Single-Point Mutations in Qß Virus-like Particles Change Binding to Cells.

Virus-like particles (VLPs) constitute large, polyvalent platforms onto which a wide variety of functional units can be grafted. Their use in biological settings often depends on their specific binding to cells or receptors of interest; this can be compromised by excessive nonspecific association with other cells. We found that lysine residues mediate such nonspecific interactions, presumably by virtue of protonation and interaction with anionic membrane lipid headgroups and/or complementary residues of cell surface proteins and polysaccharides. Chemical acylation of surface-exposed amines of the Qß VLP led to a significant reduction in the association of particles with mammalian cells. Single-point mutations of particular lysine residues to either glutamine, glutamic acid, tryptophan, or phenylalanine were mostly well-tolerated and formed intact capsids, but the introduction of double and triple mutants was far less forgiving. Introduction of glutamic acid at position 13 (K13E) led to a dramatic increase in cellular binding, whereas removal of the lysine at position 46 (K46Q) led to an equally striking reduction. Several plasma membrane components were found to specifically interact with the Qß capsid irrespective of surface charge. These results suggest that specific cellular interactions are engaged or obviated by such mutations and provide us with more "benign" particles to which can be added binding functionality for targeted delivery applications.

Metabolomics implicates altered sphingolipids in chronic pain of neuropathic origin.

Neuropathic pain is a debilitating condition for which the development of effective treatments has been limited by an incomplete understanding of its chemical basis. We show by using untargeted metabolomics that sphingomyelin-ceramide metabolism is altered in the dorsal horn of rats with neuropathic pain and that the upregulated, endogenous metabolite N,N-dimethylsphingosine induces mechanical hypersensitivity in vivo. These results demonstrate the utility of metabolomics to implicate unexplored biochemical pathways in disease.

A view from above: cloud plots to visualize global metabolomic data.

Global metabolomics describes the comprehensive analysis of small molecules in a biological system without bias. With mass spectrometry-based methods, global metabolomic data sets typically comprise thousands of peaks, each of which is associated with a mass-to-charge ratio, retention time, fold change, p-value, and relative intensity. Although several visualization schemes have been used for metabolomic data, most commonly used representations exclude important data dimensions and therefore limit interpretation of global data sets. Given that metabolite identification through tandem mass spectrometry data acquisition is a time-limiting step of the untargeted metabolomic workflow, simultaneous visualization of these parameters from large sets of data could facilitate compound identification and data interpretation. Here, we present such a visualization scheme of global metabolomic data using a so-called "cloud plot" to represent multidimensional data from septic mice. While much attention has been dedicated to lipid compounds as potential biomarkers for sepsis, the cloud plot shows that alterations in hydrophilic metabolites may provide an early signature of the disease prior to the onset of clinical symptoms. The cloud plot is an effective representation of global mass spectrometry-based metabolomic data, and we describe how to extract it as standard output from our XCMS metabolomic software.

Readily accessible fluorescent probes for sensitive biological imaging of hydrogen peroxide.

Hydrogen peroxide is a major component of oxygen metabolism in biological systems that, when present in high concentrations, can lead to oxidative stress in cells. Noninvasive molecular imaging of H(2)O(2) using fluorogenic systems represents an effective way to detect and measure the accumulation of this metabolite. Herein, we detail the development of robust H(2)O(2)-sensitive fluorescent probes using a boronic ester trigger appended to the fluorophore through a benzyl ether linkage. A major advantage of the probes presented here is their synthetic accessibility, with only one step needed to generate the probes on the gram scale. The sensitivity of the probes was evaluated in simulated physiological conditions, showing micromolar sensitivity to H(2)O(2). The probes were tested in biological model systems, demonstrating effective imaging of unstimulated, endogenous H(2)O(2) levels in RAW 264.7 cells and murine brain tissue.

Endocytic uptake pathways utilized by CPMV nanoparticles.

Cowpea mosaic virus (CPMV) has been used as a nanoparticle platform for biomedical applications including vaccine development, in vivo vascular imaging, and tissue-targeted delivery. A better understanding of the mechanisms of CPMV targeting and cell internalization would enable enhanced targeting and more effective delivery. Previous studies showed that, following binding and internalization by mammalian cells, CPMV localizes in a perinuclear late-endosome compartment where it remains for as long as several days. To further investigate endocytic trafficking of CPMV within the cell, we used multiple approaches including pharmacologic inhibition of pathways and colocalization with endocytic vesicle compartments. CPMV internalization was clathrin-independent and utilized a combination of caveolar endocytosis and macropinocytosis pathways for entry. CPMV particles colocalized with Rab5(+) early endosomes to traffic ultimately to a lysosomal compartment. These studies facilitate the further development of effective intracellular drug-delivery strategies using CPMV.

