Antifungal coatings by caspofungin immobilization onto biomaterials surfaces via a plasma polymer interlayer.

Not only bacteria but also fungal pathogens, particularly Candida species, can lead to biofilm infections on biomedical devices. By covalent grafting of the antifungal drug caspofungin, which targets the fungal cell wall, onto solid biomaterials, a surface layer can be created that might be able to provide long-term protection against fungal biofilm formation. Plasma polymerization of propionaldehyde (propanal) was used to deposit a thin (∼20 nm) interfacial bonding layer bearing aldehyde surface groups that can react with amine groups of caspofungin to form covalent interfacial bonds for immobilization. Surface analyses by x-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry confirmed the intended grafting and uniformity of the coatings, and durability upon extended washing. Testing for fungal cell attachment and ensuing biofilm formation showed that caspofungin retained activity when covalently bound onto surfaces, disrupting colonizing Candida cells. Mammalian cytotoxicity studies using human primary fibroblasts indicated that the caspofungin-grafted surfaces were selective in eliminating fungal cells while allowing attachment and spreading of mammalian cells. These in vitro data suggest promise for use as antifungal coatings, for example, on catheters, and the use of a plasma polymer interlayer enables facile transfer of the coating method onto a wide variety of biomaterials and biomedical devices.

Surface coatings with covalently attached caspofungin are effective in eliminating fungal pathogens.

In this work we have prepared surface coatings formulated with the antifungal drug caspofungin, an approved pharmaceutical lipopeptide compound of the echinocandin drug class. Our hypothesis was to test whether an antifungal drug with a known cell-wall disrupting effect could be irreversibly tethered to surface coatings and kill (on contact) biofilm-forming fungal human pathogens from Candida spp. The first aim of the study was to use surface analysis to prove that the chemical binding to the surface polymer interlayer was through specific and irreversible bonds (covalent) and not due to non-specific adsorption through weak forces that could be later reversed (physisorption). Secondly, we quantified the antifungal nature of these coatings in a biological assay showing excellent killing against C. albicans and C. tropicalis and moderate killing against C. glabrata and C. parapsilosis. We concluded that caspofungin retains antifungal activity even when it is irreversibly immobilized on a surface, providing a new insight into its mechanism of action. Thus, surface coatings that have echinocandins permanently bound will be useful in preventing the establishment of fungal biofilms on materials.

Solid-state capture and real-time analysis of individual T cell activation via self-assembly of binding multimeric proteins on functionalized materials surfaces.

Polyfunctional T cell responses are increasingly underpinning new and improved vaccination regimens. Studies of the nature and extent of these T cell responses may be facilitated if specific T cell populations can be assessed from mixed populations by ligand-mediated capture in a solid-state assay format. Accordingly, we report here the development of a novel strategy for the solid-state capture and real-time activation analyses of individual cognate T cells which utilizes a spontaneous self-assembly process for generating multimers of biotinylated class I major histocompatibility-peptide complex (MHCp) directly on the solid-state assay surface while also ensuring stability by covalent interfacial binding. The capture surface was constructed by the fabrication of multilayer coatings onto standard slides. The first layer was a thin polymer coating with surface aldehyde groups, onto which streptavidin was covalently immobilized, followed by the docking of multimers of biotinylated MHCp or biotinylated anti-CD45.1 monoclonal antibody. The high binding strength at each step of this immobilization sequence aims to ensure that artefacts such as (partial) detachment, or displacement by proteins from solution, would not interfere with the intended biological assays. The multilayer coating steps were monitored by X-ray photoelectron spectroscopy; data indicated that the MHCp proteins self-assembled in a multimeric form onto the streptavidin surface. Immobilized multimeric MHCp demonstrated the capacity to bind and retain antigen-specific T cells from mixed populations of cells onto the solid carrier. Furthermore, real-time confocal microscopic detection and quantification of subsequent calcium flux using paired fluorescent ratiometric probes facilitated the analysis of individual T cell response profiles, as well as population analyses using a combination of individual T cell events.

Enhanced molecular chaperone activity of the small heat-shock protein alphaB-cystallin following covalent immobilization onto a solid-phase support.

The well-characterized small heat-shock protein, alphaB-crystallin, acts as a molecular chaperone by interacting with unfolding proteins to prevent their aggregation and precipitation. Structural perturbation (e.g., partial unfolding) enhances the in vitro chaperone activity of alphaB-crystallin. Proteins often undergo structural perturbations at the surface of a synthetic material, which may alter their biological activity. This study investigated the activity of alphaB-crystallin when covalently bound to a support surface; alphaB-crystallin was immobilized onto a range of solid material surfaces, and its characteristics and chaperone activity were assessed. Immobilization was achieved via a plasma-deposited thin polymeric interlayer containing aldehyde surface groups and reductive amination, leading to the covalent binding of alphaB-crystallin lysine residues to the surface aldehyde groups via Schiff-base linkages. Immobilized alphaB-crystallin was characterized by X-ray photoelectron spectroscopy, atomic force microscopy, and quartz crystal microgravimetry, which showed that 300 ng cm(-2) (dry mass) of oligomeric alphaB-crystallin was bound to the surface. Immobilized alphaB-crystallin exhibited a significant enhancement (up to 5000-fold, when compared with the equivalent activity of alphaB-crystallin in solution) of its chaperone activity against various proteins undergoing both amorphous and amyloid fibril forms of aggregation. The enhanced molecular chaperone activity of immobilized alphaB-crystallin has potential applications in preventing protein misfolding, including against amyloid disease processes, such as dialysis-related amyloidosis, and for biodiagnostic detection of misfolded proteins.

Fimbrolide-coated antimicrobial lenses: their in vitro and in vivo effects.

PURPOSE: To examine the ability of contact lenses coated with fimbrolides, inhibitors of bacterial quorum sensing, to prevent microbial adhesion and their safety during short-term clinical assessment. METHODS: A fimbrolide was covalently attached to commercially available high Dk contact lenses. Subsequently Pseudomonas aeruginosa, Staphylococcus aureus, Serratia marcescens, or Acanthamoeba sp. were added to the lenses and control uncoated contact lenses. Lenses plus microbes were incubated for 24 h, then washed thoroughly to remove non-adherent microbes. Lenses were macerated and resulting slurry plated onto agar plates. After appropriate incubation, the numbers of colony forming units of bacteria (or numbers of Acanthamoeba trophozoites measured using a hemocytometer) from fimbrolide-coated and uncoated lenses were examined. A Guinea Pig model of lens wear was used to assess the safety of lenses worn on a continuous basis for 1 month. In a separate study, 10 subjects wore fimbrolide-coated lenses for 24 h. The responses of the Guinea Pigs and human volunteers to the lenses were assessed by slit lamp examination. RESULTS: The fimbrolides-coated lenses reduced the adhesion of all bacterial strains tested, with reductions occurring of between 67 and 92%. For Acanthamoeba a reduction of 70% was seen. There were no significant differences in ocular responses to fimbrolide-coated lenses compared with controls in either the 1 month animal model or overnight human trial. CONCLUSIONS: Fimbrolide-coated lenses show promise as an antibacterial and anti-acanthamoebal coating on contact lenses and appear to be safe when worn on the eye in an animal model.