Name: surface functionalization of hepatitis e virus nanoparticles using chemical conjugation methods
Uploaded: 2018-05-11T14:00:00
Description: Watch this Scientific Journal Video about Surface Functionalization of Hepatitis E Virus Nanoparticles Using Chemical Conjugation Methods at JoVE.com

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...

<ol>
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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...

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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