Viral Nanoparticles for In vivo Tumor Imaging

Summary

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.

Abstract

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.

Protocol

1. VNP (CPMV, BMV, PVX, and TMV) Propagation

1. Set the indoor plant chamber controls to 15 hr of day (100% light, 25 °C, 65% humidity) and 9 hr of night (0% light, 22 °C, 60% humidity).
2. Inoculate plants according to the timeline in Table 1.

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.

1. Homogenize 100 g of frozen leaves in a standard blender using 2 volumes of cold buffer (see Table 2). Filter through 2-3 layers of cheesecloth.
2. For PVX, adjust pH to 6.5 using 1 M HCl. Add 0.2% (w/v) ascorbic acid and 0.2% (w/v) sodium sulfite.
3. Centrifuge crude plant homogenate at 5,500 x g for 20 min. Collect supernatant.
4. For BMV, layer 25 ml of supernatant over 5 ml of 10% (w/v) sucrose solution. Centrifuge at 9,000 x g for 3 hr and resuspend pellets in 38.5% CsCl solution (w/v). Mix by shaking for 5 hr, then continue with step 2.12.
5. Extract plant material by adding 0.7 volumes of 1:1 (v/v) chloroform:1-butanol. Stir mixture for 30-60 min.
6. Centrifuge solution at 5,500 x g for 20 min. Collect the upper aqueous phase.
7. Add NaCl to 0.2 M and 8% (w/v) PEG (MW 8,000). For TMV, also add 1% (v/v) Triton X-100. Stir for at least 1 hr, then let sit for at least 1 hr.
8. Centrifuge solution at 15,000 x g for 15 min. Resuspend pellet in 10 ml of buffer. For PVX, add 0.1% β-mercaptoethanol and urea to 0.5 M.
9. Centrifuge at 8,000 x g for 30 min and collect supernatant.
10. Ultracentrifuge supernatant at 160,000 x g for 3 hr. Resuspend pellet in 5 ml buffer overnight.
11. Prepare a 10-40% sucrose gradient using equal volumes of 10%, 20%, 30%, and 40% sucrose in buffer (heaviest first). Allow the gradient to equilibrate overnight at room temperature.
12. Ultracentrifuge resuspended pellet over sucrose gradient at 100,000 x g for 2 hr (24 hr for BMV).
13. Collect light scattering band and dialyze against buffer.
14. Characterize the VNPs (below) and store at 4 °C. For long-term storage, store at -80 °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

1. Perform UV/visible spectroscopy to determine the concentration of VNPs.
   1. Measure the absorbance of 2 μl of sample using a NanoDrop spectrophotometer.
   2. Determine the concentration of particles and dyes using the Beer-Lambert law (A=εcl, where A is the absorbance, ε is the extinction coefficient, c is the concentration, and l is the path length). The path length is 0.1 cm for the NanoDrop.
      The VNP-specific extinction coefficients are:
      CPMV: 8.1 cm^-1mg^-1ml (at 260 nm)
      PVX: 2.97 cm^-1mg^-1ml (at 260 nm)
      TMV: 3.0 cm^-1mg^-1ml (at 260 nm)
      BMV: 5.15 cm^-1mg^-1ml (at 260 nm)
2. Analyze particles by size-exclusion fast protein liquid chromatography (FPLC).
   1. Using a Superose 6 size-exclusion column and the ÄKTA Explorer, load 50-100 μg of VNPs in 200 μl of 0.1 M potassium phosphate buffer (pH 7.0).
   2. Set detectors to 260 nm (nucleic acid), 280 nm (protein), and the excitation wavelength of any dyes attached.
   3. Run at a flow rate of 0.5 ml/min for 72 min.
   4. The elution profile and A260:A280 nm indicates whether the VNP preparation is pure and whether particles are intact and assembled.
      The following A260:280 ratios indicate a pure VNP preparation:
      CPMV: 1.8±0.1
      PVX: 1.2±0.1
      TMV: 1.1±0.1
      BMV: 1.7±0.1
3. Perform denaturing (pre-cast NuPAGE) Bis-Tris polyacrylamide 4-12% gradient gel electrophoresis to analyze purity of preparation and conjugation to individual coat proteins.
   1. Add 3 ml of 4x LDS sample buffer to 10 μg of the particles in 9 μl of potassium phosphate buffer. Add an additional 1 μl of 4x LDS sample buffer and 3 μl of β-mercaptoethanol to BMV to reduce the high number of disulfide bonds.
   2. Incubate in heat block for 5 min at 100 °C.
   3. Load samples onto an SDS gel.
   4. Run samples at 200 V for 1 hr in 1x MOPS running buffer.
   5. Document the gel under UV light to visualize fluorescent coat proteins.
   6. For non-fluorescent protein, stain with Coomassie blue (0.25% (w/v) Coomassie Brilliant Blue R-250, 30% (v/v) methanol, 10% (v/v) acetic acid) for 1 hr.
   7. Destain with 30% methanol, 10% acetic acid overnight. Change the solution if required.
   8. Document the gel under white light.
4. Analyze integrity of particles by transmission electron microscopy (TEM).
   1. Dilute samples to 0.1-1 mg/ml in 20 μl of DI water.
   2. Place 20 μl drops of the samples on Parafilm.
   3. Cover drops with a TEM grid and let sit for 2 min. Wick off the excess solution on the grid with filter paper.
   4. Wash grid by placing on a drop of DI water then wicking dry.
   5. Stain grid by placing on a 20 μl drop of 2% (w/v) uranyl acetate for 2 min. Wick off the excess stain with filter paper.
   6. Wash grid once more in water.
   7. Observe grid under a transmission electron microscope.

