Surface Functionalization of Hepatitis E Virus Nanoparticles Using Chemical Conjugation Methods

Summary

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.

Abstract

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.

Introduction

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 pathway^2,3,4. Nanomedicine has come to the forefront of targeted delivery because optimally sized nanoparticles have the capacity to stabilize circulation of th....

Protocol

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.

1. Culture Sf9 cells in insect cell media (see Table of Materials) to 50 - 75% confluence in 6-well plates.
2. Using insect cell transfection reagents according to the manufacturer's protocols, transfect Bacmids containing HEVNP-573C ORF2 into Sf99 cells to produce recombinant baculovirus. Incubate the transfected cells at 27 °C for 3 - 6 days, depending on the viability of cells.
3. Collect ....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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