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 border="0" cellpadding="0" cellspacing="0" width="984">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:391px;">MINI Dialysis Units, 10K MWCO</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:265px;">69572</td>
			<td style="width:167px;">mini dialysis unit</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">High Five Cells</td>
			<td>Thermo Fisher Scientific</td>
			<td>B85502</td>
			<td>Tn5 cells</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SF9 Cells&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>11496015</td>
			<td>Sf9 cells</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">Bac-to-Bac Baculovirus Expression System</td>
			<td>Thermo Fisher Scientific</td>
			<td>A11101, A11100</td>
			<td>Baculovirus expression system</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">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 height="20">
			<td height="20" style="height:20px;">ESF921 Insect Cell Media</td>
			<td>Expression Systems LLC</td>
			<td>96-001-01</td>
			<td>insect cell media</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cy5.5 NHS ester, 5mg</td>
			<td>Lumiprobe Corp</td>
			<td>27020</td>
			<td>Cy5.5 NHS ester</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Zeba Spin Desalting Columns, 40K MWCO, 0.5 mL</td>
			<td>Thermo Scientific</td>
			<td>87766</td>
			<td>spin desalting column</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MES Hydrate</td>
			<td>Sigma-Aldrich Chemical Co</td>
			<td>M8250-250G</td>
			<td>MES</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ultra-Clear Centrifuge Thinwall Ultra-Centrifuge Tubes</td>
			<td>Beckman Coulter, Inc</td>
			<td>Depends on Rotor</td>
			<td>ultracentrifuge tube</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NuPage 4-12% Bis-Tris Protein Gels</td>
			<td>Thermo Fisher Scientific</td>
			<td>NPO321BOX</td>
			<td>SDS protein gel</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cellfectin II Reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>10362100</td>
			<td>transfection reagent</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:391px;">EMS Glow Discharger</td>
			<td style="width:161px;">Electron Microscopy Science</td>
			<td style="width:265px;"></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...

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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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Research

JoVE Journal

Bioengineering

6.8K 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

Soft Lithographic Functionalization and Patterning Oxide-free Silicon and Germanium

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a stable interface that permits efficient electron transport and protects underlying substrates from oxidative degradation. Group IV semiconductors can be effectively protected with highly-ordered self-assembled monolayers (SAMs) composed of simple alkyl chains that act as impervious barriers to both organic and aqueous solutions. Simple alkyl SAMs, however, are inert and not amenable to traditional patterning techniques. The motivation for immobilizing organic molecular systems on semiconductors is to impart new functionality to the surface that can provide optical, electronic, and mechanical function, as well as chemical and biological activity. 
Microcontact printing (&mu;CP) is a soft-lithographic technique for patterning SAMs on myriad surfaces.1-9 Despite its simplicity and versatility, the approach has been largely limited to noble metal surfaces and has not been well developed for pattern transfer to technologically important substrates such as oxide-free silicon and germanium. Furthermore, because this technique relies on the ink diffusion to transfer pattern from the elastomer to substrate, the resolution of such traditional printing is essentially limited to near 1 &mu;m.10-16
In contrast to traditional printing, inkless &mu;CP patterning relies on a specific reaction between a surface-immobilized substrate and a stamp-bound catalyst. Because the technique does not rely on diffusive SAM formation, it significantly expands the diversity of patternable surfaces. In addition, the inkless technique obviates the feature size limitations imposed by molecular diffusion, facilitating replication of very small (&lt;200 nm) features.17-23 However, up till now, inkless &mu;CP has been mainly used for patterning relatively disordered molecular systems, which do not protect underlying surfaces from degradation.
Here, we report a simple, reliable high-throughput method for patterning passivated silicon and germanium with reactive organic monolayers and demonstrate selective functionalization of the patterned substrates with both small molecules and proteins. The technique utilizes a preformed NHS-reactive bilayered system on oxide-free silicon and germanium. The NHS moiety is hydrolyzed in a pattern-specific manner with a sulfonic acid-modified acrylate stamp to produce chemically distinct patterns of NHS-activated and free carboxylic acids. A significant limitation to the resolution of many &mu;CP techniques is the use of PDMS material which lacks the mechanical rigidity necessary for high fidelity transfer. To alleviate this limitation we utilized a polyurethane acrylate polymer, a relatively rigid material that can be easily functionalized with different organic moieties. Our patterning approach completely protects both silicon and germanium from chemical oxidation, provides precise control over the shape and size of the patterned features, and gives ready access to chemically discriminated patterns that can be further functionalized with both organic and biological molecules. The approach is general and applicable to other technologically-relevant surfaces.

