[show abstract][hide abstract] ABSTRACT: Detection of atherosclerosis has generally been limited to the late stages of development, after cardiovascular symptoms present or a clinical event occurs. One possibility for early detection is the use of functionalized nanoparticles. The aim of this study was the early imaging of atherosclerosis using nanoparticles with a natural affinity for inflammatory cells in the lesion.
We investigated uptake of cowpea mosaic virus by macrophages and foam cells in vitro and correlated this with vimentin expression. We also examined the ability of cowpea mosaic virus to interact with atherosclerotic lesions in a murine model of atherosclerosis.
We found that uptake of cowpea mosaic virus is increased in areas of atherosclerotic lesion. This correlated with increased surface vimentin in the lesion compared with nonlesion vasculature. In conclusion, cowpea mosaic virus and its vimentin-binding region holds potential for use as a targeting ligand for early atherosclerotic lesions, and as a probe for detecting upregulation of surface vimentin during inflammation.
[show abstract][hide abstract] ABSTRACT: Viral nanoparticles are a novel class of biomolecular agents that take advantage of the natural circulatory and targeting properties of viruses to allow the development of therapeutics, vaccines and imaging tools. We have developed a multivalent nanoparticle platform based on the cowpea mosaic virus (CPMV) that facilitates particle labeling at high density with fluorescent dyes and other functional groups. Compared with other technologies, CPMV-based viral nanoparticles are particularly suited for long-term intravital vascular imaging because of their biocompatibility and retention in the endothelium with minimal side effects. The stable, long-term labeling of the endothelium allows the identification of vasculature undergoing active remodeling in real time. In this study, we describe the synthesis, purification and fluorescent labeling of CPMV nanoparticles, along with their use for imaging of vascular structure and for intravital vascular mapping in developmental and tumor angiogenesis models. Dye-labeled viral nanoparticles can be synthesized and purified in a single day, and imaging studies can be conducted over hours, days or weeks, depending on the application.
[show abstract][hide abstract] ABSTRACT: Multivalent nanoparticle platforms are attractive for biomedical applications because of their improved target specificity, sensitivity, and solubility. However, their controlled assembly remains a considerable challenge. An efficient hydrazone ligation chemistry was applied to the assembly of Cowpea mosaic virus (CPMV) nanoparticles with individually tunable levels of a VEGFR-1 ligand and a fluorescent PEGylated peptide. The nanoparticles recognized VEGFR-1 on endothelial cell lines and VEGFR1-expressing tumor xenografts in mice, validating targeted CPMV as a nanoparticle platform in vivo.
[show abstract][hide abstract] ABSTRACT: Cowpea mosaic virus (CPMV) is a plant comovirus in the picornavirus superfamily, and is used for a wide variety of biomedical and material science applications. Although its replication is restricted to plants, CPMV binds to and enters mammalian cells, including endothelial cells and particularly tumor neovascular endothelium in vivo. This natural capacity has lead to the use of CPMV as a sensor for intravital imaging of vascular development. Binding of CPMV to endothelial cells occurs via interaction with a 54 kD cell-surface protein, but this protein has not previously been identified. Here we identify the CPMV binding protein as a cell-surface form of the intermediate filament vimentin. The CPMV-vimentin interaction was established using proteomic screens and confirmed by direct interaction of CPMV with purified vimentin, as well as inhibition in a vimentin-knockout cell line. Vimentin and CPMV were also co-localized in vascular endothelium of mouse and rat in vivo. Together these studies indicate that surface vimentin mediates binding and may lead to internalization of CPMV in vivo, establishing surface vimentin as an important vascular endothelial ligand for nanoparticle targeting to tumors. These results also establish vimentin as a ligand for picornaviruses in both the plant and animal kingdoms of life. Since bacterial pathogens and several other classes of viruses also bind to surface vimentin, these studies suggest a common role for surface vimentin in pathogen transmission.
[show abstract][hide abstract] ABSTRACT: There is a trend toward viral-based hybrid systems to furnish viral nanoparticles with enhanced features, for function beyond a delivery vehicle. Such hybrids have included Nanogold for microwave release, quantum dots and fluorescent moieties, to provide simultaneous imaging capabilities, and iron oxide particles for image enhancement in MRI. Other systems are the subject of ongoing and vigorous research. Nanogold surface decoration of cow pea mosaic virus (CPMV) to form NG-CPMV hybrids were explored to release fluorescent carriers using microwave energy as a model system in this presentation. Thus, emergent viral-based systems will have increasingly sophisticated architectures to provide versatile functions. Zeta potential (ZP) is a powerful tool to probe the electrostatic surface potential of biological materials and remains an untapped method for studying the interaction of nanoparticles with cells. An enormous effort is being made to study nanoparticle-cell interaction, but current throughput solutions (e.g., flow cytometry) cannot differentiate between surface-attached or endocytosed particles, while standard fluorescence microscopy is tedious and costly. CPMV-WT and other mutants (CPMV-T184C, CPMV-L189C) were studied using ZP methods and rationalized based on variations in their surface exposed residue character. Understanding such subtle changes can discretely alter the cell surface interactions due to charge affinity. Applying sensitive ZP measurements on viral nanoparticles is useful to elucidating the characteristics of the surface charge and the potential interaction modes with cell surfaces they may encounter. Thus, ZP can be a unique and efficient tool for studying cell-virus interactions and aid in development of future therapeutic strategies.