Lysine addressability and mammalian cell interactions of bacteriophage λ procapsids.

Chemically or genetically modified virus particles, termed viral nanoparticles (VNPs), are being explored in applications such as drug delivery, vaccine development, and materials science. Each virus platform has inherent properties and advantages based on its structure, molecular composition, and biomolecular interactions. Bacteriophage λ was studied for its lysine addressability, stability, cellular uptake, and the ability to modify its cellular uptake. λ procapsids could be labeled primarily at a single residue on the gpE capsid protein as determined by tandem mass spectrometry, providing a unique attachment site for further capsid modification. Bioconjugation of transferrin to the procapsids mediated specific interaction with transferrin receptor-expressing cells. These studies demonstrate the utility of bacteriophage λ procapsids and their potential use as targeted drug delivery vehicles.

Viral nanoparticles as tools for intravital vascular imaging.

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.

Quantifying mutant huntingtin protein in human cerebrospinal fluid to support the development of huntingtin-lowering therapies.

Huntington's disease (HD) is caused by a cytosine adenine guanine-repeat expansion in the huntingtin gene. This results in the production of toxic mutant huntingtin protein (mHTT), which has an elongated polyglutamine (polyQ) stretch near the protein's N-terminal end. The pharmacological lowering of mHTT expression in the brain targets the underlying driver of HD and is one of the principal therapeutic strategies being pursued to slow or stop disease progression. This report describes the characterisation and validation of an assay designed to quantify mHTT in the cerebrospinal fluid of individuals with HD, for use in registrational clinical trials. The assay was optimised, and its performance was characterised with recombinant huntingtin protein (HTT) varying in overall and polyQ-repeat length. The assay was successfully validated by two independent laboratories in regulated bioanalytical environments and showed a steep signal increase as the polyQ stretch of recombinant HTTs pivoted from wild-type to mutant protein forms. Linear mixed effects modelling confirmed highly parallel concentration-response curves for HTTs, with only a minor impact of individual slopes of the concentration-response for different HTTs (typically < 5% of the overall slope). This implies an equivalent quantitative signal behaviour for HTTs with differing polyQ-repeat lengths. The reported method may be a reliable biomarker tool with relevance across the spectrum of HD mutations, which can facilitate the clinical development of HTT-lowering therapies in HD.

Differential uptake of chemically modified cowpea mosaic virus nanoparticles in macrophage subpopulations present in inflammatory and tumor microenvironments.

There remains a tremendous need to develop targeted therapeutics that can both image and localize the toxic effects of chemotherapeutics and antagonists on diseased tissue while reducing adverse systemic effects. These needs have fostered the development of a nanotechnology-based approach that can combine targeting and toxicity potential. In this study, CPMV nanoparticles were chemically modified with the dye Alexa Flour 488 and were also tandemly modified with PEG1000 followed by AF488; and the derivatized nanoparticles were subsequently added to macrophages stimulated with either LPS (M1) or IL-4 (M2). Previously published studies have shown that M1/M2 macrophages are both present in an inflammatory microenvironment (such as a tumor microenvironment and atherosclerosis) and play opposing yet balancing roles; M2 macrophages have a delayed and progressive onset in the tumor microenvironment (concomitant with an immunosuppression of M1 macrophages). In this study, we show higher uptake of CPMV-AF488 and CPMV-PEG-AF488 by M2 macrophages compared to M1 macrophages. M1 macrophages showed no uptake of CPMV-PEG-AF488. More specifically, M2 macrophages are known to be up-regulated in early atherosclerosis plaque. Indeed, previous work showed that M2 macrophages in plaque also correlate with CPMV internalization. These studies emphasize the potential effectiveness of CPMV as a tailored vehicle for targeting tumor macrophages involved in cancer metastasis or vascular inflammation and further highlight the potential of CPMV in targeted therapeutics against other diseases.

Endothelial targeting of cowpea mosaic virus (CPMV) via surface vimentin.

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.

Multivalent display of proteins on viral nanoparticles using molecular recognition and chemical ligation strategies.

