The authors acknowledge the sponsorship of the funding to RHC by NIH grant #&#39;s: AI095382, EB021230, CA198880, National Institute of Food and Agriculture, as well as the Finland Distinguished Professor program.

1. HEVNP Production in Insect Cells
NOTE: All following steps should be performed in a cell culture hood. Refer to our previous publication for more detailed HEVNP production procedures11.
<ol>
	<li>Culture Sf9 cells in insect cell media (see Table of Materials) to 50 - 75% confluence in 6-well plates.</li>
	<li>Using insect cell transfection reagents according to the manufacturer&#39;s protocols, transfect Bacmids containing HEVNP-573C ORF2 into Sf99 cells to produce recombinant baculovirus. Incubate the transfected cells at 27 &#176;C for 3 - 6 days, depending on the viability of cells.</li>
	<li>Collect supernatant at 3 - 6 days post-infection (after the transfected cells are lysed due to baculovirus infection) as P0 baculovirus stock.</li>
	<li>Remove culture medium before applying 200 &#181;L of P0 stock to 50 - 75% confluent Sf9 cells in a 25 cm2 monolayer flask. Rock the flask every 15 min to ensure the full coverage of the inoculum. Repeat 4 times, for a total of 60 min.</li>
	<li>Add 2 mL of insect cell culture medium to the flask and keep at 27 &#176;C for 3 - 6 days, depending on viability of cells, to amplify baculovirus to a higher titer.</li>
	<li>Carry out plaque assays to obtain the baculovirus titer reading12.</li>
	<li>Culture the suspended Tn5 insect cells (Table of Materials) with 100 mL of insect cell medium in 250 mL flask and shake in 27 &#176;C at 150 rpm to a titer of 0.5 x 105&#160;- 1 x 106 for inoculation.</li>
	<li>Add baculovirus at a multiplicity of infection (MOI) of 5 - 10 to 100 mL Tn5 cells in a 250 mL flask. After inoculation, shake in 27 &#176;C at 150 rpm for 5 - 7 days.</li>
	<li>Once most cells appear to have vesicles and 70 - 90% of cells are dead under optical microscope observation, collect the Tn5 cells and transfer into 33 mL ultracentrifuge tubes. Place the ultracentrifuge tubes in swinging bucket rotors, balance, and spin down the cell debris and recombinant baculoviruses at 10,000 x g for 90 min at 25 &#176;C.</li>
	<li>Keep the supernatant that contains the released HEVNPs at 4 &#176;C for further purification. For extended storage of baculovirus supernatant, add protease inhibitors.</li>
</ol>
2. HEVNP Purification
<ol>
	<li>Pellet and isolate the HEVNPs using cesium chloride (CsCl) gradient separation:
	<ol>
		<li>Transfer the collected supernatant (from step 1.10) and add 20% NP-40 into each tube to make a final concentration of 0.5% NP-40 to dissolve any remaining cellular membrane. Gently mix by pipetting and incubate for at least 30 min at 25 &#176;C.</li>
		<li>Ultracentrifuge the HEVNPs at 112,400 x g in swinging bucket rotors for 2 h at 4 &#176;C to pellet down the HEVNPs from the supernatant. Discard the supernatant after centrifugation and gently re-suspend the crude pellet in 200 &#181;L of 10 mM MES buffer pH 6.2 in each tube overnight (O/N) at 4 &#176;C. Run SDS-PAGE (step 3.1) to confirm the presence of HEVNP ORF2 in the crude pellet as a 52 kDa band.</li>
		<li>Prepare a 38.5% (w/v) CsCl gradient by mixing 1.96 g CsCl, the re-suspended crude pellet, and ~4 mL of 0.01 M MES pH 6.2 in a 5 mL ultracentrifuge tube. Balance the tubes and place in a swinging bucket rotor. Ultracentrifuge at 147,000 x g for 16 h at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Collect the fractions after CsCl gradient:
	<ol>
		<li>Discard the top 500 &#181;L fraction, which is primarily light-weight cell membrane debris. Collect 500 &#181;L fractions, starting from the top of the tube, and change tips in between each fraction. Place each fraction into numbered/labeled 1.5 mL tubes. 
		NOTE: The presence of HEVNP ORF2 in fractions separated from CsCl gradient cannot be detected by running SDS PAGE gels due to the high concentration of CsCl. The CsCl in each fraction can be removed or diluted by following a CsCl clean-up procedure. Alternatively, the CsCl gradient can be replaced by a 10 - 40% sucrose gradient13 to avoid CsCl residual.</li>
		<li>Transfer each fraction to a 5 mL ultracentrifuge tube and dilute the CsCl with 4.5 mL of 10 mM MES, pH 6.2. Balance the tubes and place in a swinging bucket rotor. Ultracentrifuge at 147,000 x g for 2 h at 4 &#176;C to pellet down the HEVNPs.</li>
