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.

1. VNP (CPMV, BMV, PVX, and TMV) Propagation
<ol>
	<li>Set the indoor plant chamber controls to 15 hr of day (100% light, 25 &deg;C, 65% humidity) and 9 hr of night (0% light, 22 &deg;C, 60% humidity).</li>
	<li>Inoculate plants according to the timeline in Table 1.</li>
</ol>
<table border="1">
	<tbody>
		<tr>
			<td>CPMV</td>
			<td>PVX, TMV, and BMV</td>
		</tr>
		<tr>
			<td rowspan="2">Day 0: Plant 3 cowpea seeds/pot.</td>
			<td>Day 0: Plant ~30 N. benthamiana seeds/pot. Fertilize once a week with 1 tablespoon fertilizer/5 L water.</td>
		</tr>
		<tr>
			<td>Day 14: Re-pot N. benthamiana at 1 plant/pot.</td>
		</tr>
		<tr>
			<td>Day 10: Infect leaves primary leaves with CPMV (5 &mu;g/50 &mu;l/leaf) by mechanical inoculation using a light dusting of carborundum.</td>
			<td>Day 28: Infect three to five leaves with PVX, TMV, or BMV (5 &mu;g/50 &mu;l/leaf) by mechanical inoculation using a light dusting of carborundum.</td>
		</tr>
		<tr>
			<td>Day 20: Harvest leaves and store in -80 &deg;C.</td>
			<td>Day 42: Harvest leaves and store in -80 &deg;C.</td>
		</tr>
	</tbody>
</table>
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 &deg;C.
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Centrifuge crude plant homogenate at 5,500 x g for 20 min. Collect supernatant.</li>
	<li>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.</li>
	<li>Extract plant material by adding 0.7 volumes of 1:1 (v/v) chloroform:1-butanol. Stir mixture for 30-60 min.</li>
	<li>Centrifuge solution at 5,500 x g for 20 min. Collect the upper aqueous phase.</li>
	<li>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.</li>
	<li>Centrifuge solution at 15,000 x g for 15 min. Resuspend pellet in 10 ml of buffer. For PVX, add 0.1% &beta;-mercaptoethanol and urea to 0.5 M.</li>
	<li>Centrifuge at 8,000 x g for 30 min and collect supernatant.</li>
	<li>Ultracentrifuge supernatant at 160,000 x g for 3 hr. Resuspend pellet in 5 ml buffer overnight.</li>
	<li>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.</li>
	<li>Ultracentrifuge resuspended pellet over sucrose gradient at 100,000 x g for 2 hr (24 hr for BMV).</li>
	<li>Collect light scattering band and dialyze against buffer.</li>
	<li>Characterize the VNPs (below) and store at 4 &deg;C. For long-term storage, store at -80 &deg;C.</li>
</ol>
<table border="1">
	<tbody>
		<tr>
			<td>CPMV and TMV</td>
			<td>0.1 M potassium phosphate buffer (pH 7.0) 
			38.5 mM KH2PO4 
			61.5 mM K2HPO4</td>
		</tr>
		<tr>
			<td>PVX</td>
			<td>0.5 M borate buffer (pH 7.8) 
			0.5 M boric acid 
			Adjust pH with NaOH</td>
		</tr>
		<tr>
			<td>BMV</td>
			<td>SAMA buffer (pH 4.5) 
			250 mM sodium acetate 
			10 mM MgCl2 
			2 mM &beta;-mercaptoethanol (add fresh)</td>
		</tr>
	</tbody>
</table>
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
<ol>
	<li>Perform UV/visible spectroscopy to determine the concentration of VNPs.
	<ol>
		<li>Measure the absorbance of 2 &mu;l of sample using a NanoDrop spectrophotometer.</li>
		<li>Determine the concentration of particles and dyes using the Beer-Lambert law (A=&epsilon;cl, where A is the absorbance, &epsilon; 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)</li>
	</ol>
	</li>
	<li>Analyze particles by size-exclusion fast protein liquid chromatography (FPLC).
	<ol>
		<li>Using a Superose 6 size-exclusion column and the &Auml;KTA Explorer, load 50-100 &mu;g of VNPs in 200 &mu;l of 0.1 M potassium phosphate buffer (pH 7.0).</li>
		<li>Set detectors to 260 nm (nucleic acid), 280 nm (protein), and the excitation wavelength of any dyes attached.</li>
		<li>Run at a flow rate of 0.5 ml/min for 72 min.</li>
		<li>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&plusmn;0.1 
		PVX: 1.2&plusmn;0.1 
		TMV: 1.1&plusmn;0.1 
		BMV: 1.7&plusmn;0.1</li>
	</ol>
	</li>
	<li>Perform denaturing (pre-cast NuPAGE) Bis-Tris polyacrylamide 4-12% gradient gel electrophoresis to analyze purity of preparation and conjugation to individual coat proteins.
	<ol>
		<li>Add 3 ml of 4x LDS sample buffer to 10 &mu;g of the particles in 9 &mu;l of potassium phosphate buffer. Add an additional 1 &mu;l of 4x LDS sample buffer and 3 &mu;l of &beta;-mercaptoethanol to BMV to reduce the high number of disulfide bonds.</li>
