Virus-like particles (VLPs) are unique macromolecular structures that hold great promise in biomedical and biomaterial applications. The interior of the 30 nm-diameter Qβ VLP was functionalized by a three-step process: (1) hydrolytic removal of endogenously packaged RNA, (2) covalent attachment of initiator molecules to unnatural amino acid residues located on the interior capsid surface, and (3) atom-transfer radical polymerization of tertiary amine-bearing methacrylate monomers. The resulting polymer-containing particles were moderately expanded in size; however, biotin-derivatized polymer strands were only very weakly accessible to avidin, suggesting that most of the polymer was confined within the protein shell. The polymer-containing particles were also found to exhibit physical and chemical properties characteristic of positively charged nanostructures, including the ability to easily enter mammalian cells and deliver functional small interfering RNA.
virus-like particle; ATRP; polymerization; encapsulation; unnatural amino acid
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.
Hydrogen peroxide (H2O2) is a major component of oxygen metabolism in biological systems that, when present in high concentrations, can lead to oxidative stress in cells. Non-invasive molecular imaging of H2O2 using fluorogenic systems represents an effective way to detect and measure the accumulation of this metabolite. Herein, we detail the development of robust H2O2-sensitive fluorescent probes using a boronic ester trigger appended to the fluorophore via 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 H2O2. The probes were tested in biological model systems, demonstrating effective imaging of unstimulated, endogenous H2O2 levels in RAW 264.7 cells and murine brain tissue.
hydrogen peroxide; fluorescent probes; reactive oxygen species
Global metabolomics describes the comprehensive analysis of small molecules in a biological system without bias. With mass spectrometry-based methods, global metabolomic datasets 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 datasets. 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 by using a so-called “cloud plot” to represent multi-dimensional 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.
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 co-localization with endocytic vesicle compartments. CPMV internalization was clathrin-independent, and utilized a combination of caveolar endocytosis and macropinocytosis pathways for entry. CPMV particles co-localized 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.
Cowpea mosaic virus (CPMV); endocytosis; entry; caveolae; macropinocytosis; clathrin; Rab5; Rab11
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.
Cowpea mosaic virus (CPMV); macrophages; M1; M2; nanoparticle; internalization; therapeutics
Viral nanoparticles (VNPs) are structurally regular, highly stable, tunable nanomaterials that can be conveniently produced in high yields. Unmodified VNPs from plants and bacteria generally do not show tissue specificity or high selectivity in binding to or entry into mammalian cells. They are, however, malleable by both genetic and chemical means, making them useful scaffolds for the display of large numbers of cell- and tissue-targeting ligands, imaging moieties, and/or therapeutic agents in a well-defined manner. Capitalizing on this attribute, we modified the genetic sequence of the Cowpea mosaic virus (CPMV) coat protein to display an RGD oligopeptide sequence derived from human adenovirus type 2 (HAdV-2) Concurrently, wild-type CPMV was modified via NHS acylation and Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) chemistry to attach an integrin-binding cyclic RGD peptide. Both types of particles showed strong and selective affinity for several different cancer cell lines that express RGD-binding integrin receptors.
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.
Materials & methods
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.
Results & conclusions
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.
aorta; atherosclerosis; cowpea mosaic virus; foam cell; imaging; lesion; macrophage; surface vimentin; targeting; viral nanoparticle
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 up-regulated, endogenous metabolite N,N-dimethylsphingosine induces mechanical hypersensitivity in vivo. These results demonstrate the utility of metabolomics to implicate unexplored biochemical pathways in disease.
Lymphocytic choriomeningitis virus (LCMV) infection of mice is non-cytopathic, producing well-characterized changes reflecting host immune response. Untargeted metabolomics using mass spectrometry identified endogenous small molecule changes in blood from mice inoculated with LCMV, sampled at days 1, 3, 7 and 14 post infection. These represent well-characterized time points in acute LCMV infection and the immune response. Diverse pathways were altered, including TCA cycle intermediates, γ-glutamyl dipeptides, lysophosphatidyl cholines, and fatty acids. The kynurenine pathway was activated, surprising because it is stimulated by IFN-γ, which LCMV suppresses, thus suggesting alternative activators. In contrast, biopterin/neopterin, another IFN-γ stimulated pathway, was not activated. Many metabolites followed “response and recovery” kinetics, decreasing after infection to a minimum at days 3–7, and returning to normal by day 14. The TCA pathway followed this pattern, including citrate, cis-aconitate and α-ketoglutarate, intriguing because succinate has been shown to mediate cellular immunity. This response and recovery dynamic tracks the immune response, including the rise and fall of natural killer cell populations, serum TNF receptor concentration, and viral clearance. Metabolomics can provide target pathways for molecular diagnostics or therapeutics of viral infection and immunity.
untargeted metabolomics; systems biology; mass spectrometry; metabolism; innate immunity
Vimentin, a type III intermediate filament, is upregulated during epithelial–mesenchymal transition and tumor progression. Vimentin is surface-expressed on cells involved in inflammation; the function remains unknown. We investigated the expression of surface vimentin on cancer cells and evaluated targeting nanoparticles to tumors exploiting vimentin.