Current topics in microbiology and immunology 02/2009; 327:59-69. · 4.86 Impact Factor
[show abstract][hide abstract] ABSTRACT: A great challenge in biomedicine is the ability to target therapeutics to specific locations in the body in order to increase therapeutic benefit and minimize adverse effects. Virus-based nanotechnology takes advantage of the natural circulatory and targeting properties of viruses, in order to design therapeutics and vaccines that specifically target tissues of interest in vivo. Cowpea mosaic virus (CPMV) and flock house virus (FHV) nanoparticle-based strategies hold great promise for the design of targeted therapeutics, as well as for structure-based vaccine approaches.
Current topics in microbiology and immunology 02/2009; 327:95-122. · 4.86 Impact Factor
[show abstract][hide abstract] ABSTRACT: Extensively investigated and mutagenized Cow Pea Mosaic Virus (CPMV) has been demonstrated in a variety of nanoassemblies. [1–3] Iron Oxide (IO) has the potential to surpass limits of detection in bioimaging applications. Particularly g-Fe 2 O 3 (maghemite) is considered as one of the most desirable materials for technological and biomedical applications due to its inherent biocompatible nature. [4,5] Additionally, maghemite nanoparticles could be directed to an organ, tissue, or tumor using an external magnetic field or heated under an alternating magnetic field. [6,7] Based on the unique magnetic properties of IO nanoparticles they have been extensively used in biomedical applications, such as magnetic resonance imaging, targeting drug delivery and hyperthermia therapy detoxification and cell separation. [8–12] Combining the two systems can be devised to enhance the local magnetic field strength, by organizing monodisperse IO clusters on a CPMV-T184C mutant viral template. It is known that contrast enhancement is observed by use of super-paramagnetic iron oxide nanoparticles (SPIONs) based MRI, by creating large dipolar magnetic field gradients due to their local field inhomogeneity. However, clustering a greater number of IO nanoparticles can further improve contrast beyond free particle SPIONs enhanced MRI, by creating a cumulative dipole effect.  CPMV-T184C is a useful model that has a well characterized structure amenable to surface functionalization.  The smallest repeating structure (asymmetric unit, composed of a ''small'' (24kD) and ''large'' (42kD) subunit) displays 5 solvent exposed lysines used for IO linkage.  By insertion of a cysteine, at residue 184 of the small subunit, anchorage of CPMV to a self assembled monolayer (SAM) on gold substrate pathway can be employed. A previously reported SAM on Au stepwise assembly was used to integrate monodisperse CPMV-IO hybrids for characterization.  It is also been shown that aggregation of Iron oxide particles can exhibit a greater magnetic dipole, and can be suited for in bio-imaging, provided certain properties are met. Harris et al.,  demonstrated protease activated aggregation of pegy-lated iron oxide nanoparticles with enhanced MRI contrast to be most beneficial in improving detection limits of small tumors. Pegylation of CPMV was previously demonstrated to improve circulation times and reduce immunogenicity.  Also, based on enhanced permeability and retention effects (EPR),  the longest retention times at tumor sites for nanoparticles occurred for 60–400 nm.  Above 300 nm, there is vulnerability to macrophage phagocytosis,  and below 10 nm, nanoparticles can leave the systemic circulation via the lymph nodes.  Therefore, the IONs-CPMV nanoparticle hybrid system synthe-sized and MFM characterized in this report could be used for contrast enhanced MRI applications. In this work the local enhancement of field strength is studied and demonstrated by magnetic force microscopy (MFM) characterization of CPMV-IO hybrids bound to a substrate by a stepwise assembly process (Fig. 1). AFM was used to characterize structurally the as-synthesized IO nanoparticles on a silicon substrate (Fig. 2A). In addition, a histogram of the size distribution of the IO nanoparticles (Inset Fig. 2A) determined from 68 individual measurements on single IO nanoparticles exhibited a mean size of $11 nm.