		<li>Discard the supernatant and gently re-suspend the HEVNPs in 100 &#181;L of 10 mM MES pH 6.2 in each tube. Cover the tubes to avoid evaporation and incubate O/N at 4 &#176;C.</li>
		<li>Record the A280 reading and A260/A280 nm ratio using a spectrophotometer. Determine the approximate concentration of ORF2 as: 
		<img alt="Equation" src="/files/ftp_upload/57020/57020eq1.jpg" /> 
		Each ORF2 will contain 1 Cys site and 1 Lys site for chemical conjugation. 
		NOTE: The molar extinction coefficient of HEVNP ORF2 is 60,280, which is equivalent to 1.019 mg/mL &#215; A280. This is so close to 1:1 that the concentration of HEVNPs (in mg/mL) can be approximated by the A280, and therefore, the concentration of ORF2 by the above equation. For example, an HEVNP with an A280 reading of 1 will have a concentration of 1 mg/mL, which is equivalent to 18.8 &#181;M ORF2.</li>
		<li>Prepare an SDS-PAGE. Use a 6 &#181;L sample from each fraction to determine the fractions containing 53.3 kDa HEVNP ORF2 protein (step 3.1). The HEVNPs should be found in fractions 3 - 5, with a density of ~1.25 g/mL.</li>
		<li>Confirm the presence and the purity of HEVNPs by TEM observation. Prepare or dilute the HEVNP samples to 0.5 - 2.0 mg/mL for TEM. The HEVNPs appear in TEM as empty icosahedral proteins, ~27 nm in diameter (Figure 2). Some protein contaminants may remain in the fractions and will be observed under TEM (step 3.2).</li>
	</ol>
	</li>
	<li>In the case that impurities are present under TEM, repeat step 2.1.3 - 2.2.6 for better purity of the HEVNPs. 
	NOTE: Extra purification through CsCl gradient may cause yield loss of HEVNPs. Alternatively, the CsCl gradient purification can be replaced by a 10 - 40% sucrose gradient to avoid residual CsCl13.</li>
</ol>
3. HEVNP Characterization
<ol>
	<li>Prepare SDS PAGE 4-12% Bis-Tris Protein Gels, 1.0 mm, 17-wells (see Table of Materials) according to the user&#39;s manual14:
	<ol>
		<li>Add 2 &#181;L of 4x loading buffer to 6 &#181;L of protein sample. Incubate the sample mixture in a heat block for 10 min at 100 &#176;C to denature the protein. Load the protein samples onto the gel.</li>
		<li>Run the SDS-PAGE by setting the DC power supply at 100 V for 10 min, then 150 V for 45 min until the samples run to about 1 cm above the bottom of the gel.</li>
		<li>Stain the SDS PAGE gel with Coomassie blue (0.25% (w/v) Coomassie Brilliant Blue R250, 30% (v/v) methanol, 10% (v/v) acetic acid), for 1 h.</li>
		<li>After the staining procedure, remove the Coomassie blue stain and apply de-staining buffer (30% (v/v) methanol, 10% (v/v) acetic acid) onto the protein gel for &#62;12 h at room temperature.</li>
		<li>Document the gel under white light to confirm the presence of HEVNP ORF2 at the 52 kDa band.</li>
	</ol>
	</li>
	<li>Observe the HEVNPs using TEM.
	<ol>
		<li>Prepare or dilute the HEVNP samples to 0.5 - 2 mg/mL with 10 mM MES pH 6.2 for TEM imaging.</li>
		<li>Ionize the carbon-coated grids with 40 mA glow discharge for 30 s to produce a hydrophilic carbon surface. The glow discharge equipment is described in the Table of Materials. 
		NOTE: The hydrophilic carbon surface of the grids can only last for 30 min after glow discharge treatment.</li>
		<li>Hold in tweezers and add 2 &#181;L of HEVNP sample to the grid, wait for 15 - 30 s, and blot with filter paper.</li>
		<li>Immediately wash the grid with ddH20 and blot with filter paper.</li>
		<li>Immediately add 2 &#181;L of 2% uranyl acetate to the grid, wait 15 s, then blot with filter paper. Dry the sample grids by putting them in an electronic dehumidify dry cabinet for O/N.</li>
		<li>Transfer the grid into a TEM and image at 10-80k magnification. HEVNPs appear in TEM as empty icosahedral proteins that are ~27 nm in diameter, due to the absence of viral RNA.</li>
	</ol>
	</li>
</ol>
4. Chemical Conjugation of HEVNPs with Biotin, Cancer Targeting Ligand, and Fluorophores
<ol>
	<li>Perform one step conjugation of HEVNPs and maleimide linked biotin.
	<ol>
		<li>Buffer change: Apply the HEVNPs in mini dialysis units and dialyze against 0.01 M PBS pH 7.4 at room temperature for 1 h according to the manufacturer&#39;s protocol (Table of Materials). Transfer the HEVNPs to 1.5 mL tubes and measure the protein concentration at 280 nm using a spectrophotometer.</li>
