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Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.
The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.
1. VNP (CPMV, BMV, PVX, and TMV) Propagation
CPMV | PVX, TMV, and BMV |
Day 0: Plant 3 cowpea seeds/pot. | Day 0....... |
Figure 1. Plant virus-infected plants. Vigna unguiculata plants infected with CPMV (A). Nicotiana benthamiana plants infected with PVX (B), TMV (C), and BMV (D). The pictures were taken about 10 days post infection by mechanical inoculation.
This protocol provides an approach for the chemical modification of VNPs and their applications for in vivo tumor imaging. The animal fluorescence imaging, fluorescence quantification, and immunohistochemistry techniques presented here are useful for studying biodistribution and evaluating tumor homing. These techniques provide valuable information regarding access of the nanoparticles to the tumor via the EPR effect. By combining the results from the various analytical methods, we get a powerful approach for ev.......
This work was supported by NIH/NIBIB grants R00 EB009105 (to NFS) and P30 EB011317 (to NFS), a NIH/NIBIB training grant T32 EB007509 (to AMW), a Case Western Reserve University Interdisciplinary Alliance Investment Grant (to NFS), and a Case Comprehensive Cancer Center grant P30 CA043703 (to NFS). We thank the Steinmetz Lab undergraduate student researchers for their hands-on support: Nadia Ayat, Kevin Chen, Sourav (Sid) Dey, Alice Yang, Sam Alexander, Craig D'Cruz, Stephen Hern, Lauren Randolph, Brian So, and Paul Chariou.
....Name | Company | Catalog Number | Comments |
Material Name | Company | Catalogue number | Comments (optional) |
 |  |  | VNP production |
Indoor plant chamber | Percival Scientific | E-41L2 | Â |
V. unguiculata seeds (California black-eye no. 5) | Burpee | 51771A | Â |
N. benthamiana seeds | Â | Â | N. benthamiana seeds were a gift from Salk Institute. Seeds are produced through plant propagation. |
Carborundum | Fisher | C192-500 | Â |
Pro-mix BX potting soil | Premier Horticulture | 713400 | Â |
Jack's Professional 20-10-20 Peat-Lite Fertilizer | JR Peters | 77860 | Â |
 |  |  | Equipment |
50.2 Ti rotor | Beckman | 337901 | Â |
SW 32 Ti rotor | Beckman | 369694 | Â |
Optima L-90K ultracentrifuge | Beckman | 365672 | Â |
SLA-3000 rotor | Thermo Scientific | 07149 | Â |
SS-34 rotor | Thermo Scientific | 28020 | Â |
Sorvall RC-6 Plus centrifuge | Thermo Scientific | 46910 | Â |
Polypropylene bottle | Beckman | 355607 | For SLA-3000 rotor |
Polycarbonate bottle | Beckman | 357002 | For SS-34 rotor |
Ultra-Clear tube | Beckman | 344058 | For sucrose gradient and SW 32 Ti rotor |
Polycarbonate bottle | Beckman | 355618 | For pelleting and 50.2 Ti rotor |
NanoDrop spectrophotometer | Thermo Scientific | NanoDrop2000c | Â |
PowerEase 500 pre-cast gel system | Invitrogen | EI8675EU | Â |
Superose 6 10/300 GL (24 ml) size-exclusion column | GE Healthcare | 17-5172-01 | Â |
ÄKTA Explorer 100 Chromatograph | GE Healthcare | 28-4062-66 |  |
Allegra X-12R | Beckman | 392302 | Benchtop centrifuge |
Cryostat | Leica | CM1850 | Â |
Maestro 2 | Caliper Life Sciences | Â | In vivo imaging system |
Tissue-Tearor | Biospec Products | 985370-395 | Â |