		<li>Incubate in heat block for 5 min at 100 &deg;C.</li>
		<li>Load samples onto an SDS gel.</li>
		<li>Run samples at 200 V for 1 hr in 1x MOPS running buffer.</li>
		<li>Document the gel under UV light to visualize fluorescent coat proteins.</li>
		<li>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.</li>
		<li>Destain with 30% methanol, 10% acetic acid overnight. Change the solution if required.</li>
		<li>Document the gel under white light.</li>
	</ol>
	</li>
	<li>Analyze integrity of particles by transmission electron microscopy (TEM).
	<ol>
		<li>Dilute samples to 0.1-1 mg/ml in 20 &mu;l of DI water.</li>
		<li>Place 20 &mu;l drops of the samples on Parafilm.</li>
		<li>Cover drops with a TEM grid and let sit for 2 min. Wick off the excess solution on the grid with filter paper.</li>
		<li>Wash grid by placing on a drop of DI water then wicking dry.</li>
		<li>Stain grid by placing on a 20 &mu;l drop of 2% (w/v) uranyl acetate for 2 min. Wick off the excess stain with filter paper.</li>
		<li>Wash grid once more in water.</li>
		<li>Observe grid under a transmission electron microscope.</li>
	</ol>
	</li>
</ol>
4. Chemical Conjugation of VNPs with PEG and Fluorophores, Purification, and Characterization
<ol>
	<li>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</li>
	<li>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).</li>
	<li>Conjugate dyes and PEG to tyrosines of TMV by diazonium coupling followed by copper(I)-catalyzed azide-alkyne cycloaddition.
	<ol>
		<li>Prepare diazonium salt (alkyne) by mixing 400 &mu;l of 0.3 M p-toluenesulfonic acid monohydrate, 25 &mu;l of 3.0 M sodium nitrite, and 75 &mu;l of 0.68 M distilled 3-ethynylaniline dissolved in acetonitrile at 4 &deg;C for 1 hr.</li>
		<li>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).</li>
		<li>React the TMV with 450 &mu;l 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.</li>
		<li>Purify the final product using a sucrose cushion as described in step 4.4.</li>
		<li>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).</li>
	</ol>
	</li>
	<li>Conjugate dyes to lysines and PEG to cysteines of BMV cysteine mutant (cBMV):
	<ol>
		<li>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 &deg;C protected from light.</li>
		<li>Purify particles using centrifugal filters as described in step 4.4.</li>
		<li>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 &deg;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).</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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) .</li>
</ol>
5. Tumor Targeting and Imaging using a Mouse Xenograft Model
<ol>
	<li>Culture HT-29 human colon cancer cells in RPMI medium supplemented with 5% FBS, 1% penicillin-streptomycin, and 1% L-glutamine at 37 &deg;C in 5% CO2 using 175 cm2 cell culture flasks.</li>
	<li>Wash cells twice with sterile PBS and harvest by incubating with 5 ml of trypsin-EDTA at 37 &deg;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 &deg;C and resuspend in fresh RPMI at 5x106 cells/50 &mu;l 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).</li>
	<li>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 &mu;l/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 &mu;l of sterile PBS or 10 mg/kg VNP formulation.</li>
</ol>
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:
<ol start="4">
	<li>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 (<a href="http://imagej.nih.gov/ij" target="_blank">http://imagej.nih.gov/ij</a>). Compare the pattern of uptake of the VNPs in tumors with other major tissues with time.</li>
	<li>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 &deg;C until ready for further processing.</li>
	<li>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.</li>
	<li>Pipet 100 &mu;l 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.</li>
	<li>Immunohistochemistry: Prepare cryo-microtome sections (10 &mu;m) and store at -20 &deg;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.</li>
</ol>
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

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No conflicts of interest declared.

