1Department of Biomedical Engineering, Case Western Reserve University, 2Department of Biomedical Engineering, Radiology, and Materials Science and Engineering, Case Western Reserve University
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Wen, A. M., Lee, K. L., Yildiz, I., Bruckman, M. A., Shukla, S., Steinmetz, N. F. Viral Nanoparticles for In vivo Tumor Imaging. J. Vis. Exp. (69), e4352, doi:10.3791/4352 (2012).
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: Plant ~30 N. benthamiana seeds/pot. Fertilize once a week with 1 tablespoon fertilizer/5 L water.|
|Day 14: Re-pot N. benthamiana at 1 plant/pot.|
|Day 10: Infect leaves primary leaves with CPMV (5 μg/50 μl/leaf) by mechanical inoculation using a light dusting of carborundum.||Day 28: Infect three to five leaves with PVX, TMV, or BMV (5 μg/50 μl/leaf) by mechanical inoculation using a light dusting of carborundum.|
|Day 20: Harvest leaves and store in -80 °C.||Day 42: Harvest leaves and store in -80 °C.|
Table 1. Timeline for growing, infecting, and harvesting leaves.
Note: only CPMV propagation is demonstrated as an example.
2. VNP (CPMV, BMV, PVX, and TMV) Purification
Note: All steps are carried out on ice or at 4 °C.
|CPMV and TMV||0.1 M potassium phosphate buffer (pH 7.0)
38.5 mM KH2PO4
61.5 mM K2HPO4
|PVX||0.5 M borate buffer (pH 7.8)
0.5 M boric acid
Adjust pH with NaOH
|BMV||SAMA buffer (pH 4.5)
250 mM sodium acetate
10 mM MgCl2
2 mM β-mercaptoethanol (add fresh)
Table 2. Buffers and their recipes for each VNP.
Note: only CPMV propagation is demonstrated as an example.
3. VNP (CPMV, BMV, PVX, and TMV) Characterization
4. Chemical Conjugation of VNPs with PEG and Fluorophores, Purification, and Characterization
5. Tumor Targeting and Imaging using a Mouse Xenograft Model
Note: Tissue culture experiments and studies with live animals will not be demonstrated. Hands-on demonstration will be limited to tissue processing and data acquisition. For a reference on the HT-29 tumor xenograft model, the reader is referred to ref. 19
Three techniques are used to evaluate tumor homing of VNPs:
Note: This procedure will not be demonstrated, representative data are shown in Figure 8. For a reference on immunohistochemistry and the described staining methods, the reader is referred to ref. 19
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.
Figure 2. UV/visible spectra of CPMV, PVX, TMV, and BMV. Absorbance at 260 nm is proportional to the nucleic acid concentration, and absorbance at 280 nm is proportional to the protein concentration. The A260:A280 ratio can be used to assess purity of VNP preparations. The characteristic ratio for CPMV is 1.8, for PVX is 1.2, for BMV is 1.7, and for TMV is 1.1.
Figure 3. FPLC of CPMV, PVX, TMV, and BMV. Size-exclusion elution profiles of VNPs were obtained by using a Superose 6 column and Äkta purifier. Blue = A260 nm; red = A280 nm. As with the UV/visible spectra, the A260:A280 ratio can be used to assess purity of VNP preparations. In addition, the elution profile can be used to determine purity of the particles. CPMV typically elutes from the column at 9-10 ml and BMV around 8 ml. Due to their large size, PVX and TMV elute at the void volume of the column (approximately 7 ml). Impurities or free coat proteins would result in peaks at higher elution volumes.
Figure 4. SDS gels of CPMV, PVX, TMV, and BMV after Coomassie staining. SDS gel electrophoresis is used to separate denatured coat protein subunits. CPMV particles are formed by two proteins, a large protein (42 kDa) and a small protein (24 kDa); the small protein has two electrophoretic forms. The PVX coat protein is 25 kDa in size, and TMV consists of a 17 kDa protein. The BMV coat protein is 17 kDa in size. M = SeeBlue Plus2 protein marker.
Figure 5. TEM images of virus particles. TEM grids were negatively stained with 2% uranyl acetate, so the particles appear light on a dark background. Images are of 30 nm-sized CPMV (A), 515x13 nm-sized PVX (B), 30 nm-sized BMV (C), and 300x18 nm-sized TMV (D).