[show abstract][hide abstract] ABSTRACT: Cowpea Mosaic Virus (CPMV) is increasingly being used as a nanoparticle platform for multivalent display of molecules via chemical bioconjugation to the capsid surface. A growing variety of applications have employed the CPMV multivalent display technology including nanoblock chemistry, in vivo imaging, and materials science. CPMV nanoparticles can be inexpensively produced from experimentally infected cowpea plants at high yields and are extremely stable. Although CPMV has not been shown to replicate in mammalian cells, uptake in mammalian cells does occur in vitro and in vivo. Thus, inactivation of the virus RNA genome is important for biosafety considerations, however the surface characteristics and chemical reactivity of the particles must be maintained in order to preserve chemical and structural functionality.
Short wave (254 nm) UV irradiation was used to crosslink the RNA genome within intact particles. Lower doses of UV previously reported to inactivate CPMV infectivity inhibited symptoms on inoculated leaves but did not prohibit systemic virus spread in plants, whereas higher doses caused aggregation of the particles and an increase in chemical reactivity further indicating broken particles. Intermediate doses of 2.0-2.5 J/cm(2) were shown to maintain particle structure and chemical reactivity, and cellular binding properties were similar to CPMV-WT.
These studies demonstrate that it is possible to inactivate CPMV infectivity while maintaining particle structure and function, thus paving the way for further development of CPMV nanoparticles for in vivo applications.
PLoS ONE 02/2008; 3(10):e3315. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Cowpea mosaic virus (CPMV) is a well-characterized nanoparticle that has been used for a variety of nanobiotechnology applications. CPMV interacts with several mammalian cell lines and tissues in vivo. To overcome natural CPMV targeting and redirect CPMV particles to cells of interest, we attached a folic acid-PEG conjugate by using the copper-catalyzed azide-alkyne cycloaddition reaction. PEGylation of CPMV completely eliminated background binding of the virus to tumor cells. The PEG-folate moiety allowed CPMV-specific recognition of tumor cells bearing the folate receptor. In addition, by testing CPMV formulations with different amounts of the PEG-FA moiety displayed on the surface, we show that higher-density loading of targeting ligands on CPMV may not be necessary for efficient targeting to tumor cells. These studies help to define the requirements for efficiently targeting nanoparticles and protein cages to tumors.
[show abstract][hide abstract] ABSTRACT: Virus-based nanoparticles (VNPs) from a variety of sources are being developed for biomedical and nanotechnology applications that include tissue targeting and drug delivery. However, the fate of most of those particles in vivo has not been investigated. Cowpea mosaic virus (CPMV), a plant comovirus, has been found to be amenable to the attachment of a variety of molecules to its coat protein, as well as to modification of the coat protein sequence by genetic means. We report here the results of studies of the bio-distribution, toxicology, and pathology of CPMV in mice. Plasma clearance and tissue biodistribution were measured using CPMV particles derivatized with lanthanide metal complexes. CPMV particles were cleared rapidly from plasma, falling to undetectable levels within 20 min. By 30 min the majority of the injected VNPs were trapped in the liver and to a lesser extent the spleen with undetectable amounts in other tissues. At doses of 1 mg, 10 mg and 100 mg per kg body weight, no toxicity was noted and the mice appeared to be normal. Hematology was essentially normal, although with the highest dose examined, the mice were somewhat leukopenic with relative decreases in both neutrophils and lymphocytes. Histological examination of the spleen showed cellular infiltration, which upon flow cytometry analyses revealed elevated B lymphocytes on the first day following virus administration that subsequently subsided. Microscopic evaluation of various other tissues revealed a lack of apparent tissue degeneration or necrosis. Overall, CPMV appears to be a safe and non-toxic platform for in vivo biomedical applications.
Journal of Controlled Release 08/2007; 120(1-2):41-50. · 7.63 Impact Factor
[show abstract][hide abstract] ABSTRACT: An electrical multi stability effect was observed for a single layer device fabricated, comprising a hybrid virus-semiconducting quantum dot ( Cd Se / Zn S core/shell Qds) assembled onto icosahedral-mutant-virus template (CPMV-T184C). A substrate based bottom-up pathway was used to conjugate two different color emitting Qds for fluorescence visualization and to insert a charging/decharging factor. Pulsed wave measurements depicted distinct conductive states with repeatable and nonvolatile behavior as a functioning memory element.
[show abstract][hide abstract] ABSTRACT: A significant impediment to the widespread use of noninvasive in vivo vascular imaging techniques is the current lack of suitable intravital imaging probes. We describe here a new strategy to use viral nanoparticles as a platform for the multivalent display of fluorescent dyes to image tissues deep inside living organisms. The bioavailable cowpea mosaic virus (CPMV) can be fluorescently labeled to high densities with no measurable quenching, resulting in exceptionally bright particles with in vivo dispersion properties that allow high-resolution intravital imaging of vascular endothelium for periods of at least 72 h. We show that CPMV nanoparticles can be used to visualize the vasculature and blood flow in living mouse and chick embryos to a depth of up to 500 microm. Furthermore, we show that the intravital visualization of human fibrosarcoma-mediated tumor angiogenesis using fluorescent CPMV provides a means to identify arterial and venous vessels and to monitor the neovascularization of the tumor microenvironment.