Here we describe a simple method for patterning oxide-free silicon and germanium with reactive organic monolayers and demonstrate functionalization of the patterned substrates with small molecules and proteins. The approach completely protects surfaces from chemical oxidation, provides precise control over feature morphology, and provides ready access to chemically discriminated patterns.

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a ...

Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles

Biodegradable nanoparticles have emerged as a versatile platform for the design and implementation of new intranasal vaccines against respiratory infectious diseases. Specifically, polyanhydride nanoparticles composed of the aliphatic sebacic acid (SA), the aromatic 1,6-bis(p-carboxyphenoxy)hexane (CPH), or the amphiphilic 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) display unique bulk and surface erosion kinetics1,2 and can be exploited to slowly release functional biomolecules (e.g., protein antigens, immunoglobulins, etc.) in vivo3,4,5. These nanoparticles also possess intrinsic adjuvant activity, making them an excellent choice for a vaccine delivery platform6,7,8.
	In order to elucidate the mechanisms governing the activation of innate immunity following intranasal mucosal vaccination, one must evaluate the molecular and cellular responses of the antigen presenting cells (APCs) responsible for initiating immune responses. Dendritic cells are the principal APCs found in conducting airways, while alveolar macrophages (AM&#632;) predominate in the lung parenchyma9,10,11. AM&#632; are highly efficient in clearing the lungs of microbial pathogens and cell debris12,13. In addition, this cell type plays a valuable role in the transport of microbial antigens to the draining lymph nodes, which is an important first step in the initiation of an adaptive immune response9. AM&#632; also express elevated levels of innate pattern recognition and scavenger receptors, secrete pro-inflammatory mediators, and prime na&iuml;ve T cells12,14. A relatively pure population of AM&#632; (e.g., greater than 80%) can easily be obtained via lung lavage for study in the laboratory. Resident AM&#632; harvested from immune competent animals provide a representative phenotype of the macrophages that will encounter the particle-based vaccine in vivo. Herein, we describe the protocols used to harvest and culture AM&#632; from mice and examine the activation phenotype of the macrophages following treatment with polyanhydride nanoparticles in vitro.

Herein, we describe protocols for harvesting murine alveolar macrophages, which are resident innate immune cells in the lung, and examining their activation in response to co-culture with polyanhydride nanoparticles.

Biodegradable nanoparticles have emerged as a  versatile platform for the design and implementation of new intranasal vaccines  against respiratory ...

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells1-5. Internalization of nanoparticles (NP) by antigen presenting cells is a critical step in generating an effective immune response to the encapsulated antigen. To determine how changes in nanoparticle formulation impact function, we sought to develop a high throughput, quantitative experimental protocol that was compatible with detecting internalized nanoparticles as well as bacteria. To date, two independent techniques, microscopy and flow cytometry, have been the methods used to study the phagocytosis of nanoparticles. The high throughput nature of flow cytometry generates robust statistical data. However, due to low resolution, it fails to accurately quantify internalized versus cell bound nanoparticles. Microscopy generates images with high spatial resolution; however, it is time consuming and involves small sample sizes6-8. Multi-spectral imaging flow cytometry (MIFC) is a new technology that incorporates aspects of both microscopy and flow cytometry that performs multi-color spectral fluorescence and bright field imaging simultaneously through a laminar core. This capability provides an accurate analysis of fluorescent signal intensities and spatial relationships between different structures and cellular features at high speed.
	Herein, we describe a method utilizing MIFC to characterize the cell populations that have internalized polyanhydride nanoparticles or Salmonella enterica serovar Typhimurium. We also describe the preparation of nanoparticle suspensions, cell labeling, acquisition on an ImageStreamX system and analysis of the data using the IDEAS application. We also demonstrate the application of a technique that can be used to differentiate the internalization pathways for nanoparticles and bacteria by using cytochalasin-D as an inhibitor of actin-mediated phagocytosis.