		<li>Mix the HEVNP to 1 mg/mL, which is equivalent to 18.8 &#181;M of Cys reaction sites (see details in step 2.2.4), with an equal amount of maleimide-biotin (100 &#181;M) in 0.01 M PBS, pH 7.4, to make a 1:5 molar ratio; react O/N at 4 &#176;C. Remove unbound maleimide-biotin with a 40 K MWCO Spin Desalting column procedure according to the manufacturer&#39;s protocol (Table of Materials).</li>
		<li>Analyze the samples through a standard reducing SDS-PAGE (step 3.1).</li>
		<li>Using standard procedures, prepare a chemiluminescent Western Blot using HRP-linked Streptavidin. Capture the chemiluminescent signal by X ray film (Figure 2).</li>
	</ol>
	</li>
	<li>Perform two step LXY30 conjugation to surface-exposed cysteine on HEV NPs (Figure 5).
	<ol>
		<li>Buffer exchange: Apply HEVNPs in mini dialysis units and dialyze against 0.01 M PBS pH 7.4 at room temperature for 1 h. Transfer the HEVNPs to 1.5 mL tubes and measure protein concentration at 280 nm using a spectrophotometer.</li>
		<li>Add 650 &#181;M maleimide-azide and 650 &#181;M alkyne-LXY3010 in 0.01 M PBS pH 7.4 with 200 &#181;M CuSO4 and 1 mM ascorbic acid to form maleimide-linked LXY30 (Mal-LXY30) at 650 &#181;M. Incubate the mixture at 4 &#176;C for O/N.</li>
		<li>Mix the HEVNP to 1 mg/mL, which is equivalent to 18.8 &#181;M of Cys reaction site (see details in step 2.2.4), with about 10% volume of Mal-LXY30 (650 &#181;M) in 0.01 M PBS pH 7.4, to make a 1:3 molar ratio; react O/N at 4 &#176;C. 
		NOTE: Due to the relatively high concentration of Mal-LXY30, the final concentrations of the reactants, such as CuSO4, are reduced about 10 times after mixing, to avoid their damage to HEVNPs. Another option is the Cu-free conjugation method15.</li>
		<li>Remove unbound maleimide-click-LXY30 with a 40 K MWCO Spin Desalting column according to the manufacturer&#39;s protocol (see Table of Materials). Keep the LXY30-linked HEVNPs (LXY30-HEVNPs) at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Perform one step conjugation of the LXY30-HEVNPs and Cy5.5 NHS ester (NHS-Cy5.5)
	<ol>
		<li>Mix the LXY30-linked HEVNPs (LXY30-HEVNPs) to 1 mg/mL, which is equivalent to 18.8 &#181;M of the Cys reaction site (see details in step 2.2.4), with equal volume of NHS-Cy5.5 (100 &#181;M) in 0.01 M PBS pH 7.4 to make a 1:5 molar ratio; react O/N at 4 &#176;C.</li>
		<li>Remove unbound Cy5.5-NHS with a 40K MWCO Spin Desalting column procedure according to the manufacturer&#39;s protocol (see Table of Materials). Keep the LXY30, Cy5.5-linked HEVNPs (LXY30-HEVNP-Cy5.5) at 4 &#176;C.</li>
	</ol>
	</li>
</ol>
5. HEVNP Binding to and Internalization in MDA-MB231 Breast Cancer Cells
<ol>
	<li>Seed MDA-MB231 breast cancer cells into a 35-mm glass bottom dishes (5 x 104 per dish) O/N in a mammalian cell culture cabinet.</li>
	<li>For the cell binding experiment, prepare LXY30-HEVNP-Cy5.5 by following steps 4.2 -&#160;4.3. Dilute the LXY30-HEVNP-Cy5.5 to 0.01 mg/mL, which is equivalent to 0.188 &#181;M of HEVNP ORF2 (refer to step 2.2.4), in 250 &#181;L of 0 - 1% FBS/DMEM.
	<ol>
		<li>Prepare the negative control sample at 0.01 mg/mL HEVNP-Cy5.5 by following step 4.3 but conjugated with NHS-Cy5.5 dye only, (HEV-Cy5.5). Dilute the HEVNP-Cy5.5 to 0.01 mg/mL, which is equivalent to 0.188 &#181;M of HEVNP ORF2 (refer to step 2.2.4), in 250 &#181;L of 10% FBS supplemented with DMEM.</li>
	</ol>
	</li>
	<li>Wash the cells once by applying 250 &#181;L of 1 M PBS buffer, pH 7.4. Remove the PBS buffer after the wash, while keeping some buffer in the cell culture dish.</li>
	<li>Apply 250 &#181;L of LXY30-HEVNP-Cy5.5 in 10% FBS supplemented with DMEM or HEVNP-Cy5.5 in 10% FBS supplemented with DMEM to the cultured MDA-MB231 breast cancer cells. Shield the cell cultured dishes from light with aluminum foil.</li>
	<li>Keep the cell cultured dishes in a 37 &#176;C cell culture cabinet for 1 h for internalization.</li>
	<li>Wash the cultured cells on ice 3 times, 5 min per wash, with 250 &#181;L of 1 M PBS, pH 7.4.</li>
	<li>Fix the cells in 4% PFA in 1 M PBS, pH 7.4 for 20 min and then wash once with 250 &#181;L of 1 M PBS, pH 7.4. 
	NOTE: The cells are now ready to be imaged by confocal microscope. Representative data are shown in Figure 5.</li>
</ol>