4. Chemical Conjugation of VNPs with PEG and Fluorophores, Purification, and Characterization

1. For calculations for the reactions below, the molar mass of the VNPs are:
   CPMV: 5.6 x 10⁶ g/mol
   PVX: 35 x 10⁶ g/mol
   TMV: 41 x 10⁶ g/mol
   BMV: 4.6 x 10⁶ g/mol
2. Conjugate dyes and PEG to surface lysines of CPMV and PVX using a one-step N-hydroxy succinimide coupling reaction: Add 2,500 molar equivalents (all molar excesses refer to molar excess per VNP) of Alexa Fluor 647 succinimidyl ester and 4,500 equivalents of NHS-PEG (M.W. 5,000) dissolved in DMSO to CPMV in 0.1 M potassium phosphate buffer. When working with PVX, add a 10,000 molar excess of NHS-dye and NHS-PEG. Adjust the buffer and DMSO volumes such that the final concentration of CPMV and PVX is 2 mg/ml and DMSO content is 10% of the total reaction volume. Incubate the reaction mixture overnight at room temperature protected from light. CPMV and PVX have 300 and 1,270 addressable lysines, respectively. (The reader is referred to the following references for further reading on chemical modification of CPMV and PVX: ^13-15).
3. Conjugate dyes and PEG to tyrosines of TMV by diazonium coupling followed by copper(I)-catalyzed azide-alkyne cycloaddition.
   1. Prepare diazonium salt (alkyne) by mixing 400 μl of 0.3 M p-toluenesulfonic acid monohydrate, 25 μl of 3.0 M sodium nitrite, and 75 μl of 0.68 M distilled 3-ethynylaniline dissolved in acetonitrile at 4 °C for 1 hr.
   2. Add 3.3 ml of borate buffer, pH 8.8, containing 100 mM NaCl to 1.25 ml of TMV (20 mg/ml stock solution).
   3. React the TMV with 450 μl of the diazonium salt (alkyne) solution in an ice bath for 3 hr to add an alkyne ligation handle to TMV by diazonium coupling. The solution will turn into a light brown color. TMV has 2,140 available tyrosines for conjugation.
   4. Purify the final product using a sucrose cushion as described in step 4.4.
   5. Attach azide-functional Alexa Fluor 647 and PEG-azide (M.W. 5,000) using copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). Add 2 equivalents of dye- and PEG-azide per coat protein and incubate with 1 mM CuSO₄, 2 mM AMG, and 2 mM sodium ascorbate at room temperature for 15 min. Adjust the buffer volume such that the final reaction concentration of TMV is 2 mg/ml. (The reader is referred to the following references for further reading on chemical modification of TMV: ^16,17).
4. Conjugate dyes to lysines and PEG to cysteines of BMV cysteine mutant (cBMV):
   1. Add 2,000 molar equivalents of Oregon Green 488 succinimidyl ester dissolved in DMSO to cBMV in 0.1 M TNKM buffer (50 mM Tris base, 50 mM NaCl, 10 mM KCl, 5 mM MgCl₂, pH 7.4). Adjust the buffer and DMSO volumes such that the final concentration of BMV is 1 mg/ml and DMSO content is 10% of the total reaction volume. Incubate the reaction mixture overnight at 4 °C protected from light.
   2. Purify particles using centrifugal filters as described in step 4.4.
   3. Add 2,000 molar excess of PEG-maleimide (M.W. 2,000) using the same reaction conditions as before and incubate the reaction mixture for 2 hr at 4 °C. cBMV has 180 reactive lysines and cysteines. (The reader is referred to the following references for further reading on chemical modification of BMV:¹⁸).
5. Purification: Pass the solution through a 40% (w/v) sucrose cushion at 160,000 x g for 2.5 hr. Re-dissolve the pellet in buffer. Alternatively, dialyze against appropriate buffer using 10 kDa cut-off spin filters.
6. Characterization: PEGylated and fluorescently-labeled VNPs are analyzed using the above-described methods: UV/visible spectroscopy, SDS gel electrophoresis, FPLC, and TEM (not shown, however, refer to Figures 6 and 7) .

