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 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.
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.
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.
2. HEVNP Purification
3. HEVNP Characterization
4. Chemical Conjugation of HEVNPs with Biotin, Cancer Targeting Ligand, and Fluorophores
5. HEVNP Binding to and Internalization in MDA-MB231 Breast Cancer Cells
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. Following maleimide-biotin conjugation, Cys modified HEVNPs displayed streptavidin binding signals (Figure 2).
Effective two-step cysteine conjugation was influenced by the order in which conjugation was carried out. Because α3β1 integrin is be overexpressed in breast cancer tumor cells, a synthetic ligand, LXY30, with specific affinity to α3β110, was selected as a tumor-targeting conjugate molecule. Direct maleimide (MaI) functionalization of LXY30 was not feasible because LXY30 is cyclized through an intermolecular disulfide bond; hence, there was a concern for self-reactivity. Instead, LXY30 was functionalized with an alkyne group and bound to maleimide-azide through a click chemistry reaction. When HEV-573C NPs were bound to maleimide-azide first, followed by a click chemistry reaction to LXY30-alkyne, the HEV-573C NPs appeared to disassemble under TEM observation (Figure 4C). However, an initial click chemistry reaction between LXY30-alkyne and maleimide-azide to form MaI-LXY30, followed by MaI-LXY30 conjugation to Cys-modified HEVNPs (LXY30-Mal-Cy5.5) did not affect its structure (Figure 4B). Figure 4 depicts a schematic of the LXY30-functionalization of HEVNPs for tumor cell targeting.
HEVNPs conjugated to the breast cancer targeting ligand, LXY30 (LXY30-HEVNPs) bound and were internalized into cultured breast cancer cells. For fluorescent detection, LXY30-HEVNPs and unbound HEVNP-573Cs were labeled with NHS functionalized Cy5.5 fluorescent dye (NHS-Cy5.5) through primary amine coupling of lysine on HEVNP-573Cs. LXY30-HEVNP-Cy5.5 and HEVNP-Cy5.5 were applied to cultured breast cancer cells (MDA-MB-231), and observed by confocal microscopy. The NIR fluorescence images by confocal microscope indicate LXY30-HEVNP-Cy5.5 had higher binding affinity for and increased internalization in MDA-MB-231 breast cancer cells compared with HEVNP-Cy5.5 (Figure 5).
Figure 1: Cysteine replacement on the surface protrusion domain of hepatitis E virus nanoparticle. Cysteine mutations were proposed for residues Y485, T489, S533, N573, and T586 at the flexible coil region to minimize the disturbance to the secondary structure of HEV ORF2 (A). All the selected residues for cysteine mutation are located at the surface of the protrusion (P) domain (grey; the other domains are the shell (S)-domain in blue and middle (M)-domain in pink), with residue Y485 (cyan), T489 (yellow), T586 (orange) on the outmost surface, and residue S533 (magenta) and N573 (red) on the side facing the five-fold axis of HEVNP (B). The figure has been modified from Chen et al. 20164 with permission. Please click here to view a larger version of this figure.
Figure 2: Characterization of HEVNP carrying N573C as an example for all Cys modified HEVNPs. The HEVNP-573C appeared as spherical projections on electron micrographs (A) and remained as intact particles after Mal-biotinylating conjugation (B). Representative results of the conjugation and epitope exposure of Cys-modified HEVNPs. They were bound to maleimide-biotin via cysteine-maleimide coupling and subsequently assayed through Western blot for streptavidin binding. The cysteine mutation at N573 allowed efficient biotinylation to the HEVNP compared to the other mutation sites, i.e., Y485, T489, S533, and T586 (C). Bar equals 100 nm. Please click here to view a larger version of this figure.
Figure 3: Schematic of breast cancer cells that are specifically targeted with Cy5.5-labeled LXY30-HEVNPs (LXY30-HEVNP-Cy5.5). LXY30-HEVNP-Cy5.5 selectively bind to breast cancer cells (magenta) due to LXY30's specific affinity to α3β1, which is an over-expressed integrin on the breast cancer cell membrane. Please click here to view a larger version of this figure.
Figure 4: A schematic of the two-step chemical conjugation processes, including a thiol-selective reaction and a copper catalyzed azide-alkyne cycloaddition or 'click chemistry' reaction to build LXY30-HEVNPs. Two methods have been tested. Method 1: the copper catalyzed azide-alkyne cycloaddition reaction between Mal-azide and alkyne-LXY30 to form Mal-linked LXY30 (Mal-LXY30) was performed first. Then the Mal-LXY30 was added to react with the Cys site of HEV-573C NPs (A). The LXY30 and Cy5.5 decorated HEV-573C NPs (LXY30-HEVNP-Cy5.5) remained intact after all these chemical conjugation processes (B). Method 2: the conjugation process begins by labeling Mal-linked azide (Mal-Azide) at the Cys site of HEV-573C NPs through a thiol-selective reaction to build azide-linked HEVNPs (Azide-HEVNPs), followed by adding LXY30-alkyne, ascorbic acid, and CuSO4 to form LXY30-linked HEVNPs (LXY30-HEVNPs) (A). However, most LXY30-HEVNPs were damaged or disassembled from the conjugation processes, which may be caused by the reactive reagents, including azide, ascorbic acid, and CuSO4 (C). Mal: Maleimide. This figure has been modified from Chen et al. 20164 with permission. Please click here to view a larger version of this figure.
Figure 5: Representative results of cell binding and internalization of fluorescently labeled LXY30-HEVNPs, which binds to MDA-MB-231 breast cancer cells. NIR fluorescence images of LXY30-HEVNP-Cy5.5 targeting to MDA-MB-231 cells. The signal of the nuclei, which were stained by DAPI (blue), and the signal of Cy5.5 (red) were acquired using a confocal fluorescence microscope. This figure has been modified from Chen et al. 20164 with permission. Please click here to view a larger version of this figure.
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 time.
Unlike traditional genetic engineering, which can only insert the polypeptides into proteins of interest, the chemical conjugation can connect various materials including polypeptides, small chemical molecules, and nano-particles onto the surface of the protein of interest, as long as there is a Cys or Lys residue to be used as the reactive site. If there is no such residue at the chemical conjugation site, simple Cys/Lys replacement can be genetically modified on the protein of interest to make it chemically activatable as demonstrated in previous research4.
To control that the chemical conjugation only occurs on the selective reaction sites, the structure of both the conjugate molecule and protein to be conjugated need to be thoroughly studied to avoid intramolecular reactivity. As demonstrated herein with the breast cancer targeting peptide LXY3010, a critical disulfide bond stabilizes cyclic ligand conformation. Given the thiol-reactivity of maleimide, direct maleimide-functionalization of LXY30 ligands will likely result in intramolecular reactivity. Instead, two step conjugation (step 4.2) was performed by reacting the maleimide-linked azide with alkyne-linked LXY30 through click chemistry reaction. However, the sequence of the click chemistry reaction and thiol-selected reaction is critical for the conformational intactness of the LXY30-HEVNPs (Figure 4). Through a CuSO4 catalyzed click-chemistry reaction, LXY30 was indirectly bound to maleimide to build MaI-LXY30. Subsequently, the MaI-LXY30 are conjugated to HEVNP-573C such that the formed LXY30-HEVNPs can retain their native icosahedral structure (Figure 4).
The authors have nothing to disclose.
The authors acknowledge the sponsorship of the funding to RHC by NIH grant #'s: AI095382, EB021230, CA198880, National Institute of Food and Agriculture, as well as the Finland Distinguished Professor program.
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 |