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...

<table border="1">
	<tbody>
		<tr>
			<td>Material Name</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>VNP production</td>
		</tr>
		<tr>
			<td>Indoor plant chamber</td>
			<td>Percival Scientific</td>
			<td>E-41L2</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>V. unguiculata seeds (California black-eye no. 5)</td>
			<td>Burpee</td>
			<td>51771A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>N. benthamiana seeds</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>N. benthamiana seeds were a gift from Salk Institute. Seeds are produced through plant propagation.</td>
		</tr>
		<tr>
			<td>Carborundum</td>
			<td>Fisher</td>
			<td>C192-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pro-mix BX potting soil</td>
			<td>Premier Horticulture</td>
			<td>713400</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Jack's Professional 20-10-20 Peat-Lite Fertilizer</td>
			<td>JR Peters</td>
			<td>77860</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>50.2 Ti rotor</td>
			<td>Beckman</td>
			<td>337901</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SW 32 Ti rotor</td>
			<td>Beckman</td>
			<td>369694</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Optima L-90K ultracentrifuge</td>
			<td>Beckman</td>
			<td>365672</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SLA-3000 rotor</td>
			<td>Thermo Scientific</td>
			<td>07149</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SS-34 rotor</td>
			<td>Thermo Scientific</td>
			<td>28020</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sorvall RC-6 Plus centrifuge</td>
			<td>Thermo Scientific</td>
			<td>46910</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Polypropylene bottle</td>
			<td>Beckman</td>
			<td>355607</td>
			<td>For SLA-3000 rotor</td>
		</tr>
		<tr>
			<td>Polycarbonate bottle</td>
			<td>Beckman</td>
			<td>357002</td>
			<td>For SS-34 rotor</td>
		</tr>
		<tr>
			<td>Ultra-Clear tube</td>
			<td>Beckman</td>
			<td>344058</td>
			<td>For sucrose gradient and SW 32 Ti rotor</td>
		</tr>
		<tr>
			<td>Polycarbonate bottle</td>
			<td>Beckman</td>
			<td>355618</td>
			<td>For pelleting and 50.2 Ti rotor</td>
		</tr>
		<tr>
			<td>NanoDrop spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>NanoDrop2000c</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PowerEase 500 pre-cast gel system</td>
			<td>Invitrogen</td>
			<td>EI8675EU</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Superose 6 10/300 GL (24 ml) size-exclusion column</td>
			<td>GE Healthcare</td>
			<td>17-5172-01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&Auml;KTA Explorer 100 Chromatograph</td>
			<td>GE Healthcare</td>
			<td>28-4062-66</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Allegra X-12R</td>
			<td>Beckman</td>
			<td>392302</td>
			<td>Benchtop centrifuge</td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica</td>
			<td>CM1850</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Maestro 2</td>
			<td>Caliper Life Sciences</td>
			<td>&nbsp;</td>
			<td>In vivo imaging system</td>
		</tr>
		<tr>
			<td>Tissue-Tearor</td>
			<td>Biospec Products</td>
			<td>985370-395</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>Tecan</td>
			<td>Infinite-200</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Transmission electron microscope</td>
			<td>ZEISS</td>
			<td>Libra 200FE</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FluoView laser scanning confocal microscope</td>
			<td>Olympus</td>
			<td>FV1000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Chemicals and Reagents</td>
		</tr>
		<tr>
			<td>3-ethynylaniline</td>
			<td>Sigma Aldrich</td>
			<td>498289-5G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>384 well black plate</td>
			<td>BD Biosciences</td>
			<td>353285</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>4-12% Bis-Tris NuPAGE SDS gel</td>
			<td>Invitrogen</td>
			<td>NP0321BOX</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>4X LDS sample buffer</td>
			<td>Invitrogen</td>
			<td>NP0008</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>Fisher</td>
			<td>A385-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Sigma Aldrich</td>
			<td>271004-1L</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 azide</td>
			<td>Invitrogen</td>
			<td>A10277</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 carboxylic acid, succinimidyl ester</td>
			<td>Invitrogen</td>
			<td>A20006</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Amicon Ultra-0.5 ml Centrifugal Filters</td>
			<td>Millipore</td>
			<td>UFC501096</td>
			<td>10 kDa cut-off</td>
		</tr>
		<tr>
			<td>Aminoguanidine hydrochloride</td>
			<td>Acros Organics</td>
			<td>36891-0250</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>Fisher</td>
			<td>A74-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Coomassie Brilliant Blue R-250</td>
			<td>Fisher</td>