5. Tumor Targeting and Imaging using a Mouse Xenograft Model

1. Culture HT-29 human colon cancer cells in RPMI medium supplemented with 5% FBS, 1% penicillin-streptomycin, and 1% L-glutamine at 37 °C in 5% CO₂ using 175 cm² cell culture flasks.
2. Wash cells twice with sterile PBS and harvest by incubating with 5 ml of trypsin-EDTA at 37 °C for 5 min. Inactivate the trypsin with 5 ml of RPMI medium. Collect cells by centrifuging at 500 x g for 5 min. at 4 °C and resuspend in fresh RPMI at 5x10⁶ cells/50 μl medium (determine total cell count using trypan blue and a hemocytometer). Mix with an equal volume of matrigel prior to injection (keep all solutions and reagents sterile).
3. Procure six-week old NCR nu/nu mice and maintain them on an alfalfa free diet for 2 weeks. [Note: all animal procedures must be IACUC approved.] Induce tumor xenografts by subcutaneous injection of 5x10⁶ cells/100 μl/tumor in the flanks (2 tumors/mouse) using an 18 1/2 gauge sterile needle. Monitor the animals regularly. Measure the tumor size using calipers and allow the tumors to grow to an average volume of 20 mm³ (within the next 12 days). Assign mice to two different groups randomly: PBS and VNP (n = 3 animals/group/time point). Using a 1 ml 28 gauge insulin syringe, administer by intravenous tail vein injection 100 μl of sterile PBS or 10 mg/kg VNP formulation.

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:

4. Fluorescence imaging using Maestro Imaging System: Sacrifice mice at different time points (2, 24, and 72 hr) using CO₂ gas. Dissect the animals and excise all major organs (brain, heart, lungs, spleen, kidneys and liver) along with the tumors on the flanks, place the tissues on parafilm, and analyze with fluorescence imaging instrument using yellow excitation and emission filters (800 ms exposure) to detect the presence of fluorescent signals in the tissues (derived from A647 label conjugated to the VNPs). Save the images and analyze fluorescent intensities using ImageJ 1.44o software (http://imagej.nih.gov/ij). Compare the pattern of uptake of the VNPs in tumors with other major tissues with time.
5. After imaging, cut each tissue in half and embed one half in OCT compound for cryo-sectioning and confocal analysis. Collect the other half in pre-weighed cryo-vials and immediately freeze them using liquid N₂. Store at -80 °C until ready for further processing.
6. Fluorescence quantification: Record tissues weights. Thaw frozen tissues at room temperature and place them in separate 50 ml Falcon tubes containing 1 ml of PBS. Using a handheld tissue homogenizer, homogenize the tissues for 2-3 min in PBS then transfer the homogenate to microfuge tubes. Centrifuge the homogenates for 10 min at 13,000 x g to remove non-homogenized tissue.
7. Pipet 100 μl of the supernatant from the tissues from each group (PBS and VNP formulations/time points) into a 384 well black UV plate. Evaluate fluorescence intensity (Ex/Em wavelengths 600/665) using a plate reader. Normalize the obtained fluorescent values by the tissue weights.
8. Immunohistochemistry: Prepare cryo-microtome sections (10 μm) and store at -20 °C. Stain tissue sections for cell nuclei (DAPI) and endothelial cell marker (FITC-labeled anti-mouse CD31 antibody). Carry out confocal microscopy analysis to map the vascular and intra-tumoral localization of fluorescently-labeled 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

