et al. Boosting immunity to small tumor-associated carbohydrates with bacteriophage qbeta capsids. ACS Chem Biol 2013; 8:1253-62. Yin Z, Nguyen HG, Chowdhury S, Bentley P, Bruckman MA, Miermont A, et al. Tobacco mosaic virus as a new carrier for tumor associated carbohydrate antigens. Bioconjug Chem 2012; 23:1694-703. Yonesaka K, Zejnullahu K, Okamoto I, Satoh T, Cappuzzo F, Souglakos J, et al. Activation of ERBB2 signaling causes resistance to the EGFR-directed therapeutic antibody cetuximab. Science translational medicine 2011; 3:99ra86. Yoo HS, Lee KH, Oh JE, Park TG. In vitro and in vivo anti-tumor activities of nanoparticles based on doxorubicin-PLGA conjugates. J Control Release 2000; 68:419-31. Yoshimura M, Itasaka S, Harada H, Hiraoka M. Microenvironment and radiation therapy. Biomed Res Int 2013; 2013:685308. Younes A, Bartlett NL, Leonard JP, Kennedy DA, Lynch CM, Sievers EL, et al. Brentuximab vedotin (SGN-35) for relapsed CD30-positive lymphomas. N Engl J Med 2010; 363:1812-21. Yusibov V, Modelska A, Steplewski K, Agadjanyan M, Weiner D, Hooper DC, et al. Antigens produced in plants by infection with chimeric plant viruses immunize against rabies virus and HIV-1. Proc Natl Acad Sci U S A 1997; 94:5784-8. Zagar TM, Vujaskovic Z, Formenti S, Rugo H, Muggia F, O'Connor B, et al. Two phase I dose-escalation/pharmacokinetics studies of low temperature liposomal doxorubicin (LTLD) and mild local hyperthermia in heavily pretreated patients with local regionally recurrent breast cancer. Int J Hyperthermia 2014; 30:285-94. Zamora E, Handisurya A, Shafti-Keramat S, Borchelt D, Rudow G, Conant K, et al. Papillomavirus-like particles are an effective platform for amyloid-beta immunization in rabbits and transgenic mice. J Immunol 2006; 177:2662-70. Zeitouni NC, Shieh S, Oseroff AR. Laser and photodynamic therapy in the management of cutaneous malignancies. Clin Dermatol 2001; 19:328-38. Zeng Q, Wen H, Wen Q, Chen X, Wang Y, Xuan W, et al. Cucumber mosaic virus as drug delivery vehicle for doxorubicin. Biomaterials 2013; 34:4632-42. Zhang BL, Fang CY, Chang CC, Peterson R, Maswadi S, Glickman RD, et al. Photoacoustic emission from fluorescent nanodiamonds enhanced with gold nanoparticles. Biomedical Optics Express 2012; 3:1662-9. Zhang Q, Xiang G, Zhang Y, Yang K, Fan W, Lin J, et al. Increase of doxorubicin sensitivity for folate receptor positive cells when given as the prodrug N- (phenylacetyl) doxorubicin in combination with folate-conjugated PGA. J Pharm Sci 2006; 95:2266-75. Zhang W, Wang L, Liu Y, Chen X, Liu Q, Jia J, et al. Immune responses to vaccines involving a combined antigen-nanoparticle mixture and nanoparticle-encapsulated antigen formulation. Biomaterials 2014; 35:6086-97. Zhang XD, Wu D, Shen X, Liu PX, Yang N, Zhao B, et al. Size-dependent in vivo toxicity of PEG-coated gold nanoparticles. Int J Nanomedicine 2011; 6:2071-81.
364 BIBLIOGRAPHY
Zhang Y, Zhang H, Wang X, Wang J, Zhang X, Zhang Q. The eradication of breast cancer and cancer stem cells using octreotide modified paclitaxel active targeting micelles and salinomycin passive targeting micelles. Biomaterials 2012; 33:679- 91. Zhao D, Zhao X, Zu Y, Li J, Zhang Y, Jiang R, et al. Preparation, characterization, and in vitro targeted delivery of folate-decorated paclitaxel-loaded bovine serum albumin nanoparticles. Int J Nanomedicine 2010; 5:669-77. Zhao L, Seth A, Wibowo N, Zhao CX, Mitter N, Yu C, et al. Nanoparticle vaccines. Vaccine 2014; 32:327-37. Zhao Q, Chen W, Chen Y, Zhang L, Zhang J, Zhang Z. Self-assembled virus-like particles from rotavirus structural protein VP6 for targeted drug delivery. Bioconjug Chem 2011; 22:346-52. Zhao Y, Alakhova DY, Kim JO, Bronich TK, Kabanov AV. A simple way to enhance Doxil(R) therapy: drug release from liposomes at the tumor site by amphiphilic block copolymer. J Control Release 2013; 168:61-9. Zhu Q, Jia L, Gao Z, Wang C, Jiang H, Zhang J, et al. A tumor environment responsive doxorubicin-loaded nanoparticle for targeted cancer therapy. Mol Pharm 2014; 11:3269-78. Zou W. Immunosuppressive networks in the tumour environment and their therapeutic relevance. Nat Rev Cancer 2005; 5:263-74. zur Hausen H. Papillomaviruses causing cancer: evasion from host-cell control in early events in carcinogenesis. J Natl Cancer Inst 2000; 92:690-8. Zurbriggen R. Immunostimulating reconstituted influenza virosomes. Vaccine 2003; 21:921-4.
365