Nature Medicine 04/2006; 12(3):354-60. · 22.86 Impact Factor
[show abstract][hide abstract] ABSTRACT: Specific targeting of tumor cells is an important goal for the design of nanotherapeutics for the treatment of cancer. Recently, viruses have been explored as nano-containers for specific targeting applications, however these systems typically require modification of the virus surface using chemical or genetic means to achieve tumor-specific delivery. Interestingly, there exists a subset of viruses with natural affinity for receptors on tumor cells that could be exploited for nanotechnology applications. For example, the canine parvovirus (CPV) utilizes transferrin receptors (TfRs) for binding and cell entry into canine as well as human cells. TfRs are over-expressed by a variety of tumor cells and are widely being investigated for tumor-targeted drug delivery. We explored whether the natural tropism of CPV to TfRs could be harnessed for targeting tumor cells. Towards this goal, CPV virus-like particles (VLPs) produced by expression of the CPV-VP2 capsid protein in a baculovirus expression system were examined for attachment of small molecules and delivery to tumor cells. Structural modeling suggested that six lysines per VP2 subunit are presumably addressable for bioconjugation on the CPV capsid exterior. Between 45 and 100 of the possible 360 lysines/particle could be routinely derivatized with dye molecules depending on the conjugation conditions. Dye conjugation also demonstrated that the CPV-VLPs could withstand conditions for chemical modification on lysines. Attachment of fluorescent dyes neither impaired binding to the TfRs nor affected internalization of the 26 nm-sized VLPs into several human tumor cell lines. CPV-VLPs therefore exhibit highly favorable characteristics for development as a novel nanomaterial for tumor targeting.
Journal of Nanobiotechnology 02/2006; 4:2. · 5.09 Impact Factor
[show abstract][hide abstract] ABSTRACT: The plant virus, cowpea mosaic virus (CPMV), is increasingly being used as a nanoparticle platform for multivalent display of peptides. A growing variety of applications have employed the CPMV display technology including vaccines, antiviral therapeutics, nanoblock chemistry, and materials science. CPMV chimeras can be inexpensively produced from experimentally infected cowpea plants and are completely stable at 37 degrees C and low pH, suggesting that they could be used as edible or mucosally-delivered vaccines or therapeutics. However, the fate of CPMV particles in vivo, or following delivery via the oral route, is unknown. To address this question, we examined CPMV in vitro and in vivo. CPMV was shown to be stable under simulated gastric conditions in vitro. The pattern of localization of CPMV particles to mouse tissues following oral or intravenous dosing was then determined. For several days following oral or intravenous inoculation, CPMV was found in a wide variety of tissues throughout the body, including the spleen, kidney, liver, lung, stomach, small intestine, lymph nodes, brain, and bone marrow. CPMV particles were detected after cardiac perfusion, suggesting that the particles entered the tissues. This pattern was confirmed using methods to specifically detect the viral capsid proteins and the internal viral RNA. The stability of CPMV virions in the gastrointestinal tract followed by their systemic dissemination supports their use as orally bioavailable nanoparticles.
[show abstract][hide abstract] ABSTRACT: Successful postexposure treatment for inhalation anthrax is thought to include neutralization of anthrax toxin. The soluble anthrax toxin receptor/tumor endothelial marker 8 and capillary morphogenesis protein 2 (sATR/TEM8 and sCMG2, respectively) receptor decoys bind to anthrax toxin protective antigen (PA) and compete with cellular receptors for binding. Here, we show that, in a tissue-culture model of intoxication, sCMG2 is a 11.4-fold more potent antitoxin than sATR/TEM8 and that this increased activity corresponds to an approximately 1000-fold higher PA-binding affinity. Stoichiometric concentrations of sCMG2 protect rats against lethal toxin challenge, making sCMG2 one of the most effective anthrax antitoxins described to date.
The Journal of Infectious Diseases 10/2005; 192(6):1047-51. · 5.85 Impact Factor
[show abstract][hide abstract] ABSTRACT: Viruses are exemplary models in nanoassembly for their regular geometries, well characterized surface properties, and nanoscale dimensions. Armed with versatile tools aimed at site-directed mutagenesis to modify the virion's surface, conjugation chemistry for capsid coupling, and manipulation of nanoparticles, we have demonstrated nanoscale assembly of inorganic carbon nanotubes and quantum dots with engineered viruses to produce an intimate array of hybrid structures.