Materials & methods
Cowpea mosaic virus nanoparticles that interact with surface vimentin were used as probes. Tumor homing was tested using the chick chorioallantoic membrane model with human tumor xenografts.
Results & discussion
Surface vimentin levels varied during cell cycle and among the cell lines tested. Surface vimentin expression correlated with cowpea mosaic virus uptake, underscoring the utility of cowpea mosaic virus to detect invasive cancer cells. Targeting to tumor xenografts was observed; homing was based on the enhanced permeability and retention effect. Our data provide novel insights into the role of surface vimentin in cancer and targeting nanoparticles in vivo.
CAM model; cancer; cell cycle; CPMV; EPR effects; PEGylation; vimentin; viral nanoparticles
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 bio-imaging. 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 covalently display 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.
Viral nanoparticle; Flock House virus; virus-like particle; anthrax protective antigen; expressed protein ligation; aniline-catalyzed oxime ligation; vaccine development
Soluble receptor decoy inhibitors, including receptor-immunogloubulin (Ig) fusion proteins, have shown promise as candidate anthrax toxin therapeutics. These agents act by binding to the receptor-interaction site on the protective antigen (PA) toxin subunit, thereby blocking toxin binding to cell surface receptors. Here we have made the surprising observation that co-administration of receptor decoy-Ig fusion proteins significantly delayed, but did not protect, rats challenged with anthrax lethal toxin. The delayed toxicity was associated with the in vivo assembly of a long-lived complex comprised of anthrax lethal toxin and the receptor decoy-Ig inhibitor. Intoxication in this system presumably results from the slow dissociation of the toxin complex from the inhibitor following their prolonged circulation. We conclude that while receptor decoy-Ig proteins represent promising candidates for the early treatment of B. anthracis infection, they may not be suitable for therapeutic use at later stages when fatal levels of toxin have already accumulated in the bloodstream.
Next-generation targeted nanodevices are currently under development for imaging and therapeutic applications. We engineered HK97 viral nanoparticles (VNPs) for tumor cell-specific targeting.
A combination of genetic and chemical engineering methods were developed and applied to generate dual-labeled HK97 cysteine mutant particles displaying transferrin and fluorescent labels. The targeting properties of transferrin-conjugated VNPs were evaluated by in vitro experiments using different cancer cell lines.
We found that HK97–tranferrin formulations were indeed targeted to cancer cells in vitro via the transferrin receptor. These studies highlight the utility and facilitate the further development of HK97-based VNPs.
cancer; HK97; protein conjugation; targeted cellular entry; transferrin; viral nanoparticles
Cholesterol is an essential component of cellular membranes that is required for normal lipid organization and cell signaling. While the mechanisms associated with maintaining cholesterol homeostasis in the plasma and peripheral tissues have been well studied, the role and regulation of cholesterol biosynthesis in normal brain function and development have proven much more challenging to investigate. Smith-Lemli-Opitz syndrome (SLOS) is a disorder of cholesterol synthesis characterized by mutations of DHCR7 (7-dehydrocholesterol reductase) that impair the reduction of 7-dehydrocholesterol (7DHC) to cholesterol and lead to neurocognitive deficits, including cerebellar hypoplasia and austism behaviors. Here we have used a novel mass spectrometry-based imaging technique called cation-enhanced nanostructure-initiator mass spectrometry (NIMS) for the in situ detection of intact cholesterol molecules from biological tissues. We provide the first images of brain sterol localization in a mouse model for SLOS (Dhcr7−/−). In SLOS mice, there is a striking localization of both 7DHC and residual cholesterol in the abnormally developing cerebellum and brainstem. In contrast, the distribution of cholesterol in 1-day old healthy pups was diffuse throughout the cerebrum and comparable to that of adult mice. This study represents the first application of NIMS to localize perturbations in metabolism within pathological tissues and demonstrates that abnormal cholesterol biosynthesis may be particularly important for the development of these brain regions.