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The authors declare that they have no competing interests.

In contrast to the time consuming genetic engineering procedure, which usually takes weeks, here we demonstrate simple two-step and one-step chemical conjugation procedures, which can be completed within 3 days, of adding the cancer targeting ligand and/or fluorescence detection dye to the Cys/Lys sites of HEVNPs. The technique can be used to screen for the best ligand target from a pool of candidates, and thus takes advantage of the available peptide/small molecule synthesis services at a reasonable cost and delivery ti...

The development of nano-sized vectors in therapeutic and diagnostic delivery, known as nanotheranostics, has shifted much of the biomedical field away from generalized treatments towards targeted delivery1. Targeted nanotheranostic delivery integrates nano-sized vectors (nanoparticles) with theranostic molecules to stably direct theranostic molecules to a specific diseased tissue or biochemical pathway2,3,4. Nanomedicine has come to the forefront of targeted delivery because optimally sized nanoparticles have the capacity to stabilize circulation of theranostic molecules and selectively target cell surface molecules presented on diseased tissues. Many nanotheranostic platforms still suffer from passive cell uptake, pre-mature degradation, toxicity, and insufficient association with theranostic molecules. VLPs overcome many of these obstacles in targeted delivery. They have been used as nanocarriers to display foreign epitopes and/or deliver small molecules: a regimen that can be used to combat many diseases1. This application relies mainly on the property of self-assembly as well as the ease of genetic modifications, to fulfill the designed application for the given VLP. Compared to genetic engineering, chemical conjugation of foreign peptides to VLP displays a significant advantage because it allows a great variety of entities, such as peptides or oligosaccharides, to be conjugated to the surface of VLPs in a modulated and flexible manner without alteration of VLP assembly.
HEVNPs, derived from the recombinant HEV capsid protein, 2nd open reading frame (ORF2), are non-infectious, self-assembling capsids capable of cell-binding and entry. Because HEV evolved for mucosal transmission, the assembled capsid protein is similarly stable in proteolytic and acidic mucosal conditions5. HEVNPs form a hollow, T = 1 icosahedral capsid, composed of 60 identical units6,7 of ORF2, rendering it highly stable both in storage and in harsh physiological conditions. Lacking any viral genetic elements, the efficient, high yield production is achieved through baculovirus expression system in insect cells. Because of their proteolytic stability, self-assembled HEVNPs are extracted and purified from cell supernatant, substantially reducing necessary purification steps. Additionally, HEVNPs possess a surface exposed protrusion domain (P domain) connected through a flexible hinge to a stable icosahedral base. The P domain forms surface-exposed spikes atop the icosahedral base while the flexible hinge makes it possible to significantly modify the P domain without compromising the base icosahedral structure. With 60 repeated units, single site-specific modification results in 60 symmetric sites for chemical modulation. Recently, we proposed a nano-platform using HEVNP that can chemically conjugate ligands or small molecules for theranostic applications. This was achieved by replacing a single amino acid with cysteine on the protrusion domain of HEV-VLP as a reaction site with maleimide-linked peptides or molecules. Based on previous structural analysis of HEV-VLP and well-studied immunogenic epitopes8,9, the following five HEV-VLP amino acids were replaced with cysteine as potential candidates: Y485C, T489C, S533C, N573C, and T586C (Figure 1). After expression and purification from insect cells, their VLP formations were confirmed by transmission electron microscopy (TEM) observation (Figure 2), and the exposed cysteine sites were analyzed by Western blot after maleimide-linked biotin conjugation (Figure 2). Among the five mutants, HEVNP-573C displayed the strongest signal of maleimide-biotin conjugation (Figure 2) and was used for follow up demonstration as the nanocarrier for breast cancer cell targeting4 (Figure 3).
This protocol depicts chemical conjugation methods to attach tumor-targeting molecules to HEVNPs through surface cysteine conjugation. We detail the conjugation of tumor targeting and detection molecules for tumor delivery with recombinant HEVNPs containing a cysteine at N573 (HEVNP-573C). We focused on a two-step click chemistry conjugation process to bind a breast cancer tumor targeting peptide, LXY3010 to HEVNPs to form LXY30-HEVNP (Figure 4). Subsequently, N-hydroxysuccimide (NHS)-Cy5.5 were conjugated to the separate Lys site on HEVNPs to build LXY30-HEVNP-Cy5.5 for fluorescent detection both in vitro (Figure 5) and in vivo4.

<table><tbody><tr><td>MINI Dialysis Units, 10K MWCO</td><td>Thermo Fisher Scientific</td><td>69572</td><td>mini dialysis unit</td></tr><tr><td>High Five Cells</td><td>Thermo Fisher Scientific</td><td>B85502</td><td>Tn5 cells</td></tr><tr><td>SF9 Cells&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>11496015</td><td>Sf9 cells</td></tr><tr><td>Bac-to-Bac Baculovirus Expression System</td><td>Thermo Fisher Scientific</td><td>A11101, A11100</td><td>Baculovirus expression system</td></tr><tr><td>Bac-to-Bac Baculovirus Expression System</td><td>Life Technologies</td><td>10359-016, 10360-014, 10584-027, 10712-024</td><td>Bacmid</td></tr><tr><td>ESF921 Insect Cell Media</td><td>Expression Systems LLC</td><td>96-001-01</td><td>insect cell media</td></tr><tr><td>Cy5.5 NHS ester, 5mg</td><td>Lumiprobe Corp</td><td>27020</td><td>Cy5.5 NHS ester</td></tr><tr><td>Zeba Spin Desalting Columns, 40K MWCO, 0.5 mL</td><td>Thermo Scientific</td><td>87766</td><td>spin desalting column</td></tr><tr><td>MES Hydrate</td><td>Sigma-Aldrich Chemical Co</td><td>M8250-250G</td><td>MES</td></tr><tr><td>Ultra-Clear Centrifuge Thinwall Ultra-Centrifuge Tubes</td><td>Beckman Coulter, Inc</td><td>Depends on Rotor</td><td>ultracentrifuge tube</td></tr><tr><td>NuPage 4-12% Bis-Tris Protein Gels</td><td>Thermo Fisher Scientific</td><td>NPO321BOX</td><td>SDS protein gel</td></tr><tr><td>Cellfectin II Reagent</td><td>Thermo Fisher Scientific</td><td>10362100</td><td>transfection reagent</td></tr><tr><td>EMS Glow Discharger</td><td>Electron Microscopy Science</td><td></td><td>glow discharger</td></tr></tbody></table>

surface functionalization of hepatitis e virus nanoparticles using chemical conjugation methods

