Cowpea mosaic virus capsid: a promising carrier for the development of carbohydrate based antitumor vaccines

Immunotherapy targeting tumor cell surface carbohydrates is a promising approach for cancer treatment. However, the low immunogenecity of carbohydrates presents a formidable challenge. We describe here the enhancement of carbohydrate immunogenicity by an ordered display on the surface of the cowpea mosaic virus (CPMV) capsid. The Tn glycan, which is overexpressed on numerous cancer cell surfaces, was selected as the model antigen for our study. Previously it has been shown that it is difficult to induce a strong T cell-dependent immune response against the monomeric form of Tn presented in several ways on different carriers. In this study, we first synthesized Tn antigens derivatized with either a maleimide or a bromoacetamide moiety that was conjugated selectively to a cysteine mutant of CPMV. The glycoconjugate was then injected into mice and pre- and post-immune antibody levels in the mice sera were measured by enzyme-linked immunosorbant assays. High total antibody titers and, more importantly, high IgG titers specific for Tn were obtained in the post-immune day 35 serum, suggesting the induction of T cell-dependent antibody isotype switching by the glycoconjugate. The antibodies generated were able to recognize Tn antigens presented in their native conformations on the surfaces of both MCF-7 breast cancer cells and the multidrug resistant breast cancer cell line NCI-ADR RES. These results suggest that the CPMV capsid can greatly enhance the immunogenicity of weak antigens such as Tn and this can provide a promising tool for the development of carbohydrate based anti-cancer vaccines.     

Decoration of discretely immobilized cowpea mosaic virus with luminescent quantum dots

This report describes two related methods for decorating cowpea mosaic virus (CPMV) with luminescent semiconductor nanocrystals (quantum dots, QDs). Variants of CPMV are immobilized on a substrate functionalized with NeutrAvidin using modifications of biotin-avidin binding chemistry in combination with metal affinity coordination. For example, using CPMV mutants expressing available 6-histidine sequences inserted at loops on the viral coat protein, we show that these virus particles can be specifically immobilized on NeutrAvidin functionalized substrates in a controlled fashion via metal-affinity coordination. To accomplish this, a hetero-bifunctional biotin-NTA moiety, activated with nickel, is used as the linker for surface immobilization of CPMV (bridging the CPMVs' histidines to the NeutrAvidin). Two linking chemistries are then employed to achieve CPMV decoration with hydrophilic CdSe-ZnS core-shell QDs; they target the histidine or lysine residues on the exterior virus surface and utilize biotin-avidin interactions. In the first scheme, QDs are immobilized on the surface-tethered CPMV via electrostatic attachment to avidin previously bound to the virus particle. In the second strategy, the lysine residues common to each viral surface asymmetric unit are chemically functionalized with biotin groups and the biotinylated CPMV is discretely immobilized onto the substrate via NeutrAvidin-biotin interactions. The biotin units on the upper exposed surface of the immobilized CPMV then serve as capture sites for QDs conjugated with a mixture of avidin and a second protein, maltose binding protein, which is also used for QD-protein conjugate purification. Characterization of the assembled CPMV and QD structures is presented, and the potential uses for protein-coated QDs functionalized onto this symmetrical virion nanoscaffold are discussed.     

Generation and structural analysis of reactive empty particles derived from an icosahedral virus

Chemical and genetic modifications on the surface of viral protein cages confer unique properties to the virus particles with potential nano and biotechnological applications. The enclosed space in the interior of the virus particles further increases its versatility as a nanomaterial. In this paper, we report a simple method to generate a high yield of stable cowpea mosaic virus (CPMV) empty capsids from their native nucleoprotein counterparts by removing the encapsidated viral genome without compromising the integrity of the protein coat. Biochemical and structural comparison of artificially generated empty particles did not reveal any distinguishable differences from CPMV particles containing viral RNA. Preliminary results on the use of artificially produced empty CPMV capsids as a carrier capsule are described.     