			<td>BP101-25</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CsCl</td>
			<td>Acros Organics</td>
			<td>42285-1000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>MP Biomedicals</td>
			<td>157574</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Fisher</td>
			<td>BP231-100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Fisher</td>
			<td>09-801K</td>
			<td>P5 grade</td>
		</tr>
		<tr>
			<td>FITC anti-mouse CD31</td>
			<td>BioLegend</td>
			<td>102406</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Invitrogen</td>
			<td>16210-064</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Fisher</td>
			<td>BP366-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>L-ascorbic acid sodium salt</td>
			<td>Acros Organics</td>
			<td>35268-0050</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher</td>
			<td>A412P-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Fisher</td>
			<td>BP214-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Fisher</td>
			<td>12-544-3</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microscope cover glass</td>
			<td>VWR</td>
			<td>48366-277</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MOPS buffer</td>
			<td>Invitrogen</td>
			<td>NP0001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>mPEG-mal</td>
			<td>Nanocs</td>
			<td>PG1-ML-2k</td>
			<td>MW 2000</td>
		</tr>
		<tr>
			<td>mPEG-N3</td>
			<td>Nanocs</td>
			<td>PG1-AZ-5k</td>
			<td>MW 5000</td>
		</tr>
		<tr>
			<td>mPEG-NHS</td>
			<td>Nanocs</td>
			<td>PG1-SC-5k</td>
			<td>MW 5000</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher</td>
			<td>BP358-212</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Oregon Green 488 succinimidyl ester *6-isomer*</td>
			<td>Invitrogen</td>
			<td>O-6149</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>p-toluenesulfonic acid monohydrate</td>
			<td>Acros Organics</td>
			<td>13902-0050</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Permount</td>
			<td>Fisher</td>
			<td>SP15-100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Potassium phosphate dibasic</td>
			<td>Fisher</td>
			<td>BP363-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Fisher</td>
			<td>BP362-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Fisher</td>
			<td>BP333-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium nitrite</td>
			<td>Acros Organics</td>
			<td>42435-0050</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium sulfite</td>
			<td>Amresco</td>
			<td>0628-500G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher</td>
			<td>S6-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>TEM grid</td>
			<td>Ted Pella</td>
			<td>FCF-400Cu</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Fisher</td>
			<td>BP152-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>EMD Chemicals</td>
			<td>TX1568-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&beta;-mercaptoethanol</td>
			<td>Fisher</td>
			<td>O3446I-100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Tissue Culture</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Invitrogen</td>
			<td>12483-020</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Fisher</td>
			<td>0267110</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HT-29 cells</td>
			<td>ATCC</td>
			<td>HTB-38</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Invitrogen</td>
			<td>25030-080</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Cellgro</td>
			<td>21-040-CV</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Invitrogen</td>
			<td>10378-016</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>Invitrogen</td>
			<td>31800-089</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tissue culture flasks</td>
			<td>Corning</td>
			<td>431080</td>
			<td>175 cm2</td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Thermo Scientific</td>
			<td>SV30084.01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Trypsin, 0.05% (1X) with EDTA 4Na, liquid</td>
			<td>Invitrogen</td>
			<td>25300-054</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Animal Studies</td>
		</tr>
		<tr>
			<td>18% Protein Rodent Diet</td>
			<td>Harlan Teklad</td>
			<td>Teklad Global 2018S</td>
			<td>Alfalfa free diet</td>
		</tr>
		<tr>
			<td>Insulin syringe</td>
			<td>BD Biosciences</td>
			<td>329410</td>
			<td>28 gauge</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Baxter</td>
			<td>AHN3637</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Matrigel Matrix basement membrane</td>
			<td>BD Biosciences</td>
			<td>356234</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NCR nu/nu mice</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>CWRU School 
			of Medicine Athymic Animal and Xenograft Core Facility</td>
		</tr>
		<tr>
			<td>Sterile syringe</td>
			<td>BD Biosciences</td>
			<td>305196</td>
			<td>18 1/2 gauge</td>
		</tr>
		<tr>
			<td>Tissue-Tek CRYO-OCT Compound</td>
			<td>Andwin Scientific</td>
			<td>4583</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

