In the absence of aqueous buffer, most enzymes retain little or no activity; however, "water-free" enzymes would have many diverse applications. Here, we describe the chemically precise immobilization of an enzyme on an engineered surface designed to support catalytic activity in air at ambient humidity. Covalent immobilization of haloalkane dehalogenase on a surface support displaying poly(sorbitol methacrylate) chains resulted in ∼40-fold increase in activity over lyophilized enzyme powders for the gas-phase dehalogenation of 1-bromopropane. The activity of the immobilized enzyme in air approaches 25% of the activity obtained in buffer for the immobilized enzyme. Poly(sorbitol methacrylate) appears to enhance activity by replacing protein-water interactions, thereby preserving the protein structure.

Silver is a widely used antimicrobial agent, yet when impregnated in macroscopic dressings, it stains wounds, can lead to tissue toxicity and can inhibit healing. Recently, we reported that polymeric nanofilms containing silver nanoparticles exhibit antimicrobial activity at loadings and release rates of silver that are 100x lower than conventional dressings. Here we report fabrication of composite microfilm constructs that provide a facile way to transfer the silver-loaded polymeric nanofilms onto wounds in vivo. The construct is fabricated from a silver nanoparticle-loaded polymeric nanofilm that is laminated with a micrometer-thick soluble film of polyvinylalcohol (PVA). When placed on a moist wound, the PVA dissolves, leaving the silver-loaded nanofilm immobilized on the wound-bed. In vitro, the immobilized nanofilms release <1 μg cm−2/day of silver over 30 days from skin-dermis and they kill 5 log10 CFUs of Staphylococcus aureus in 24 h. In mice, wounds inoculated with 105 CFU S. aureus presented up to 3 log10 less bacterial burden when treated with silver/nanofilms for 3 days, as compared to unmodified wounds. In uncontaminated wounds, silver/nanofilms allow normal and complete wound closure by re-epithelialization. We conclude that dissolvable microfilm constructs may overcome key limitations associated with current uses of silver in wound healing.

The persistence of bacterial biofilms in chronic wounds delays wound healing. Although Ga 3+ can inhibit or kill biofilms, precipitation as Ga(OH) 3 has prevented its use as a topical wound treatment. We report the design of a microfilm construct comprising a polyelectrolyte film that releases non-cytotoxic concentrations of Ga 3+ over 20 days and a dissolvable micrometer-thick film of polyvinylalcohol that enables facile transfer onto biomedically important surfaces. By using infrared spectroscopy, we show that the density of free carboxylate/carboxylic acid and amine groups within the polyelectrolyte film regulates the capacity of the construct to be loaded with Ga 3+ , and that the density of covalent cross-links introduced into the polyelectrolyte film (amide-bonds) controls the release rate of Ga 3+ . Following transfer onto the wound-contact surface of a biologic wound dressing, an optimized construct is demonstrated to release of ~0.7 µg cm −2Correspondence to: Jonathan F. McAnulty, mcanultj@svm.vetmed.wisc.edu; Charles J. Czuprynski, czuprync@svm.vetmed.wisc.edu; Nicholas L. Abbott, abbott@engr.wisc.edu. HHS Public Access Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript day −1 of Ga 3+ over 3 weeks, thus continuously replacing Ga 3+ lost to precipitation. The optimized construct inhibited formation of P. aeruginosa (two strains; ATCC 27853 and PA01) biofilms for up to 4 days and caused pre-existing biofilms to disperse. Overall, this study provides designs of polymeric constructs that permit facile modification of the wound-contacting surfaces of dressings and biomaterials to manage biofilms.

We report a general and facile method that permits the transfer (stacking) of multiple independently fabricated and nanoscopically thin polymeric films, each containing a distinct bioactive agent, onto soft biomedically relevant surfaces (e.g., collagen-based wound dressings). By using polyelectrolyte multilayer films (PEMs) formed from poly(allyl amine hydrochloride) and poly(acrylic acid) as representative polymeric nanofilms and micrometer-thick water-soluble poly(vinyl alcohol) sacrificial films to stack the PEMs, we demonstrate that it is possible to create stacked polymeric constructs containing multiple bioactive agents (e.g., antimicrobial and antibiofilm agents) on soft and chemically complex surfaces onto which PEMs cannot be routinely transferred by stamping. We illustrate the characteristics and merits of the approach by fabricating stacks of Ga (antibiofilm agent)- and Ag (antimicrobial agent)-loaded PEMs as prototypical examples of agent-containing PEMs and demonstrate that the stacked PEMs incorporate precise loadings of the agents and provide flexibility in terms of tuning release rates. Specifically, we show that simultaneous release of Ga and Ag from the stacked PEMs on collagen-based wound dressings can lead to synergistic effects on bacteria, killing and dispersing biofilms formed by Pseudomonas aeruginosa (two strains: ATCC 27853 and MPAO1) at sufficiently low loadings of agents such that cytotoxic effects on mammalian cells are avoided. The approach is general (a wide range of bioactive agents other than Ga and Ag can be incorporated into PEMs), and the modular nature of the approach potentially allows end-user functionalization of soft biological surfaces for programmed release of multiple bioactive agents.

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