Plant viral capsids as nanobuilding blocks: construction of arrays on solid supports

The virions of Cowpea mosaic virus (CPMV) can be regarded as programmable nanobuilding blocks with a diameter of approximately 28 nm. The particles display a number of features that can be exploited for nanoscale material fabrication. In this study we use the virus-derived building blocks for construction of arrays on solid supports. Biotin-modified CPMV particles are used with Streptavidin as a linker molecule in order to enable self-assembly of arrays from the surface up by a layer-by-layer approach. CPMV particles with different fluorescent labels, which enable differential detection of each layer, have been immobilized on surfaces and arranged in defined layers. This approach provides novel structured arrays which have the potential for development as functional devices at the nanoscale.     

Natural supramolecular building blocks. Wild-type cowpea mosaic virus

Cowpea mosaic virus (CPMV) can be isolated in gram quantities, possesses a structure that is known to atomic resolution, and is quite stable. It is therefore of potential use as a molecular entity in synthesis, particularly as a building block on the nanochemical scale. CPMV was found to possess a lysine residue with enhanced reactivity in each asymmetric unit, and thus 60 such lysines per virus particle. The identity of this residue was established by a combination of acylation, protein digestion, and mass spectrometry. Under forcing conditions, up to four lysine residues per asymmetric unit can be addressed. In combination with engineered cysteine reactivity described in the accompanying paper, this provides a powerful platform for the alteration of the chemical and physical properties of CPMV particles.     

Self-Assembly of Mitochondria-Targeted Photosensitizer to Increase Photostability and Photodynamic Therapeutic Efficacy in Hypoxia

The development of photosensitizers for cancer photodynamic therapy has been challenging due to their low photostability and therapeutic inefficacy in hypoxic tumor microenvironments. To overcome these issues, we have developed a mitochondria-targeted photosensitizer consisting of an indocyanine moiety with triphenylphosphonium arms, which can self-assemble into spherical micelles directed to mitochondria. Self-assembly of the photosensitizer resulted in a higher photostability by preventing free rotation of the indoline ring of the indocyanine moiety. The mitochondria targeting capability of the photosensitizer allowed it to utilize intramitochondrial oxygen. We found that the mitochondria-targeted photosensitizer localized to mitochondria and induced apoptosis of cancer cells both normoxic and hypoxic conditions through generation of ROS. The micellar self-assemblies of the photosensitizer were further confirmed to selectively localize to tumor tissues in a xenograft tumor mouse model through passive targeting and showed efficient tumor growth inhibition.     

Fluorescent In Situ Targeting Probes for Rapid Imaging of Ovarian‐Cancer‐Specific γ‐Glutamyltranspeptidase

γ‐Glutamyltranspeptidase (GGT) is a tumor biomarker that selectively catalyzes the cleavage of glutamate overexpressed on the plasma membrane of tumor cells. Here, we developed two novel fluorescent in situ targeting (FIST) probes that specifically target GGT in tumor cells, which comprise 1) a GGT‐specific substrate unit (GSH), and 2) a boron–dipyrromethene (BODIPY) moiety for fluorescent signalling. In the presence of GGT, sulfur‐substituted BODIPY was converted to amino‐substituted BODIPY, resulting in dramatic fluorescence variations. By exploiting this enzyme‐triggered photophysical property, we employed these FIST probes to monitor the GGT activity in living cells, which showed remarkable differentiation between ovarian cancer cells and normal cells. These probes represent two first‐generation chemodosimeters featuring enzyme‐mediated rapid, irreversible aromatic hydrocarbon transfer between the sulfur and nitrogen atoms accompanied by switching of photophysical properties.     