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of various diseases. This application relies on genetic modification, self-assembly, and cysteine conjugation to fulfill the tumor-targeting application of recombinant VLPs. Compared with genetic modification alone, chemical conjugation of foreign peptides to VLPs offers a significant advantage because it allows a variety of entities, such as synthetic peptides or oligosaccharides, to be conjugated to the surface of VLPs in a modulated and flexible manner without alteration of the VLP assembly.
Here, we demonstrate how to use the hepatitis E virus nanoparticle (HEVNP), a modularized theranostic capsule, as a multifunctional delivery carrier. Functions of HEVNPs include tissue-targeting, imaging, and therapeutic delivery. Based on the well-established structural research of HEVNP, the structurally independent and surface-exposed residues were selected for cysteine replacement as conjugation sites for maleimide-linked chemical groups via thiol-selective linkages. One particular cysteine-modified HEVNP (a Cys replacement of the asparagine at 573 aa (HEVNP-573C)) was conjugated to a breast cancer cell-specific ligand, LXY30 and labeled with near-infrared (NIR) fluorescence dye (Cy5.5), rendering the tumor-targeted HEVNPs as effective diagnostic capsules (LXY30-HEVNP-Cy5.5). Similar engineering strategies can be employed with other macromolecular complexes with well-known atomic structures to explore potential applications in theranostic delivery.

Akin to HEV-VLPs, all Cys modified HEVNPs formed soluble icosahedral capsids and did not aggregate in solution during production or purification. Before and after single-step maleimide-biotin conjugation, each of the Cys modified HEVNPs were indistinguishable from HEV-VLPs in the negative stain EM (Figure 2). Maleimide-biotin conjugation efficiency to Cys modified HEVNPs was first tested with Western blotting via chemiluminescent streptavidin binding. Followi...

Watch this Scientific Journal Video about Surface Functionalization of Hepatitis E Virus Nanoparticles Using Chemical Conjugation Methods at JoVE.com

Surface Functionalization of Hepatitis E Virus Nanoparticles Using Chemical Conjugation Methods

We have engineered the capsid protein of hepatitis E virus as a theranostic nanoparticle (HEVNP). HEVNP self-assembles into a stable icosahedral cage in mucosal delivery. Here, we describe the modification of HEVNPs for tumor targeting by mutating surface-exposed residues to cysteines, which conjugate synthetic ligands that specifically bind tumor cells.

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of ...

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JoVE Journal

Bioengineering

6.9K Views.  University of California Davis. We have engineered the capsid protein of hepatitis E virus as a theranostic nanoparticle (HEVNP). HEVNP self-assembles into a stable icosahedral cage in mucosal delivery. Here, we describe the modification of HEVNPs for tumor targeting by mutating surface-exposed residues to cysteines, which conjugate synthetic ligands that specifically bind tumor cells.

Video: Surface Functionalization of Hepatitis E Virus Nanoparticles Using Chemical Conjugation Methods

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.

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.

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

Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in human non-hepatic and murine cell lines, the efficiency was very low and required long-term selection procedures using HCV replicon constructs expressing dominant antibiotic-selectable markers1-5. HCV in vitro research is therefore limited to human hepatoma cell lines permissive for virus entry and completion of the viral life cycle. Due to HCVs narrow species tropism, there is no immunocompetent small animal model available that sustains the complete HCV replication cycle 6-8. Inefficient replication of HCV in non-human cells e.g. of mouse origin is likely due to lack of genetic incompatibility of essential host dependency factors and/or expression of restriction factors.
We investigated whether HCV propagation is suppressed by dominant restriction factors in either human cell lines derived from non-hepatic tissues or in mouse liver cell lines. To this end, we developed two independent conditional trans-complementation methods relying on somatic cell fusion. In both cases, completion of the viral replication cycle is only possible in the heterokaryons. Consequently, successful trans-complementation, which is determined by measuring de novo production of infectious viral progeny, indicates absence of dominant restrictions.
Specifically, subgenomic HCV replicons carrying a luciferase transgene were transfected into highly permissive human hepatoma cells (Huh-7.5 cells). Subsequently, these cells were co-cultured and fused to various human and murine cells expressing HCV structural proteins core, envelope 1 and 2 (E1, E2) and accessory proteins p7 and NS2. Provided that cell fusion was initiated by treatment with polyethylene-glycol (PEG), the culture released infectious viral particles which infected na&iuml;ve cells in a receptor-dependent fashion.
To assess the influence of dominant restrictions on the complete viral life cycle including cell entry, RNA translation, replication and virus assembly, we took advantage of a human liver cell line (Huh-7 Lunet N cells 9) which lacks endogenous expression of CD81, an essential entry factor of HCV. In the absence of ectopically expressed CD81, these cells are essentially refractory to HCV infection 10 . Importantly, when co-cultured and fused with cells that express human CD81 but lack at least another crucial cell entry factor (i.e. SR-BI, CLDN1, OCLN), only the resulting heterokaryons display the complete set of HCV entry factors requisite for infection. Therefore, to analyze if dominant restriction factors suppress completion of the HCV replication cycle, we fused Lunet N cells with various cells from human and mouse origin which fulfill the above mentioned criteria. When co-cultured cells were transfected with a highly fusogenic viral envelope protein mutant of the prototype foamy virus (PFV11) and subsequently challenged with infectious HCV particles (HCVcc), de novo production of infectious virus was observed. This indicates that HCV successfully completed its replication cycle in heterokaryons thus ruling out expression of dominant restriction factors in these cell lines. These novel conditional trans-complementation methods will be useful to screen a large panel of cell lines and primary cells for expression of HCV-specific dominant restriction factors.