In this article, we describe a method utilizing multi-spectral imaging flow cytometry to quantify the internalization of polyanhydride nanoparticles or bacteria by RAW 264.7 cells.

Nanoparticulate  systems have emerged as valuable tools in vaccine delivery through their  ability to efficiently deliver cargo, including proteins, to ...

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design efficacious vaccines carriers. Nanoparticle-based platforms can prolong the persistence of vaccine antigens, which could improve vaccine immunogenicity1. Several biodegradable polymers have been studied as vaccine delivery vehicles1; in particular, polyanhydride particles have demonstrated the ability to provide sustained release of stable protein antigens and to activate antigen presenting cells and modulate immune responses2-12.
The molecular design of these vaccine carriers needs to integrate the rational selection of polymer properties as well as the incorporation of appropriate targeting agents. High throughput automated fabrication of targeting ligands and functionalized particles is a powerful tool that will enhance the ability to study a wide range of properties and will lead to the design of reproducible vaccine delivery devices.
The addition of targeting ligands capable of being recognized by specific receptors on immune cells has been shown to modulate and tailor immune responses10,11,13 C-type lectin receptors (CLRs) are pattern recognition receptors (PRRs) that recognize carbohydrates present on the surface of pathogens. The stimulation of immune cells via CLRs allows for enhanced internalization of antigen and subsequent presentation for further T cell activation14,15. Therefore, carbohydrate molecules play an important role in the study of immune responses; however, the use of these biomolecules often suffers from the lack of availability of structurally well-defined and pure carbohydrates. An automation platform based on iterative solution-phase reactions can enable rapid and controlled synthesis of these synthetically challenging molecules using significantly lower building block quantities than traditional solid-phase methods16,17.
Herein we report a protocol for the automated solution-phase synthesis of oligosaccharides such as mannose-based targeting ligands with fluorous solid-phase extraction for intermediate purification. After development of automated methods to make the carbohydrate-based targeting agent, we describe methods for their attachment on the surface of polyanhydride nanoparticles employing an automated robotic set up operated by LabVIEW as previously described10. Surface functionalization with carbohydrates has shown efficacy in targeting CLRs10,11 and increasing the throughput of the fabrication method to unearth the complexities associated with a multi-parametric system will be of great value (Figure 1a).

In this article, a high throughput method is presented for the synthesis of oligosaccharides and their attachment to the surface of polyanhydride nanoparticles for further use in targeting specific receptors on antigen presenting cells.

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design ...

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 ...

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, ...

Flash NanoPrecipitation for the Encapsulation of Hydrophobic and Hydrophilic Compounds in Polymeric Nanoparticles

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can improve bioavailability and modify drug distribution within the body. For hydrophilic drugs like peptides or proteins, encapsulation within NPs can also provide protection from natural clearance mechanisms. There are few techniques for the production of polymeric NPs that are scalable. Flash NanoPrecipitation (FNP) is a process that uses engineered mixing geometries to produce NPs with narrow size distributions and tunable sizes between 30 and 400 nm. This protocol provides instructions on the laboratory-scale production of core-shell polymeric nanoparticles of a target size using FNP. The protocol can be implemented to encapsulate either hydrophilic or hydrophobic compounds with only minor modifications. The technique can be readily employed in the laboratory at milligram scale to screen formulations. Lead hits can directly be scaled up to gram- and kilogram-scale. As a continuous process, scale-up involves longer mixing process run time rather than translation to new process vessels. NPs produced by FNP are highly loaded with therapeutic, feature a dense stabilizing polymer brush, and have a size reproducibility of &#177; 6%.

Flash NanoPrecipitation (FNP) is a scalable approach to produce polymeric core-shell nanoparticles. Lab-scale formulations for the encapsulation of hydrophobic or hydrophilic therapeutics are described.

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can ...