Microplate reader | Tecan | Infinite-200 | Â |
Transmission electron microscope | ZEISS | Libra 200FE | Â |
FluoView laser scanning confocal microscope | Olympus | FV1000 | Â |
 |  |  | Chemicals and Reagents |
3-ethynylaniline | Sigma Aldrich | 498289-5G | Â |
384 well black plate | BD Biosciences | 353285 | Â |
4-12% Bis-Tris NuPAGE SDS gel | Invitrogen | NP0321BOX | Â |
4X LDS sample buffer | Invitrogen | NP0008 | Â |
Acetic Acid | Fisher | A385-500 | Â |
Acetonitrile | Sigma Aldrich | 271004-1L | Â |
Alexa Fluor 647 azide | Invitrogen | A10277 | Â |
Alexa Fluor 647 carboxylic acid, succinimidyl ester | Invitrogen | A20006 | Â |
Amicon Ultra-0.5 ml Centrifugal Filters | Millipore | UFC501096 | 10 kDa cut-off |
Aminoguanidine hydrochloride | Acros Organics | 36891-0250 | Â |
Boric acid | Fisher | A74-500 | Â |
Coomassie Brilliant Blue R-250 | Fisher | BP101-25 | Â |
CsCl | Acros Organics | 42285-1000 | Â |
DAPI | MP Biomedicals | 157574 | Â |
Dimethyl sulfoxide | Fisher | BP231-100 | Â |
Filter paper | Fisher | 09-801K | P5 grade |
FITC anti-mouse CD31 | BioLegend | 102406 | Â |
Goat serum | Invitrogen | 16210-064 | Â |
KCl | Fisher | BP366-500 | Â |
L-ascorbic acid sodium salt | Acros Organics | 35268-0050 | Â |
Methanol | Fisher | A412P-4 | Â |
MgCl2 | Fisher | BP214-500 | Â |
Microscope slides | Fisher | 12-544-3 | Â |
Microscope cover glass | VWR | 48366-277 | Â |
MOPS buffer | Invitrogen | NP0001 | Â |
mPEG-mal | Nanocs | PG1-ML-2k | MW 2000 |
mPEG-N3 | Nanocs | PG1-AZ-5k | MW 5000 |
mPEG-NHS | Nanocs | PG1-SC-5k | MW 5000 |
NaCl | Fisher | BP358-212 | Â |
Oregon Green 488 succinimidyl ester *6-isomer* | Invitrogen | O-6149 | Â |
p-toluenesulfonic acid monohydrate | Acros Organics | 13902-0050 | Â |
Permount | Fisher | SP15-100 | Â |
Potassium phosphate dibasic | Fisher | BP363-1 | Â |
Potassium phosphate monobasic | Fisher | BP362-1 | Â |
Sodium acetate | Fisher | BP333-500 | Â |
Sodium nitrite | Acros Organics | 42435-0050 | Â |
Sodium sulfite | Amresco | 0628-500G | Â |
Sucrose | Fisher | S6-500 | Â |
TEM grid | Ted Pella | FCF-400Cu | Â |
Tris base | Fisher | BP152-500 | Â |
Triton X-100 | EMD Chemicals | TX1568-1 | Â |
β-mercaptoethanol | Fisher | O3446I-100 |  |
 |  |  | Tissue Culture |
Fetal bovine serum | Invitrogen | 12483-020 | Â |
Hemocytometer | Fisher | 0267110 | Â |
HT-29 cells | ATCC | HTB-38 | Â |
L-glutamine | Invitrogen | 25030-080 | Â |
PBS | Cellgro | 21-040-CV | Â |
Penicillin-streptomycin | Invitrogen | 10378-016 | Â |
RPMI-1640 | Invitrogen | 31800-089 | Â |
Tissue culture flasks | Corning | 431080 | 175 cm2 |
Trypan Blue | Thermo Scientific | SV30084.01 | Â |
Trypsin, 0.05% (1X) with EDTA 4Na, liquid | Invitrogen | 25300-054 | Â |
 |  |  | Animal Studies |
18% Protein Rodent Diet | Harlan Teklad | Teklad Global 2018S | Alfalfa free diet |
Insulin syringe | BD Biosciences | 329410 | 28 gauge |
Isoflurane | Baxter | AHN3637 | Â |
Matrigel Matrix basement membrane | BD Biosciences | 356234 | Â |
NCR nu/nu mice | Â | Â | CWRU School of Medicine Athymic Animal and Xenograft Core Facility |
Sterile syringe | BD Biosciences | 305196 | 18 1/2 gauge |
Tissue-Tek CRYO-OCT Compound | Andwin Scientific | 4583 | Â |
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