Author(s): McKinley C. Lawson*, Kevin B. Hoth, Kristi S. Anseth, Christopher N. Bowman; University of Colorado Boulder, CO
Title: Living Radical Photopolymerization and the Rational Design of Surface-Active Antibiotic Polymers.
Purpose: The purpose of this report is to describe experiments in which living-radical surface-basedphotopolymerization was utilized to evaluate the activity of a polymerizable antibiotic species, vancomycin- PEG-acrylate, useful for biomaterial coatings.
Methods: First, a polymerizable vancomycin derivative was synthesized (vancomycin-PEG(3400)- acrylate) by reaction of vancomycin hydrochloride with N-hydroxysuccinimide-PEG(3400)-acrylate in an aprotic solvent. The product was recovered by gel filtration chromatography and chemically characterized by NMR spectroscopy and MALDI mass spectrometry. Subsequently, VPA(3400) was co-grafted in varying proportions with PEG(375)-acrylate and PEG(3400)- acrylate using a surface-mediated living-radical polymerization (LRP) dithiocarbamate substrate. Substrates were prepared from monomer formulations consisting of aromatic urethane diacrylate, triethylene glycol diacrylate, 1 wt% tetraethylthiuram disulfide, and 1.5 wt% 2,2-dimethoxy-2-phenylacetophenone initiator. Grafting solutions were formed by diluting VPA(3400) into PEG(375)-acrylate at varying concentrations, and surfaces were grafted by exposing solutions to 365 nm UV light using standard photomasking techniques. Grafted surfaces were washed copiously until elution of unattached antibacterial species had stopped. Surfaces were then challenged with bacterial suspensions of S. epidermidis ATCC 12228. Following incubation, suspensions were sampled, serially diluted, and plated for CFU counts.
Results: Vancomycin-PEG-acrylate (VPA) was purified by gel filtration chromatography and found to be of ca. 5,000 molecular weight based on MALDI-MS results (single adduct product). The product demonstrated structural features of vancomycin and PEG-acrylate by 1H NMR. When grafted from an LRP substrate, VPA demonstrated a dose-response trend, with no detectable bacteria on a 100 mg/mL graft (true bactericidal activity). This shows that VPA maintains activity when polymerized from a solid support and that LRP-type surfaces can be used to assess the surface-based activity of antibacterial polymers derived from polymerizable antibiotic species.
Discussion: Since polymerizable antibiotics are designed with the ultimate goal of modifying orthopaedicimplant materials, it is necessary to systematically study design factors that affect surface activity once these species have been polymerized to solid substrates. Living radical photopolymerization provides a uniquely efficient means of studying such parameters. LRP functionalized substrates allow surface mediated polymerizations with spatial and temporal control of polymer chain growth that traditional methods cannot duplicate. Typical LRP species are dithiocarbamate derivatives that, upon ultraviolet photolysis, yield a surfacebound carbon radical and a free dithiocarbamyl radical. The carbon radical can react with vinyl monomers (e.g., acrylates). The dithiocarbamyl radical can reversibly terminate growing polymer chains and allow for controlled free-radical polymerization, thus permitting precise control of polymer architecture on a substrate surface. Through this approach, the chain density, polymer chain length, and chain architecture can be varied. Here, we demonstrate that LRP methods are useful for studying polymerizable antibiotics that have promise for coating orthopaedic biomaterials.