Figure 6. Reaction scheme for conjugation to VNPs. Three bioconjugation methods are used to conjugate PEG and dyes to the VNPs. CPMV, BMV, and PVX have lysines that can be used to react with functionalized NHS esters.20 BMV cysteine mutants also have reactive thiols that can be chemically modified using a maleimide group.18 Lastly, the tyrosines of TMV can be functionalized through a two-step reaction, where an alkyne handle is first installed via diazonium coupling and then CuAAC is performed with an azide-modified fluorophore or PEG.16
Figure 7. Representative data of fluorescently-labeled and PEGylated VNPs. UV/visible spectroscopy of CPMV-A647-PEG5000 (A). The absorbance peak at 650 nm is indicative of conjugated A647. The concentration of CPMV and A647 can be calculated using the Beer-Lambert law, from which the number of dyes per particle can be determined (about 60). FPLC of fluorescently-labeled PVX (here Oregon Green 488) (B). Black = A260 nm; red = A280 nm; green = A496 nm. Co-elution of PVX and dye confirmed covalent attachment. SDS gel of cBMV and cBMV-PEG2000 (C). PEG was conjugated to the cBMV cysteines. PEGylated CPs have lower mobility due to higher molecular weight, so the upper band is the conjugated CPs. ImageJ software and band analysis tool was used to quantify the number of PEG chains per cBMV. Approximately 40% (i.e., 70 CPs per cBMV) of the CPs were labeled with PEG. Negatively-stained TEM of TMV-A647 (D). The majority of particles are shown to be intact after modification.
Figure 8. Biodistribution of CPMV in a mouse xenograft tumor model. Nude mice were implanted with human HT-29 xenografts. Biodistribution and tumor homing was visualized using Maestro animal imaging of tissues ex vivo (A). No fluorescence was observed for the PBS control, while for CPMV conjugated with A647 and PEG, there was prominent fluorescence in the liver and some in the spleen due to clearing by the RES. There was also fluorescence seen in the tumor, indicating tumor homing via the EPR effect. Quantitative data were obtained via fluorescence measurement of homogenized tissues (normalized by tissue weight) using a plate reader assay (B). As expected, the majority of the CPMV was found in the liver and spleen, but there was also a trace in the tumor. Intratumoral localization was studied using cryo-sectioned tissues and immunostaining (C). Red = fluorescently-labeled CPMV-PEG; green = CD31 marker for endothelial cells; blue = DAPI for cell nuclei. The CPMV was mainly found near the vasculature of the tumor. 20
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 evaluating localization and biodistribution of the VNPs.
Before these studies can be performed however, it is essential to obtain a pure virus preparation. Apart from mechanical inoculation, a possible alternative for VNP propagation is agroinfiltration, which can have a high transformation and accumulation rate. When propagating the VNPs, care should be taken to keep plants infected with different viruses separate as to avoid cross-contamination. In particular, TMV is readily spread and transmitted mechanically. Another critical step to be mindful of is carrying out virus purification on ice or at 4 °C. All buffers should be used ice cold. The lower temperature is necessary to prevent any damage to the VNPs as a result of proteases and oxidizing enzymes present in the plant material.
Low yields of purified VNPs may arise from multiple factors. A possible cause could be the age of the VNPs used for propagation. It is preferable to use more recent virus preparations. For example, the C-terminal 24 amino acids of the S coat protein of CPMV are necessary for the suppression of gene silencing. Deletion of this surface-exposed region occurs over time due to proteolysis. Although the structure and stability of CPMV are not affected, growth retardation and delayed spread of the virus results. Another source of low yields is loss of the VNPs during purification. Particular attention should be given to the resuspension step after PEG precipitation. Incomplete resuspension of the pellet would result in VNP lost in the pellet of the subsequent centrifugation step.
Overall, the bioconjugation chemistries presented here are straightforward.14,17,18,21 The characterization methods described are valuable for ensuring particle integrity and determining extent of conjugation. Molar excesses can be adjusted accordingly depending on the application and the degree of functionalization desired.
When working with the mouse xenograft model, the most essential practice is following aseptic technique. In addition, the tail vein injection is a critical step to ensure a uniform sample dose for each mouse. It may help to place the tail in warm water beforehand to dilate the vein. Another consideration is autofluorescence for fluorescence imaging and measurements. Generally, tissue autofluorescence is minimized beyond 600 nm, making long wavelength dyes with emission maxima beyond 600 nm optimal as the imaging probe. However, normal mouse diets consist of chlorophyll-containing alfalfa, which also fluoresces red. As a result, it is necessary to maintain the mice on an alfalfa free diet for at least 2 weeks to reduce background signals. Finally, it should be noted that fluorescence detection using fluorescence imaging is limited due to differences in the scattering and absorption environments of the various tissues. Consequently, imaging is used for visualization and as an initial method for monitoring biodistribution, while quantification is performed using tissue homogenates.