Smith-Lemli-Opitz syndrome; brain imaging; nanostructure-initiator mass spectrometry; cholesterol; metabolism; mass spectrometry
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.
click chemistry; cell labeling; metabolic labeling; bioconjugation
Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system and a leading cause of neurological disability. The complex immunopathology and variable disease course of multiple sclerosis have limited effective treatment of all patients. Altering the metabolism of immune cells may be an attractive strategy to modify their function during autoimmunity. We examined the effect of inhibiting fatty acid metabolism in experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. Mice treated with an inhibitor of carnitine palmitoyltransferase 1 (CPT-1), the rate-limiting enzyme in the beta-oxidation of fatty acids, showed a reduction in disease severity as well as less inflammation and demyelination. Inhibition of CPT-1 in encephalitogenic T-cells resulted in increased apoptosis and reduced inflammatory cytokine production. These results suggest that disruption of fatty acid metabolism promotes downregulation of inflammation in the CNS and that this metabolic pathway is a potential therapeutic target for multiple sclerosis.
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.
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 AgNO3 on the NIMS porous silicon surface to provide a uniform environment rich with cationization agents prior to desporption 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 μM). 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.
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.
Fullerenes such as C60 show promise as functional components in several emerging technologies. For biomedical applications, C60 has been used in gene- and drug-delivery vectors, as imaging agents, and as photosensitizers in cancer therapy. A major drawback of C60 for bioapplications is its insolubility in water. To overcome this limitation, we covalently attached C60 derivatives to Cowpea mosaic virus and bacteriophage Qβ virus-like particles, as 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 and stable VNP-C60 and VNP-PEG-C60 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 PEG-modified propargyl-O-PEG-C60 derivative gave rise to high loadings of fullerene on the VNP surface, indicated by the imaging of individual C60 units by STEM. The cellular uptake of dye-labeled VNP-PEG-C60 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 C60 units. These results open the door for the development of novel therapeutic devices with potential applications in photo-activated tumor therapy.
Targeted treatment of inflammatory diseases of the central nervous system (CNS) remains problematic due to the complex pathogenesis of these disorders and difficulty in drug delivery. The plant virus, cow pea mosaic virus (CPMV), has recently been explored as a nanoparticle delivery system for therapeutics targeting a number of diseases including cancer and neurodegeneration. To understand the biodistribution of CPMV in the CNS, we examined CPMV uptake during infection of mice with neurotropic mouse hepatitis virus (MHV). CPMV localized mainly to the CNS endothelium in areas that contained an intact blood brain barrier. However, in inflammatory lesions containing macrophage/microglial cell infiltration and IgG, CPMV could be detected in the brain parenchyma. Furthermore, CPMV showed rapid internalization in an in vitro model of the BBB. These results suggest that CPMV particles could be used to a vehicle to deliver therapeutics to the damaged CNS during neurodegenerative and infectious diseases of the CNS.
CPMV; blood brain barrier; nanoparticles; microglial cells; central nervous system; targeting
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.
Viral nanoparticles; Cowpea mosaic virus; PEGylation; Nanotechnology; Biomedicine
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.
The CDC recommend 60 days of oral antibiotics combined with a three-dose series of the anthrax vaccine for prophylaxis after potential exposure to aerosolized Bacillus anthracis spores. The anthrax vaccine is currently not licensed for anthrax postexposure prophylaxis and has to be made available under an Investigational New Drug protocol. Postexposure prophylaxis based on antibiotics can be problematic in cases where the use of antibiotics is contraindicated. Furthermore, there is a concern that an exposure could involve antibiotic-resistant strains of B. anthracis. Availability of alternate treatment modalities that are effective in prophylaxis of inhalation anthrax is therefore highly desirable. A major research focus toward this end has been on passive immunization using polyclonal and monoclonal antibodies against B. anthracis toxin components. Since 2001, significant progress has been made in isolation and commercial development of monoclonal and polyclonal antibodies that function as potent neutralizers of anthrax lethal toxin in both a prophylactic and therapeutic setting. Several new products have completed Phase I clinical trials and are slated for addition to the National Strategic Stockpile. These rapid advances were possible because of major funding made available by the US government through programs such as Bioshield and the Biomedical Advanced Research and Development Authority. Continued government funding is critical to support the development of a robust biodefense industry.
antibiotic treatment; biodefense funding; inhalation anthrax; lethal factor; medical countermeasures; prophylactic antibodies; protective antigen; vaccination