Multivalent display of heterologous proteins on viral nanoparticles forms a basis for numerous applications in nanotechnology, including vaccine development, targeted therapeutic delivery, and tissue-specific bioimaging. In many instances, precise placement of proteins is required for optimal functioning of the supramolecular assemblies, but orientation- and site-specific coupling of proteins to viral scaffolds remains a significant technical challenge. We have developed two strategies that allow for controlled attachment of a variety of proteins on viral particles using covalent and noncovalent principles. In one strategy, an interaction between domain 4 of anthrax protective antigen and its receptor was used to display multiple copies of a target protein on virus-like particles. In the other, expressed protein ligation and aniline-catalyzed oximation was used to display covalently a model protein. The latter strategy, in particular, yielded nanoparticles that induced potent immune responses to the coupled protein, suggesting potential applications in vaccine development.

Potato virus X as a novel platform for potential biomedical applications.

We demonstrate that nanoparticles formed from the rod-shaped plant virus Potato virus X (PVX) can serve as a novel platform for biomedical applications. Bioconjugation protocols including amine modification and "click" chemistry allowed the efficient functionalization of PVX with biotins, dyes, and PEGs. Fluorescent-labeled and PEGylated PVX particles revealed that different fluorescent labels have a profound effect on PVX-cell interactions. Applying bioconjugation chemistries to PVX opens the door for chemical functionalization with targeting and therapeutic molecules.

Hydrazone ligation strategy to assemble multifunctional viral nanoparticles for cell imaging and tumor targeting.

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.

Detection of carbohydrates and steroids by cation-enhanced nanostructure-initiator mass spectrometry (NIMS) for biofluid analysis and tissue imaging.

Nanostructure-initiator mass spectrometry (NIMS) is a highly sensitive, matrix-free technique that is well suited for biofluid analysis and imaging of biological tissues. Here we provide a new technical variation of NIMS to analyze carbohydrates and steroids, molecules that are challenging to detect with traditional mass spectrometric approaches. Analysis of carbohydrates and steroids was accomplished by spray depositing NaCl or AgNO(3) on the NIMS porous silicon surface to provide a uniform environment rich with cationization agents prior to desorption of the fluorinated polymer initiator. Laser desorption/ionization of the ion-coated NIMS surface allowed for Na(+) cationization of carbohydrates and Ag(+) cationization of steroids. The reliability of the approach is quantitatively demonstrated with a calibration curve over the physiological range of glucose and cholesterol concentrations in human serum (1-200 microM). Additionally, we illustrate the sensitivity of the method by showing its ability to detect carbohydrates and steroids down to the 800-amol and 100-fmol levels, respectively. The technique developed is well suited for tissue imaging of biologically significant metabolites such as sucrose and cholesterol. To highlight its applicability, we used cation-enhanced NIMS to image the distribution of sucrose in a Gerbera jamesonii flower stem and the distribution of cholesterol in a mouse brain. The flower stem and brain sections were placed directly on the ion-coated NIMS surface without further preparation and analyzed directly. The overall results reported underscore the potential of NIMS to analyze and image chemically diverse compounds that have been traditionally challenging to observe with mass spectrometry-based techniques.

Labeling live cells by copper-catalyzed alkyne--azide click chemistry.

The copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, optimized for biological molecules in aqueous buffers, has been shown to rapidly label mammalian cells in culture with no loss in cell viability. Metabolic uptake and display of the azide derivative of N-acetylmannosamine developed by Bertozzi, followed by CuAAC ligation using sodium ascorbate and the ligand tris(hydroxypropyltriazolyl)methylamine (THPTA), gave rise to abundant covalent attachment of dye-alkyne reactants. THPTA serves both to accelerate the CuAAC reaction and to protect the cells from damage by oxidative agents produced by the Cu-catalyzed reduction of oxygen by ascorbate, which is required to maintain the metal in the active +1 oxidation state. This procedure extends the application of this fastest of azide-based bioorthogonal reactions to the exterior of living cells.

Buckyballs meet viral nanoparticles: candidates for biomedicine.