We describe two methods for conditional trans-complementation of hepatitis C virus (HCV) assembly and the completion of the full viral life cycle, which rely on heterokaryon formation. These techniques are suitable to screen for cell lines that express dominant restriction factors, which preclude production of infectious HCV progeny.

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in ...

Immunology and Infection

Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry

The modification of virus particles has received a significant amount of attention for its tremendous potential for impacting gene therapy, oncolytic applications and vaccine development.1,2,3 Current approaches to modifying viral surfaces, which are mostly genetics-based, often suffer from attenuation of virus production, infectivity and cellular transduction.4,5 Using chemoselective click chemistry, we have developed a straightforward alternative approach which sidesteps these issues while remaining both highly flexible and accessible.1,2
The goal of this protocol is to demonstrate the effectiveness of using bioorthogonal click chemistry to modify the surface of adenovirus type 5 particles. This two-step process can be used both therapeutically1 or analytically,2,6 as it allows for chemoselective ligation of targeting molecules, dyes or other molecules of interest onto proteins pre-labeled with azide tags. The three major advantages of this method are that (1) metabolic labeling demonstrates little to no impact on viral fitness,1,7 (2) a wide array of effector ligands can be utilized, and (3) it is remarkably fast, reliable and easy to access.1,2,7
In the first step of this procedure, adenovirus particles are produced bearing either azidohomoalanine (Aha, a methionine surrogate) or the unnatural sugar O-linked N-azidoacetylglucosamine (O-GlcNAz), both of which contain the azide (-N3) functional group. After purification of the azide-modified virus particles, an alkyne probe containing the fluorescent TAMRA moiety is ligated in a chemoselective manner to the pre-labeled proteins or glycoproteins. Finally, an SDS-PAGE analysis is performed to demonstrate the successful ligation of the probe onto the viral capsid proteins. Aha incorporation is shown to label all viral capsid proteins (Hexon, Penton and Fiber), while O-GlcNAz incorporation results in labeling of Fiber only.
In this evolving field, multiple methods for azide-alkyne ligation have been successfully developed; however only the two we have found to be most convenient are demonstrated herein &#x2013; strain-promoted azide-alkyne cycloaddition (SPAAC) and copper-catalyzed azide-alkyne cycloaddition (CuAAC) under deoxygenated atmosphere.

Adenovirus particles are engineered to contain either the unnatural amino acid analogue azidohomoalanine or the azido sugar O-GlcNAz. The azide group of each is chemoselectively ligated via "click" chemistry reactions as a means of viral surface modification.

The modification of virus particles has received a significant amount of attention for its tremendous potential for impacting gene therapy, oncolytic ...

Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo

Targeted antigen delivery to cross-presenting dendritic cells (DC) in vivo efficiently induces T effector cell responses and displays a valuable approach in&#160;vaccine design. Antigen is delivered to DC via antibodies specific for endocytosis receptors such as DEC-205 that induce uptake, processing, and MHC class I- and II-presentation.
Efficient and reliable conjugation of the desired antigen to a suitable antibody is a critical step in DC targeting and among other factors depends on the format of the antigen. Chemical conjugation of&#160;full-length protein to purified antibodies is one possible strategy. In the past, we have successfully established cross-linking of the model antigen ovalbumin (OVA) and a DEC-205-specific IgG2a antibody (&#945;DEC-205)&#160;for in vivo DC targeting studies in mice. The first step of the protocol is the purification of the antibody from the supernatant of the NLDC (non-lymphoid dendritic cells)-145 hybridoma by affinity chromatography. The purified antibody is activated for chemical conjugation by sulfo-SMCC (sulfosuccinimidyl 4-[N-maleimidomethyl] cyclohexane-1-carboxylate) while at the same time the sulfhydryl-groups of the OVA protein are exposed through incubation with TCEP-HCl (tris (2-carboxyethyl) phosphine hydrochloride). Excess TCEP-HCl and sulfo-SMCC are removed and the antigen is mixed with the activated antibody for overnight coupling. The resulting &#945;DEC-205/OVA conjugate is concentrated and freed from unbound OVA. Successful conjugation of OVA to &#945;DEC-205 is verified by western blot analysis and enzyme-linked immunosorbent assay (ELISA).
We have successfully used chemically crosslinked &#945;DEC-205/OVA to induce cytotoxic T cell responses in the liver and to compare different adjuvants for their potential in inducing humoral and cellular immunity following in vivo targeting of DEC-205+ DC. Beyond that, such chemically coupled antibody/antigen conjugates offer valuable tools for the efficient induction of vaccine responses to tumor antigens and have been proven to be superior to classical immunization approaches regarding the prevention and therapy of various types of tumors.

Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo

We describe a protocol for the chemical conjugation of the model antigen ovalbumin to an endocytosis receptor-specific antibody for in vivo dendritic cell targeting. The protocol includes purification of the antibody, chemical conjugation of the antigen, as well as purification of the conjugate and the verification of efficient conjugation.

Targeted antigen delivery to cross-presenting dendritic cells (DC) in vivo efficiently induces T effector cell responses and displays a valuable approach ...