促性腺激素释放激素类似物-紫杉醇靶向抗肿瘤缀合物的合成及评价

以促性腺激素释放激素类似物(GnRHa)为靶向配体, 以紫杉醇为抗癌因子, 分别以硫醚键和二硫键为连接臂, 设计合成了2个靶向抗肿瘤缀合物. 研究了缀合物的肿瘤细胞增殖抑制活性和GnRH受体结合活性, 结果表明, 2个缀合物均具有较强的抗肿瘤活性和GnRH受体亲和力; 另外, 血浆稳定性实验结果显示, 以硫醚键偶联的缀合物1在血浆中孵育24 h, 原型保留仍在50%以上, 具有较高的稳定性.     

Design and validation of a bifunctional ligand display system for receptor targeting

Here we developed a bacteriophage display particle designed to serve as a bifunctional entity that can target tumors while delivering an agent. We engineered a chimera phage vector containing a pIII-displayed alphav integrins-targeting moiety and a pVIII-displayed streptavidin binding adaptor moiety. By using the chimeric phage particle, targeting of alphav integrins on cells in culture and tumor-related blood vessels was shown through different applications, including luminescent quantum dots localization, surface plasmon resonance-based binding detection, and an in vivo tumor model. The strategy validated here will accelerate the discovery and characterization of receptor-ligand binding events in high throughput, and cell-specific delivery of diagnostics or therapeutics to organs of choice without the need for chemical conjugation.     

New addresses on an addressable virus nanoblock; uniquely reactive Lys residues on cowpea mosaic virus

Cowpea mosaic virus (CPMV) is a robust, icosahedrally symmetric platform successfully used for attaching a variety of molecular substrates including proteins, fluorescent labels, and metals. The symmetric distribution and high local concentration of the attached molecules generates novel properties for the 30 nm particles. We report new CPMV reagent particles generated by systematic replacement of surface lysines with arginine residues. The relative reactivity of each lysine on the native particle was determined, and the two most reactive lysine residues were then created as single attachment sites by replacing all other lysines with arginine residues. Structural analysis of gold derivatization not only corroborated the specific reactivity of these unique lysine residues but also demonstrated their dramatically different presentation environment. Combined with site-directed cystine mutations, it is now possible to uniquely double label CPMV, expanding its use as an addressable nanoblock.     

Biologically active polymer - targeting polymeric antitumor agents-

To synthesize targeting polymeric antitumor agents, poly[(2-acetoxy-3,4-dihydro-2H-pyran)-alt-(maleic anhydride)] as polymer backbone, chlorophyll derivatives(CpD) extracted from silkworm excreta as targeting moiety and 5-fluorouracil and 6-mercaptopurine as pharmacons were selected. The polymer exhibited an antitumor activity comparable to that of cyclophosphamide in vivo. CpD as well as CpD-polymer conjugate were localized on tumor cells in vitro and delivered to tumor tissues in vivo specifically. The targeting polymeric antitumor agents were synthesized by coupling pharmacons and CpD on the polymer chain with the aid of reactions between functionalities of the reactants and anhydride groups on the polymer backbone. Various tests of their biological activities in vivo are in progress.     

An Activatable Photosensitizer Targeted to γ‐Glutamyltranspeptidase

We adopted a spirocyclization‐based strategy to design γ‐glutamyl hydroxymethyl selenorhodamine green (gGlu‐HMSeR) as a photo‐inactive compound that would be specifically cleaved by the tumor‐associated enzyme γ‐glutamyltranspeptidase (GGT) to generate the potent photosensitizer HMSeR. gGlu‐HMSeR has a spirocyclic structure and is colorless and does not show marked phototoxicity toward low‐GGT‐expressing cells or normal tissues upon irradiation with visible light. In contrast, HMSeR predominantly takes an open structure, is colored, and generates reactive oxygen species upon irradiation. The γ‐glutamyl group thus serves as a tumor‐targeting moiety for photodynamic therapy (PDT), switching on tumor‐cell‐specific phototoxicity. To validate this system, we employed chick chorioallantoic membrane (CAM), a widely used model for preliminary evaluation of drug toxicity. Photoirradiation after gGlu‐HMSeR treatment resulted in selective ablation of implanted tumor spheroids without damage to healthy tissue.     