viral nanoparticles for in vivo tumor 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.

<img alt="Figure 1" src="/files/ftp_upload/4352/4352fig1.jpg" /> 
 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.
<img alt="Figure 2" fo:content-width="3in" fo:src="/files/ftp_upload/4352...

Watch this Scientific Journal Video about Viral Nanoparticles for In vivo Tumor Imaging at JoVE.com

Viral Nanoparticles for In vivo Tumor Imaging

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.

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

viral-nanoparticles-for-in-vivo-tumor-imaging

Research

JoVE Journal

Bioengineering

17.1K Views.  Case Western Reserve University. 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.

Video: Viral Nanoparticles for In vivo Tumor Imaging

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques 1-3. Microfluidic devices have been used for sub-cellular imaging 4,5, in vivo laser microsurgery 2,6 and cellular imaging 4,7. In vivo imaging requires immobilization of organisms. This has been achieved using suction 5,8, tapered channels 6,7,9, deformable membranes 2-4,10, suction with additional cooling 5, anesthetic gas 11, temperature sensitive gels 12, cyanoacrylate glue 13 and anesthetics such as levamisole 14,15. Commonly used anesthetics influence synaptic transmission 16,17 and are known to have detrimental effects on sub-cellular neuronal transport 4. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from ~10 &mu;m to ~800 &mu;m for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min.
We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F 4).
Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in ~30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

Analysis of Targeted Viral Protein Nanoparticles Delivered to HER2+ Tumors

The HER2+ tumor-targeted nanoparticle, HerDox, exhibits tumor-preferential accumulation and tumor-growth ablation in an animal model of HER2+ cancer. HerDox is formed by non-covalent self-assembly of a tumor targeted cell penetration protein with the chemotherapy agent, doxorubicin, via a small nucleic acid linker. A combination of electrophilic, intercalation, and oligomerization interactions facilitate self-assembly into round 10-20 nm particles. HerDox exhibits stability in blood as well as in extended storage at different temperatures. Systemic delivery of HerDox in tumor-bearing mice results in tumor-cell death with no detectable adverse effects to non-tumor tissue, including the heart and liver (which undergo marked damage by untargeted doxorubicin). HER2 elevation facilitates targeting to cells expressing the human epidermal growth factor receptor, hence tumors displaying elevated HER2 levels exhibit greater accumulation of HerDox compared to cells expressing lower levels, both in vitro and in vivo. Fluorescence intensity imaging combined with in situ confocal and spectral analysis has allowed us to verify in vivo tumor targeting and tumor cell penetration of HerDox after systemic delivery. Here we detail our methods for assessing tumor targeting via multimode imaging after systemic delivery.

This article details the procedures for optical imaging analysis of the tumor-targeted nanoparticle, HerDox. In particular, detailed use of the multimode imaging device for detecting tumor targeting and assessing tumor penetration is described here.

The HER2+ tumor-targeted nanoparticle, HerDox, exhibits tumor-preferential accumulation and tumor-growth ablation in an animal model of HER2+ cancer. ...

