The immobilization of antimicrobial peptides (AMPs) to surfaces, enabling their utilization in biosensor and antibacterial/antifouling coating applications, is typically performed using rigid, solid support materials such as glass or gold and may require lengthy, temperamental protocols. Here, we employ a hydrogel immobilization platform to afford facile fabrication and surface functionalization while offering improved biocompatibility for evaluating the influence of linker length, surface density, and AMP conjugation site on retained peptide activity. Rapid, interfacial photo-polymerization using the radical-mediated thiol-ene addition mechanism was used to generate cross-linked, polymeric coatings bearing residual thiol moieties on prefabricated poly(ethylene glycol) (PEG)-based hydrogel supports. The photo-polymerized coatings were 60 μm thick and contained 0.55 nmol of unreacted free thiols, corresponding to a concentration of 410 μM, for use as cecropin A (CPA) immobilization handles via thiol-maleimide conjugation, where the CPA-bound maleimide moiety was localized at either the carboxyl terminus or midsequence between Ala22 and Gly23. Surface presentation of the thiol handles was controlled by varying the thiolated PEG monomer (PEGSH) used in the photo-polymerizable formulation. Bactericidal activity of CPA functionalized hydrogels against E. coli K235 indicated that CPA immobilized at the carboxyl terminus killed 94 ± 6% of the inoculated pathogens when coatings were prepared with high molecular weight PEGSH and 99 ± 1% when prepared with low molecular weight PEGSH. E. coli cell death demonstrated a stronger dependence on peptide concentration than PEG linker length or degree of thiol functionalization, with activity ranging from 34 ± 13% to 99 ± 1% bacterial cells killed as the prefunctionalization thiol concentration in the coatings was increased from 90 to 990 μM. Finally, the immobilization site on the surface-bound CPA strongly affected antibacterial activity; when midsequence modified CPA was bound to a hydrogel coating bearing 990 μM thiol, only 20 ± 4% of the E. coli population was killed.
Keywords: E. coli; antimicrobial peptide; cecropin A; hydrogel; immobilization.