Fullerenes such as C(60) show promise as functional components in several emerging technologies. For biomedical applications, C(60) has been used in gene- and drug-delivery vectors, as imaging agents, and as photosensitizers in cancer therapy. A major drawback of C(60) for bioapplications is its insolubility in water. To overcome this limitation, we covalently attached C(60) derivatives to Cowpea mosaic virus and bacteriophage Qbeta virus-like particles, which are examples of naturally occurring viral nanoparticle (VNP) structures that have been shown to be promising candidates for biomedicine. Two different labeling strategies were employed, giving rise to water-soluble, stable VNP-C(60) and VNP-PEG-C(60) conjugates. Samples were characterized using a combination of transmission electron microscopy, scanning transmission electron microscopy (STEM), gel electrophoresis, size-exclusion chromatography, dynamic light scattering, and Western blotting. "Click" chemistry bioconjugation using a poly(ethylene glycol) (PEG)-modified propargyl-O-PEG-C(60) derivative gave rise to high loadings of fullerene on the VNP surface, as indicated by the imaging of individual C(60) units using STEM. The cellular uptake of dye-labeled VNP-PEG-C(60) complexes in a human cancer cell line was found by confocal microscopy to be robust, showing that cell internalization was not inhibited by the attached C(60) units. These results open the door for the development of novel therapeutic devices with potential applications in photoactivated tumor therapy.

Efficient neutralization of antibody-resistant forms of anthrax toxin by a soluble receptor decoy inhibitor.

A soluble receptor decoy inhibitor (RDI), comprised of the extracellular I domain of ANTXR2, is a candidate anthrax therapeutic. Here we show that RDI can effectively neutralize altered forms of the protective antigen toxin subunit that are resistant to 14B7 monoclonal antibody neutralization. These data highlight the potential of RDI to act as an adjunct to existing antibody-based therapies and indicate that inhibitors based on RDI might be useful as a stand-alone treatment against specifically engineered strains of Bacillus anthracis.

Nanostructure initiator mass spectrometry: tissue imaging and direct biofluid analysis.

Nanostructure initiator mass spectrometry (NIMS) is a recently introduced matrix-free desorption/ionization platform that requires minimal sample preparation. Its application to xenobiotics and endogenous metabolites in tissues is demonstrated, where clozapine and N-desmethylclozapine were observed from mouse and rat brain sections. It has also been applied to direct biofluid analysis where ketamine and norketamine were observed from plasma and urine. Detection of xenobiotics from biofluids was made even more effective using a novel NIMS on-surface extraction method taking advantage of the hydrophobic nature of the initiator. Linear response and limit of detection were also evaluated for xenobiotics such as methamphetamine, codeine, alprazolam, and morphine, revealing that NIMS can be used for quantitative analysis. Overall, our results demonstrate the capacity of NIMS to perform sensitive, simple, and rapid analyses from highly complex biological tissues and fluids.

A viral nanoparticle with dual function as an anthrax antitoxin and vaccine.

The recent use of Bacillus anthracis as a bioweapon has stimulated the search for novel antitoxins and vaccines that act rapidly and with minimal adverse effects. B. anthracis produces an AB-type toxin composed of the receptor-binding moiety protective antigen (PA) and the enzymatic moieties edema factor and lethal factor. PA is a key target for both antitoxin and vaccine development. We used the icosahedral insect virus Flock House virus as a platform to display 180 copies of the high affinity, PA-binding von Willebrand A domain of the ANTXR2 cellular receptor. The chimeric virus-like particles (VLPs) correctly displayed the receptor von Willebrand A domain on their surface and inhibited lethal toxin action in in vitro and in vivo models of anthrax intoxication. Moreover, VLPs complexed with PA elicited a potent toxin-neutralizing antibody response that protected rats from anthrax lethal toxin challenge after a single immunization without adjuvant. This recombinant VLP platform represents a novel and highly effective, dually-acting reagent for treatment and protection against anthrax.

PEGylated viral nanoparticles for biomedicine: the impact of PEG chain length on VNP cell interactions in vitro and ex vivo.

PEGylation is an effective strategy for reducing biospecific interactions for pharmaceuticals. The plant virus Cowpea mosaic virus (CPMV) has been studied for potential nanobiomedical applications by virtue of its natural interactions with mammalian endothelial cells. To investigate the degree of PEGylation required to retarget CPMV-based formulations to other destinations, two CPMV-PEG formulations, CPMV-PEG1000 (P1) and CPMV-PEG2000 (P2) were tested. Modeling suggested that the PEG chains were displayed as flattened mushrooms on the particle with an estimated surface grafting area of 0.53% for P1 and 0.83% for P2. Only the P2 formulation effectively shielded the particles from interacting with cells or tissues, suggesting that either key interacting regions on the particle surface were blocked or that a sufficient hydration shell had been generated to inhibit cellular interactions. The large CPMV surface area available after PEGylation allows further attachment of imaging and therapeutic molecules to the particle to generate multifunctionality.