Results

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.

Discussion

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...

Materials

Name	Company	Catalog Number	Comments
Material Name	Company	Catalogue number	Comments (optional)
			VNP production
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.
Carborundum	Fisher	C192-500
Pro-mix BX potting soil	Premier Horticulture	713400
Jack's Professional 20-10-20 Peat-Lite Fertilizer	JR Peters	77860
			Equipment
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
Cryostat	Leica	CM1850
Maestro 2	Caliper Life Sciences		In vivo imaging system
Tissue-Tearor	Biospec Products	985370-395
Microplate reader	Tecan	Infinite-200
Transmission electron microscope	ZEISS	Libra 200FE
FluoView laser scanning confocal microscope	Olympus	FV1000
			Chemicals and Reagents
3-ethynylaniline	Sigma Aldrich	498289-5G
384 well black plate	BD Biosciences	353285
4-12% Bis-Tris NuPAGE SDS gel	Invitrogen	NP0321BOX
4X LDS sample buffer	Invitrogen	NP0008
Acetic Acid	Fisher	A385-500
Acetonitrile	Sigma Aldrich	271004-1L
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
Boric acid	Fisher	A74-500
Coomassie Brilliant Blue R-250	Fisher	BP101-25
CsCl	Acros Organics	42285-1000
DAPI	MP Biomedicals	157574
Dimethyl sulfoxide	Fisher	BP231-100
Filter paper	Fisher	09-801K	P5 grade
FITC anti-mouse CD31	BioLegend	102406
Goat serum	Invitrogen	16210-064
KCl	Fisher	BP366-500
L-ascorbic acid sodium salt	Acros Organics	35268-0050
Methanol	Fisher	A412P-4
MgCl2	Fisher	BP214-500
Microscope slides	Fisher	12-544-3
Microscope cover glass	VWR	48366-277
MOPS buffer	Invitrogen	NP0001
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
Oregon Green 488 succinimidyl ester *6-isomer*	Invitrogen	O-6149
p-toluenesulfonic acid monohydrate	Acros Organics	13902-0050
Permount	Fisher	SP15-100
Potassium phosphate dibasic	Fisher	BP363-1
Potassium phosphate monobasic	Fisher	BP362-1
Sodium acetate	Fisher	BP333-500
Sodium nitrite	Acros Organics	42435-0050
Sodium sulfite	Amresco	0628-500G
Sucrose	Fisher	S6-500
TEM grid	Ted Pella	FCF-400Cu
Tris base	Fisher	BP152-500
Triton X-100	EMD Chemicals	TX1568-1
β-mercaptoethanol	Fisher	O3446I-100
			Tissue Culture
Fetal bovine serum	Invitrogen	12483-020
Hemocytometer	Fisher	0267110
HT-29 cells	ATCC	HTB-38
L-glutamine	Invitrogen	25030-080
PBS	Cellgro	21-040-CV
Penicillin-streptomycin	Invitrogen	10378-016
RPMI-1640	Invitrogen	31800-089
Tissue culture flasks	Corning	431080	175 cm2
Trypan Blue	Thermo Scientific	SV30084.01
Trypsin, 0.05% (1X) with EDTA 4Na, liquid	Invitrogen	25300-054
			Animal Studies
18% Protein Rodent Diet	Harlan Teklad	Teklad Global 2018S	Alfalfa free diet
Insulin syringe	BD Biosciences	329410	28 gauge
Isoflurane	Baxter	AHN3637
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