Preparation of Neutrally-charged, pH-responsive Polymeric Nanoparticles for Cytosolic siRNA Delivery

The success of siRNA as a targeted molecular medicine is dependent upon its efficient cytosolic delivery to cells within the tissue of pathology. Clinical success for treating previously &#8216;undruggable&#8217; hepatic disease targets with siRNA has been achieved. However, efficient tumor siRNA delivery necessitates additional pharmacokinetic design considerations, including long circulation time, evasion of clearance organs (e.g., liver and kidneys), and tumor penetration and retention. Here, we describe the preparation and in vitro physicochemical/biological characterization of polymeric nanoparticles designed for efficient siRNA delivery, particularly to non-hepatic tissues such as tumors. The siRNA nanoparticles are prepared by electrostatic complexation of siRNA and the diblock copolymer poly(ethylene glycol-b-[2-(dimethylamino)ethyl methacrylate-co-butyl methacrylate]) (PEG-DB) to form polyion complexes (polyplexes) where siRNA is sequestered within the polyplex core and PEG forms a hydrophilic, neutrally-charged corona. Moreover, the DB block becomes membrane-lytic as vesicles of the endolysosomal pathway acidify (&#60; pH 6.8), triggering endosomal escape and cytosolic delivery of siRNA. Methods to characterize the physicochemical characteristics of siRNA nanoparticles such as size, surface charge, particle morphology, and siRNA loading are described. Bioactivity of siRNA nanoparticles is measured using luciferase as a model gene in a rapid and high-throughput gene silencing assay. Designs which pass these initial tests (such as PEG-DB-based polyplexes) are considered appropriate for translation to preclinical animal studies assessing the delivery of siRNA to tumors or other sites of pathology.

Methods to prepare and characterize the physicochemical properties and bioactivity of neutrally-charged, pH-responsive siRNA nanoparticles are presented. Criteria for successful siRNA nanomedicines such as size, morphology, surface charge, siRNA loading, and gene silencing are discussed.

The success of siRNA as a targeted molecular medicine is dependent upon its efficient cytosolic delivery to cells within the tissue of pathology. Clinical ...

Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

Self-assembling protein nanoparticles (SAPNs) function as repetitive antigen displays and can be used to develop a wide range of vaccines for different infectious diseases. In this article we demonstrate a method to produce a SAPN core containing a six-helix bundle (SHB) assembly that is capable of presenting antigens in a trimeric conformation. We describe the expression of the SHB-SAPN in an E. coli system, as well as the necessary protein purification steps. We included an isopropanol wash step to reduce the residual bacterial lipopolysaccharide. As an indication of the protein identity and purity, the protein reacted with known monoclonal antibodies in Western blot analyses. After refolding, the size of the particles fell in the expected range (20 to 100 nm), which was confirmed by dynamic light scattering, nanoparticle tracking analysis, and transmission electron microscopy. The methodology described here is optimized for the SHB-SAPN, however, with only slight modifications it can be applied to other SAPN constructs. This method is also easily transferable to large scale production for GMP manufacturing for human vaccines.

Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

A detailed method is provided here describing the purification, refolding, and characterization of self-assembling protein nanoparticles (SAPNs) for use in vaccine development.

Self-assembling protein nanoparticles (SAPNs) function as repetitive antigen displays and can be used to develop a wide range of vaccines for different ...

Formulating and Characterizing Lipid Nanoparticles for Gene Delivery using a Microfluidic Mixing Platform

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high encapsulation efficiency. Use of lipid nanoparticles (LNPs) to encapsulate nucleic acids is advantageous to protect the RNA or DNA from degradation, while also promoting cellular uptake. LNPs often contain multiple lipid components including an ionizable lipid, helper lipid, cholesterol, and polyethylene glycol (PEG) conjugated lipid. LNPs can readily encapsulate nucleic acids due to the ionizable lipid presence, which at low pH is cationic and allows for complexation with negatively charged RNA or DNA. Here LNPs are formed by encapsulating messenger RNA (mRNA) or plasmid DNA (pDNA) using rapid mixing of the lipid components in an organic phase and the nucleic acid component in an aqueous phase. This mixing is performed using a precise microfluidic mixing platform, allowing for nanoparticle self-assembly while maintaining laminar flow. The hydrodynamic size and polydispersity are measured using dynamic light scattering (DLS). The effective surface charge on the LNP is determined by measuring the zeta potential. The encapsulation efficiency is characterized using a fluorescent dye to quantify entrapped nucleic acid. Representative results demonstrate the reproducibility of this method and the influence that different formulation and process parameters have on the developed LNPs.

Lipid nanoparticles are developed using a microfluidic mixing platform approach for mRNA and DNA encapsulation.

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high ...