Cancer Research

Synthesis of Functionalized Magnetic Nanoparticles, Their Conjugation with the Siderophore Feroxamine and its Evaluation for Bacteria Detection

In the present work, the synthesis of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with (3-aminopropyl)triethoxysilane (APTES) and its conjugation with deferoxamine, a siderophore recognized by Yersinia enterocolitica, using a succinyl moiety as a linker are described.
Magnetic nanoparticles (MNP) of magnetite (Fe3O4) were prepared by solvothermal method and coated with SiO2 (MNP@SiO2) using the St&#246;ber process followed by functionalization with APTES (MNP@SiO2@NH2). Then, feroxamine was conjugated with the MNP@SiO2@NH2 by carbodiimide coupling to give MNP@SiO2@NH2@Fa. The morphology and properties of the conjugate and intermediates were examined by eight different methods including powder X-Ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM) and energy dispersive X-Ray (EDX) mapping. This exhaustive characterization confirmed the formation of the conjugate. Finally, in order to evaluate the capacity and specificity of the nanoparticles, they were tested in a capture bacteria assay using Yersinia enterocolitica.

This work describes protocols for the preparation of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with (3-aminopropyl)triethoxysilane (APTES) and its conjugation with deferoxamine using a succinyl moiety as a linker. A deep structural characterization description and a capture bacteria assay using Y. enterocolitica for all the intermediate nanoparticles and the final conjugate are also described in detail.

In the present work, the synthesis of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with ...

Chemistry

A Competent Hepatocyte Model Examining Hepatitis B Virus Entry through Sodium Taurocholate Cotransporting Polypeptide as a Therapeutic Target

Hepatitis B virus (HBV) infection has been considered a crucial risk factor for hepatocellular carcinoma. Current treatment can only lessen the viral load but not result in complete remission. An efficient hepatocyte model for HBV infection would offer a true-to-life viral life cycle that would be crucial for the screening of therapeutic agents. Most available anti-HBV agents target lifecycle stages post viral entry but not before viral entry. This protocol details the generation of a competent hepatocyte model capable of screening for therapeutic agents targeting pre-viral entry and post viral entry lifecycle stages. This includes the targeting of sodium taurocholate cotransporting polypeptide (NTCP) binding, cccDNA formation, transcription, and viral assembly based on imHC or HepaRG as host cells. Here, the HBV entry inhibition assay used curcumin to inhibit HBV binding and transporting functions via NTCP. The inhibitors were evaluated for binding affinity (KD) with NTCP using isothermal titration calorimetry (ITC)-a universal tool for HBV drug screening based on thermodynamic parameters.

We present a protocol to screen anti-hepatitis B virus (HBV) compounds targeting pre- and post-viral entry lifecycle stages, using isothermal titration calorimetry to measure binding affinity (KD) with host sodium taurocholate cotransporting polypeptide. Antiviral efficacy was determined through the suppression of viral lifecycle markers (cccDNA formation, transcription, and viral assembly).

Hepatitis B virus (HBV) infection has been considered a crucial risk factor for hepatocellular carcinoma. Current treatment can only lessen the viral load ...

Biochemistry

Fabrication and Characterization of Griffithsin-modified Fiber Scaffolds for Prevention of Sexually Transmitted Infections

Electrospun fibers (EFs) have been widely used in a variety of therapeutic applications; however, they have only recently been applied as a technology to prevent and treat sexually transmitted infections (STIs). Moreover, many EF technologies focus on encapsulating the active agent, relative to utilizing the surface to impart biofunctionality. Here we describe a method to fabricate and surface-modify poly(lactic-co-glycolic) acid (PLGA) electrospun fibers, with the potent antiviral lectin Griffithsin (GRFT). PLGA is an FDA-approved polymer that has been widely used in drug delivery due to its outstanding chemical and biocompatible properties. GRFT is a natural, potent, and safe lectin that possesses broad activity against numerous viruses including human immunodeficiency virus type 1 (HIV-1). When combined, GRFT-modified fibers have demonstrated potent inactivation of HIV-1 in vitro. This manuscript describes the methods to fabricate and characterize GRFT-modified EFs. First, PLGA is electrospun to create a fiber scaffold. Fibers are subsequently surface-modified with GRFT using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS)chemistry. Scanning electron microscopy (SEM) was used to assess the size and morphology of surface-modified formulations. Additionally, a gp120 or hemagglutinin (HA)-based ELISA may be used to quantify the amount of GRFT conjugated to, as well as GRFT desorption from the fiber surface. This protocol can be more widely applied to fabricate fibers that are surface-modified with a variety of different proteins.

This manuscript describes the procedure to fabricate and characterize Griffithsin-modified poly(lactic-co-glycolic acid) electrospun fibers that demonstrate potent adhesive and antiviral activity against human immunodeficiency virus type 1 infection in vitro. Methods used to synthesize, surface-modify, and characterize the resulting morphology, conjugation, and desorption of Griffithsin from surface-modified fibers are described.

Electrospun fibers (EFs) have been widely used in a variety of therapeutic applications; however, they have only recently been applied as a technology to ...

Making Conjugation-induced Fluorescent PEGylated Virus-like Particles by Dibromomaleimide-disulfide Chemistry

The recent rise in virus-like particles (VLPs) in biomedical and materials research can be attributed to their ease of biosynthesis, discrete size, genetic programmability, and biodegradability. While they&#39;re highly amenable to bioconjugation reactions for adding synthetic ligands onto their surface, the range in bioconjugation methodologies on these aqueous born capsids is relatively limited. To facilitate the direction of functional biomaterials research, non-traditional bioconjugation reactions must be considered. The reaction described in this protocol uses dibromomaleimides to introduce new functionality in the solvent exposed disulfide bonds of a VLP based upon Bacteriophage Q&#946;. Furthermore, the final product is fluorescent, which has the added benefit of generating a trackable in vitro probe using a commercially available filter set.