Natural supramolecular building blocks. Cysteine-added mutants of cowpea mosaic virus

Wild-type Cowpea mosaic virus (CPMV) displays no cysteine side chains on the exterior capsid surface and is therefore relatively unreactive with thiol-selective reagents. Four CPMV mutants bearing cysteine residues in one of two exterior positions of the asymmetric unit were created. The mutants were shown to aggregate by virtue of disulfide bond formation in the absence of added reducing agent, bind to metallic gold, and undergo selective reactions at the introduced thiol residues. Controlled aggregation by virtue of biotin-avidin interactions was demonstrated, as was the independent derivatization of reactive lysine and cysteine positions. The ability to introduce such reactivity into a system that can be readily prepared and isolated in gram quantities should open new doors to applications in biochemistry, materials science, and catalysis.     

Novel types of carborane-carrier hyaluronan derivatives via "click chemistry"

Two new HA derivatives bearing carborane rings were synthesized by click chemistry. The optimal conditions were assessed for the preparation of biocompatible boron carriers, potentially suitable for application in BNCT and capable of targeting the CD44 antigen. The new polymeric samples were characterized by means of NMR-spectroscopy techniques that gave degrees of 17 and 8% for HAAACB and HapACB, respectively. Both HAAACB and HApACB turned out to be nontoxic for colorectal, ovarian and bladder tumor cell lines, to disclose a specific interaction with the CD44 antigen as the native hyaluronan moiety, and to deliver boron-atom concentrations largely sufficient for BNCT therapy when accumulated in cancer cells.     

Rapidly reversible hydrophobization: an approach to high first-pass drug extraction

We have investigated a rapidly reversible hydrophobization of therapeutic agents for improving first-pass uptake in locoregional drug therapy. This approach involves the attachment of a hydrophobic moiety to the drug by highly labile chemical linkages that rapidly hydrolyze upon injection. Hydrophobization drastically enhances cell-membrane association of the prodrug and, consequently, drug uptake, while the rapid lability protects nontargeted tissues from exposure to the highly active agent. Using the membrane-impermeable DNA intercalator propidium iodide, and melphalan, we report results from in vitro cellular internalization and toxicity studies. Additionally, we report in vivo results after a single liver arterial bolus injection, demonstrating both tumor targeting and increased survival in a mouse tumor model.     

in vitro biological evaluation of folate-functionalized block copolymer micelles for selective anti-cancer drug delivery

The main objective of this study was to evaluate the ability of folic acid-functionalized diblock copolymer micelles to improve the delivery and uptake of two poorly water-soluble anti-tumor drugs, tamoxifen and paclitaxel, to cancer cells through folate receptor targeting. The diblock copolymer used in this study comprised a hydrophilic poly[2-(methacryloyloxy)ethyl phosphorylcholine] (MPC) block, carrying at the chain end the folate targeting moiety, and a pH-sensitive hydrophobic poly[2-(diisopropylamino)ethyl methacrylate] (DPA) block (FA-MPC-DPA). The drug-loading capacities of tamoxifen- and paclitaxel-loaded micelles were determined by high performance liquid chromatography and the micelle dimensions were determined by dynamic light scattering and transmission electron microscopy. Cell viability studies were carried out on human chronic myelogenous leukaemia (K-562) and colon carcinoma cell lines (Caco-2) in order to demonstrate that drug-loaded FA-MPC-DPA micelles exhibited higher cytotoxicities toward cancer cells than unfunctionalized MPC-DPA micelles. Uptake studies confirmed that folate-conjugated micelles led to increased drug uptake within cancer cells, demonstrating the expected selectivity toward these tumor cells.     