PLGA Nanoparticles Formed by Single- or Double-emulsion with Vitamin E-TPGS

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular choice for drug delivery applications, particularly since it is already FDA-approved for use in humans in the form of resorbable sutures. Hydrophobic and hydrophilic drugs are encapsulated in PLGA particles via single- or double-emulsion. Briefly, the drug is dissolved with polymer or emulsified with polymer in an organic phase that is then emulsified with the aqueous phase. After the solvent has evaporated, particles are washed and collected via centrifugation for lyophilization and long term storage. PLGA degrades slowly via hydrolysis in aqueous environments, and encapsulated agents are released over a period of weeks to months. Although PLGA is a material that possesses many advantages for drug delivery, reproducible formation of nanoparticles can be challenging; considerable variability is introduced by the use of different equipment, reagents batch, and precise method of emulsification. Here, we describe in great detail the formation and characterization of microparticles and nanoparticles formed by single- or double-emulsion using the emulsifying agent vitamin E-TPGS. Particle morphology and size are determined with scanning electron microscopy (SEM). We provide representative SEM images for nanoparticles produced with varying emulsifier concentration, as well as examples of imaging artifacts and failed emulsifications. This protocol can be readily adapted to use alternative emulsifiers (e.g. poly(vinyl alcohol), PVA) or solvents (e.g.&#160;dichloromethane, DCM).

We describe the production and characterization of nanoparticles and microparticles composed of poly(lactic-co-glycolic acid) using vitamin E-TPGS as an emulsifier. By varying formulation parameters such as the concentration of emulsifier, it is possible to produce nanoparticles with mean diameters ranging from 220 nm to&#160;1.98 &#181;m.

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular ...

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an integral part of normal tissue renewal, excessive amount of remodeling activity is involved in tumors, arthritis, and many other pathological conditions. During collagen remodeling, the triple helical structure of collagen molecules is disrupted by proteases in the extracellular environment. In addition, collagens present in many histological tissue samples are partially denatured by the fixation and preservation processes. Therefore, these denatured collagen strands can serve as effective targets for biological imaging. We previously developed a caged collagen mimetic peptide (CMP) that can be photo-triggered to hybridize with denatured collagen strands by forming triple helical structure, which is unique to collagens. The overall goals of this procedure are i)&#160;to image denatured collagen strands resulting from normal remodeling activities in vivo, and ii) to visualize collagens in ex vivo tissue sections using the photo-triggered caged CMPs. To achieve effective hybridization and successful in vivo and ex vivo imaging, fluorescently labeled caged CMPs are either photo-activated immediately before intravenous injection, or are directly activated on tissue sections. Normal skeletal collagen remolding in nude mice and collagens in prefixed mouse cornea tissue sections are imaged in this procedure. The imaging method based on the CMP-collagen hybridization technology presented here could lead to deeper understanding of the tissue remodeling process, as well as allow development of new diagnostics for diseases associated with high collagen remodeling activity.

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

This procedure demonstrates in vivo near IR fluorescence imaging of collagen remodeling activities in mice as well as ex vivo staining of collagens in tissue sections using caged collagen mimetic peptides that can be photo-triggered to hybridize with denatured collagen strands.

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an ...

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, complementary imaging techniques. These imaging agents facilitate the real-time assessment of pathways and mechanisms in vivo, which enhance both diagnostic and therapeutic efficacy. This article presents the protocol for the synthesis of biofunctionalized Prussian blue nanoparticles (PB NPs) - a novel class of agents for use in multimodal, molecular imaging applications. The imaging modalities incorporated in the nanoparticles, fluorescence imaging and magnetic resonance imaging (MRI), have complementary features. The PB NPs possess a core-shell design where gadolinium and manganese ions incorporated within the interstitial spaces of the PB lattice generate MRI contrast, both in T1 and T2-weighted sequences. The PB NPs are coated with fluorescent avidin using electrostatic self-assembly, which enables fluorescence imaging. The avidin-coated nanoparticles are modified with biotinylated ligands that confer molecular targeting capabilities to the nanoparticles. The stability and toxicity of the nanoparticles are measured, as well as their MRI relaxivities. The multimodal, molecular imaging capabilities of these biofunctionalized PB NPs are then demonstrated by using them for fluorescence imaging and molecular MRI in vitro.

This protocol describes the synthesis of biofunctionalized Prussian blue nanoparticles and their use as multimodal, molecular imaging agents. The nanoparticles have a core-shell design where gadolinium or manganese ions within the nanoparticle core generate MRI contrast. The biofunctional shell contains fluorophores for fluorescence imaging and targeting ligands for molecular targeting.

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, ...