Here, we present a procedure to fluorescently functionalize the disulfides on Q&#946; VLP with dibromomaleimide. We describe Q&#946; expression and purification, the synthesis of dibromomaleimide-functionalized molecules, and the conjugation reaction between dibromomaleimide and Q&#946;. The resulting yellow fluorescent conjugated particle can be used as a fluorescence probe inside cells.

The recent rise in virus-like particles (VLPs) in biomedical and materials research can be attributed to their ease of biosynthesis, discrete size, ...

Biofunctionalization of Magnetic Nanomaterials

Magnetic nanomaterials have received great attention in different biomedical applications. Biofunctionalizing these nanomaterials with specific targeting agents is a crucial aspect to enhance their efficacy in diagnostics and treatments while minimizing the side effects. The benefit of magnetic nanomaterials compared to non-magnetic ones is their ability to respond to magnetic fields in a contact-free manner and over large distances. This allows to guide or accumulate them, while they can also be monitored. Recently, magnetic nanowires (NWs) with unique features were developed for biomedical applications. The large magnetic moment of these NWs enables a more efficient remote control of their movement by a magnetic field. This has been utilized with great success in cancer treatment, drug delivery, cell tracing, stem cell differentiation or magnetic resonance imaging. In addition, the NW fabrication by template-assisted electrochemical deposition provides a versatile method with tight control over the NW properties. Especially iron NWs and iron-iron oxide (core-shell) NWs are suitable for biomedical applications, due to their high magnetization and low toxicity.
In this work, we provide a method to biofunctionalize iron/iron oxide NWs with specific antibodies directed against a specific cell surface marker that is overexpressed in a large number of cancer cells. Since the method utilizes the properties of the iron oxide surface, it is also applicable to superparamagnetic iron oxide nanoparticles. The NWs are first coated with 3-aminopropyl-tri-ethoxy-silane (APTES) acting as a linker, which the antibodies are covalently attached to. The APTES coating and the antibody biofunctionalization are proven by electron energy loss spectroscopy (EELS) and zeta potential measurements. In addition, the antigenicity of the antibodies on the NWs is tested by using immunoprecipitation and western blot. The specific targeting of the biofunctionalized NWs and their biocompatibility are studied by confocal microscopy and a cell viability assay.

In this work, we provide a protocol to biofunctionalize magnetic nanomaterials with antibodies for specific cell targeting. As examples, we utilize iron nanowires to target cancer cells.

Magnetic nanomaterials have received great attention in different biomedical applications. Biofunctionalizing these nanomaterials with specific targeting ...

Engineering Antiviral Agents via Surface Plasmon Resonance

Surface plasmon resonance (SPR) is used to measure hemagglutinin (HA) binding to domain-swapped Cyanovirin-N (CV-N) dimer and to monitor interactions between mannosylated peptides and CV-N's high-affinity binding site. Virus envelope spikes gp120, HA, and Ebola glycoprotein (GP) 1,2 have been reported to bind both high- and low-affinity binding sites on dimeric CVN2. Dimannosylated HA peptide is also bound at the two low-affinity binding sites to an engineered molecule of CVN2, which is bearing a high-affinity site for the respective ligand and mutated to replace a stabilizing disulfide bond in the carbohydrate-binding pocket, thus confirming multivalent binding. HA binding is shown to one high-affinity binding site of pseudo-antibody CVN2 at a dissociation constant (KD) of 275 nM that further neutralizes human immunodeficiency virus type 1 (HIV-1) through oligomerization. Correlating the number of disulfide bridges in domain-swapped CVN2, which are decreased from 4 to 2 by substituting cystines into polar residue pairs of glutamic acid and arginine, results in reduced binding affinity to HA. Among the strongest interactions, Ebola GP1,2 is bound by CVN2 with two high-affinity binding sites in the lower nanomolar range using the envelope glycan without a transmembrane domain. In the present study, binding of the multispecific monomeric CV-N to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein is measured at KD = 18.6 &#181;M as compared with nanomolar KD to those other virus spikes, and via its receptor-binding domain in the mid-&#181;-molar range.

Engineering Antiviral Agents via Surface Plasmon Resonance

The present protocol describes new tools for SPR binding assays to examine CV-N binding to HA, S glycoprotein, related hybrid-type glycans, and high-mannose oligosaccharides. SPR is used to determine the KD for binding either dimeric or monomeric CV-N to these glycans.

Surface plasmon resonance (SPR) is used to measure hemagglutinin (HA) binding to domain-swapped Cyanovirin-N (CV-N) dimer and to monitor interactions ...

Surface Functionalization of Hepatitis E Virus Nanoparticles Using Chemical Conjugation Methods

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Viral Nanoparticles for In vivo Tumor Imaging

Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors

Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry

Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo

Synthesis of Functionalized Magnetic Nanoparticles, Their Conjugation with the Siderophore Feroxamine and its Evaluation for Bacteria Detection

A Competent Hepatocyte Model Examining Hepatitis B Virus Entry through Sodium Taurocholate Cotransporting Polypeptide as a Therapeutic Target

Fabrication and Characterization of Griffithsin-modified Fiber Scaffolds for Prevention of Sexually Transmitted Infections

Making Conjugation-induced Fluorescent PEGylated Virus-like Particles by Dibromomaleimide-disulfide Chemistry

Biofunctionalization of Magnetic Nanomaterials

Engineering Antiviral Agents via Surface Plasmon Resonance