Separation and recovery of intact gold-virus complex by agarose electrophoresis and electroelution: application to the purification of cowpea mosaic virus and colloidal gold complex

Colloidal gold has been coupled to a mutant cowpea mosaic virus (CPMV), which contains 60 cysteine residues on the surface. A purification process was developed to separate the gold-containing viral nanoblocks (VNBs) from the free gold. Agarose electrophoresis was utilized to separate the mixture followed by electroelution of the desired sample to recover the intact virus. Mobility of Au-VNB and free colloidal gold was facilitated by the addition of thioctic acid (TA). 30% of the gold-containing virus was recovered after electroelution as determined by absorbance measurements. Histogram analysis of transmission electron microscopy (TEM) images demonstrated the efficient separation of gold-containing virus from free gold. TEM and scanning electron microscopy (SEM) images indicated that the virus was recovered intact. Monodisperse spherical particles of nominal size of 45 nm were observed under SEM.     

Targeting of cancer cells with ferrimagnetic ferritin cage nanoparticles

Protein cage architectures such as virus capsids and ferritins are versatile nanoscale platforms amenable to both genetic and chemical modification. Incorporation of multiple functionalities within these nanometer-sized protein architectures demonstrate their potential to serve as functional nanomaterials with applications in medical imaging and therapy. In the present study, we synthesized an iron oxide (magnetite) nanoparticle within the interior cavity of a genetically engineered human H-chain ferritin (HFn). A cell-specific targeting peptide, RGD-4C which binds alphavbeta3 integrins upregulated on tumor vasculature, was genetically incorporated on the exterior surface of HFn. Both magnetite-containing and fluorescently labeled RGD4C-Fn cages bound C32 melanoma cells in vitro. Together these results demonstrate the capability of a genetically modified protein cage architecture to serve as a multifunctional nanoscale container for simultaneous iron oxide loading and cell-specific targeting.     

Comparison of electrical properties of viruses studied by AC capacitance scanning probe microscopy

Capacitances of five types of viruses, adenovirus type 5 (AV5), herpes simplex virus type 1 (HSV1), simian virus 40 (SV40), vaccinia (MVA), and cowpea mosaic virus (CPMV), were compared by AC capacitance scanning probe microscopy. This technique, using a Pt-coated AFM tip as an electrode to probe capacitance of materials between the tip and a bottom electrode, has been applied to study surface structures of semiconductors and polymers with nanometer spatial resolution; however, biological samples at the nanoscale have not been explored by this technique yet. Because most biological cells are poor conductors, this approach to probe electric properties of cells by capacitance is logical. This scanning probe technique showed that each virus has distinguishable and characteristic capacitance. A series of control experiments were carried out using mutant viruses to validate the origin of the characteristic capacitance responses for different viruses. A mutation on the capsid in HSV1 with green fluorescence proteins increased capacitance from 9 x 10(-6) to 1 x 10(-5) F/cm2 at the frequency of 10(4) Hz. Herpes simplex virus type 2 (HSV2) decreased capacitance when its envelope and glycoproteins were chemically extracted. These control experiments indicate that dielectric properties of capsid proteins and envelope glycoproteins significantly influence overall dielectric constants of viruses. Because those capsid proteins and glycoproteins are characteristic of the virus strain, this technique could be applied to detect and identify viruses at the single viron level using their distinct capacitance spectra as fingerprints without labeling.     

Bio‐orthogonal AIE Dots Based on Polyyne‐Bridged Red‐emissive AIEgen for Tumor Metabolic Labeling and Targeted Imaging

In this work, we aim to develop cancer cell‐targeting AIE dots based on a polyyne‐bridged red‐emissive AIEgen, 2TPE‐4E, through the combination of metabolic engineering and bio‐orthogonal reactions. Azide groups on a tumor were efficiently produced by intravenous injection of Ac4ManNAz and glycol‐metabolic engineering. These bio‐orthogonal azide groups could facilitate the specific targeting of DBCO‐AIE dots to the tumor cells undergoing metal‐free click reaction in vivo. The efficiency of this targeting strategy could be further improved with the development of new bio‐orthogonal chemical groups with higher reactivity and a large amount of AIEgens could be delivered to the tumor for diagnosis.