Some advantages of using VNPs as the platform technology are their monodispersity, biocompatibility, capability for scale-up production, and amenability to multiple functionalization strategies. In addition to chemical engineering, genetic engineering is illustrated here with the introduction of cysteines to BMV as a target for bioconjugation. Genetic engineering could also be used to introduce targeting peptides or affinity tags. While the protocol described utilizes dual-labeled (dye and PEG) particles, ongoing studies are looking at incorporating additional functionalities (i.e., therapeutics and targeting) to enhance tissue specificity and therapeutic efficacy. Thus, this approach lays the groundwork for future biomedical applications.
No conflicts of interest declared.
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.
|Indoor plant chamber||Percival Scientific||E-41L2|
|V. unguiculata seeds (California black-eye no. 5)||Burpee||51771A|
|N. benthamiana seeds||N. benthamiana seeds were a gift from Salk Institute. Seeds are produced through plant propagation.|
|Pro-mix BX potting soil||Premier Horticulture||713400|
|Jack’s Professional 20-10-20 Peat-Lite Fertilizer||JR Peters||77860|
|50.2 Ti rotor||Beckman||337901|
|SW 32 Ti rotor||Beckman||369694|
|Optima L-90K ultracentrifuge||Beckman||365672|
|SLA-3000 rotor||Thermo Scientific||07149|
|SS-34 rotor||Thermo Scientific||28020|
|Sorvall RC-6 Plus centrifuge||Thermo Scientific||46910|
|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|
|PowerEase 500 pre-cast gel system||Invitrogen||EI8675EU|
|Superose 6 10/300 GL (24 ml) size-exclusion column||GE Healthcare||17-5172-01|
|ÄKTA Explorer 100 Chromatograph||GE Healthcare||28-4062-66|
|Allegra X-12R||Beckman||392302||Benchtop centrifuge|
|Maestro 2||Caliper Life Sciences||In vivo imaging system|
|Transmission electron microscope||ZEISS||Libra 200FE|
|FluoView laser scanning confocal microscope||Olympus||FV1000|
|Chemicals and Reagents|
|384 well black plate||BD Biosciences||353285|
|4-12% Bis-Tris NuPAGE SDS gel||Invitrogen||NP0321BOX|
|4X LDS sample buffer||Invitrogen||NP0008|
|Alexa Fluor 647 azide||Invitrogen||A10277|
|Alexa Fluor 647 carboxylic acid, succinimidyl ester||Invitrogen||A20006|
|Amicon Ultra-0.5 ml Centrifugal Filters||Millipore||UFC501096||10 kDa cut-off|
|Aminoguanidine hydrochloride||Acros Organics||36891-0250|
|Coomassie Brilliant Blue R-250||Fisher||BP101-25|
|Filter paper||Fisher||09-801K||P5 grade|
|FITC anti-mouse CD31||BioLegend||102406|
|L-ascorbic acid sodium salt||Acros Organics||35268-0050|
|Microscope cover glass||VWR||48366-277|
|Oregon Green 488 succinimidyl ester *6-isomer*||Invitrogen||O-6149|
|p-toluenesulfonic acid monohydrate||Acros Organics||13902-0050|
|Potassium phosphate dibasic||Fisher||BP363-1|
|Potassium phosphate monobasic||Fisher||BP362-1|
|Sodium nitrite||Acros Organics||42435-0050|
|TEM grid||Ted Pella||FCF-400Cu|
|Triton X-100||EMD Chemicals||TX1568-1|
|Fetal bovine serum||Invitrogen||12483-020|
|Tissue culture flasks||Corning||431080||175 cm2|
|Trypan Blue||Thermo Scientific||SV30084.01|
|Trypsin, 0.05% (1X) with EDTA 4Na, liquid||Invitrogen||25300-054|
|18% Protein Rodent Diet||Harlan Teklad||Teklad Global 2018S||Alfalfa free diet|
|Insulin syringe||BD Biosciences||329410||28 gauge|
|Matrigel Matrix basement membrane||BD Biosciences||356234|
|NCR nu/nu mice||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|