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 μm. 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.Intravital vascular imaging has the potential to be a powerful tool for the noninvasive detection and visualization of disease. The resolution of functionally significant changes in structure in the endothelium of microvasculature using fluorescence imaging in live animals has proven challenging, however, because of the inadequate tissue penetration of fluorescent signal 1 .Current agents for fluorescence imaging of microvasculature include microspheres or nanospheres 2 , iron oxide particles 3 , liposomes 4 , dextrans 5 , lectins 6 , antibodies 7 and, more recently, quantum dots 8 . Although many of these particles have specific strengths, issues related to toxicity, stability, bioavailability, cost or chemical flexibility have yet to be overcome. Inorganic synthetic particles tend to aggregate under physiological conditions and can be toxic upon exposure to ultraviolet light 9 . Multivalency with respect to fluorochrome is crucial for achieving the requisite sensitivity for adequate tissue penetration. Thus, a multivalent, biologically compatible platform for the development of fluorescent and magnetic resonance imaging agents is still much needed for both clinical and research applications.

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 minutes. By 30 minutes 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 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.

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.