Microwave-driven Synthesis of Iron Oxide Nanoparticles for Fast Detection of Atherosclerosis

A fast and reproducible microwave-driven protocol has been developed for the synthesis of neridronate-functionalized nanoparticles. Starting from the synthesis of hydrophobic nanoparticles, our method is based on an adaptation from thermal decomposition method to microwave driven synthesis. The new methodology produces a decrease in the reaction times in comparison with traditional procedures. Moreover, the use of the microwave technology increases the reproducibility of the reactions, something important from the point of view of clinical applications. The novelty of this iron oxide nanoparticle is the attachment of Neridronate. The use of this molecule leads a bisphosphonate moiety towards the outside of the nanoparticle that provides Ca2+ binding properties in vitro and selective accumulation in vivo in the atheroma plaque. The protocol allows the synthesis and plaque detection in about 3 hr since the initial synthesis from organic precursors. Their accumulation in the atherosclerotic area in less than 1 hr provides a contrast agent particularly suitable for clinical applications.

Microwave technology enables extremely fast synthesis of iron oxide nanoparticles for atherosclerosis plaque characterization. The use of an aminobisphosphonate in the external side of the nanoparticle provides a fast accumulation in the atherosclerotic area.

A fast and reproducible microwave-driven protocol has been developed for the synthesis of neridronate-functionalized nanoparticles. Starting from the ...

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds during repair, which gives rise to a lack of sufficient blood supply and causes necrosis of the new tissues. Rapid vascularization is a vital prerequisite for new tissue survival and integration with existing host tissue. The de novo generation of vasculature in scaffolds is one of the most important steps in making bone regeneration more efficient, allowing repairing tissue to grow into a scaffold. To tackle this problem, the genetic modification of a biomaterial scaffold is used to accelerate angiogenesis and osteogenesis. However, visualizing and tracking in vivo blood vessel formation in real-time and in three-dimensional (3D) scaffolds or new bone tissue is still an obstacle for bone tissue engineering. Multiphoton microscopy (MPM) is a novel bio-imaging modality that can acquire volumetric data from biological structures in a high-resolution and minimally-invasive manner. The objective of this study was to visualize angiogenesis with multiphoton microscopy in vivo in a genetically modified 3D-PLGA/nHAp scaffold for calvarial critical bone defect repair. PLGA/nHAp scaffolds were functionalized for the sustained delivery of a growth factor pdgf-b gene carrying lentiviral vectors (LV-pdgfb) in order to facilitate angiogenesis and to enhance bone regeneration. In a scaffold-implanted calvarial critical bone defect mouse model, the blood vessel areas (BVAs) in PHp scaffolds were significantly higher than in PH scaffolds. Additionally, the expression of pdgf-b and angiogenesis-related genes, vWF and VEGFR2, increased correspondingly. MicroCT analysis indicated that the new bone formation in the PHp group dramatically improved compared to the other groups. To our knowledge, this is the first time multiphoton microscopy was used in bone tissue-engineering to investigate angiogenesis in a 3D bio-degradable scaffold in vivo and in real-time.

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

Here, we present a protocol to visualize blood vessel formation in vivo and in real-time in 3D scaffolds by multiphoton microscopy. Angiogenesis in genetically modified scaffolds was studied in a murine calvarial critical bone defect model. More new blood vessels were detected in the treatment group than in controls.

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds ...

Initial Evaluation of Antibody-conjugates Modified with Viral-derived Peptides for Increasing Cellular Accumulation and Improving Tumor Targeting

Antibody-conjugates (ACs) modified with virus-derived peptides are a potentially powerful class of tumor cell delivery agents for molecular payloads used in cancer treatment and imaging due to increased cellular accumulation over current ACs. During early AC in vitro development, fluorescence techniques and radioimmunoassays are sufficient for determining intracellular localization, accumulation efficiency, and target cell specificity. Currently, there is no consensus on standardized methods for preparing cells for evaluating AC intracellular accumulation and localization. The initial testing of ACs modified with virus-derived peptides is critical especially if several candidates have been constructed. Determining intracellular accumulation by fluorescence can be affected by background signal from ACs at the cell surface and complicate the interpretation of accumulation. For radioimmunoassays, typically treated cells are fractionated and the radioactivity in different cell compartments measured. However, cell lysis varies from cell to cell and often nuclear and cytoplasmic compartments are not adequately isolated. This can produce misleading data on payload delivery properties. The intravenous injection of radiolabeled virus-derived peptide-modified ACs in tumor bearing mice followed by radionuclide imaging is a powerful method for determining tumor targeting and payload delivery properties at the in vivo phase of development. However, this is a relatively recent advancement and few groups have evaluated virus-derived peptide-modified ACs in this manner. We describe the processing of treated cells to more accurately evaluate virus-derived peptide-modified AC accumulation when using confocal microscopy and radioimmunoassays. Specifically, a method for trypsinizing cells to remove cell surface bound ACs. We also provide a method for improving cellular fractionation. Lastly, this protocol provides an in vivo method using positron emission tomography (PET) for evaluating initial tumor targeting properties in tumor-bearing mice. We use the radioisotope 64Cu (t1/2 = 12.7 h) as an example payload in this protocol.

Viral-derived peptides coupled to antibody-conjugates (ACs) is an approach gaining momentum due to the potential of delivering molecular payloads with increased tumor cell accumulation. Utilizing common methods to evaluate peptide conjugation, AC and payload intracellular accumulation, and tumor targeting, this protocol helps researchers during the key initial development phases.

Antibody-conjugates (ACs) modified with virus-derived peptides are a potentially powerful class of tumor cell delivery agents for molecular payloads used ...

Correlative Light Electron Microscopy (CLEM) for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing virus-infected or transfected cells via EM are the low efficiencies of infection or transfection, hindering the examination of these cells. In order to overcome this difficulty, light microscopy (LM) can be performed first to allocate the subpopulation of infected or transfected cells. Thus, taking advantage of the use of fluorescent proteins (FPs) fused to viral proteins, LM is used here to record the positions of the "positive-transfected" cells, expressing a FP and growing on a support with an alphanumeric pattern. Subsequently, cells are further processed for EM via high pressure freezing (HPF), freeze substitution (FS) and resin embedding. The ultra-rapid freezing step ensures excellent membrane preservation of the selected cells that can then be analyzed at the ultrastructural level by transmission electron microscopy (TEM). Here, a step-by-step correlative light electron microscopy (CLEM) workflow is provided, describing sample preparation, imaging and correlation in detail. The experimental design can be also applied to address many cell biology questions.

Correlative Light Electron Microscopy CLEM for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

A correlative light electron microscopy (CLEM) method is applied to image virus-induced intracellular structures via electron microscopy (EM) in cells that are previously selected by light microscopy (LM). LM and EM are combined as a hybrid imaging approach to achieve an integrated view of virus-host interactions.

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing ...

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in every tissue to be identified and characterized. Our laboratory is actively working to make technological advances in X-ray micro-computed tomography (micro-CT) that will bring the diagnostic power of histology to the study of full tissue volumes at cellular resolution (i.e., an X-ray Histo-tomography modality). Toward this end, we have made targeted improvements to the sample preparation pipeline. One key optimization, and the focus of the present work, is a straightforward method for rigid embedding of fixed and stained millimeter-scale samples. Many of the published methods for sample immobilization and correlative micro-CT imaging rely on placing the samples in paraffin wax, agarose, or liquids such as alcohol. Our approach extends this work with custom procedures and the design of a 3-dimensional printable apparatus to embed the samples in an acrylic resin directly into polyimide tubing, which is relatively transparent to X-rays. Herein, sample preparation procedures are described for the samples from 0.5 to 10 mm in diameter, which would be suitable for whole zebrafish larvae and juveniles, or other animals and tissue samples of similar dimensions. As proof of concept, we have embedded the specimens from Danio, Drosophila, Daphnia, and a mouse embryo; representative images from 3-dimensional scans for three of these samples are shown. Importantly, our methodology leads to multiple benefits including rigid immobilization, long-term preservation of laboriously-created resources, and the ability to re-interrogate samples.

We developed protocols and designed a custom apparatus to enable embedding of millimeter-scale specimens. We present sample preparation procedures with an emphasis on embedding in acrylic resin and polyimide tubing to achieve rigid immobilization and long-term storage of specimens for the interrogation of tissue architecture and cell morphology by micro-CT.

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...