Surface Functionalization of Ti6Al4V-ELI Alloy with Antimicrobial Peptide Nisin

Implant-associated infections are a severe global concern, especially in the case of orthopedic implants intended for long-term or permanent use. The traditional treatment through systemic antibiotic administration is often inefficient due to biofilm formation, and concerns regarding the development of highly resistant bacteria. Therefore, there is an unfulfilled need for antibiotic-free alternatives that could simultaneously support bone regeneration and prevent bacterial infection. This study aimed to perform, optimize, and characterize the surface functionalization of Ti6Al4V-ELI discs by an FDA-approved antimicrobial peptide, nisin, known to hold a broad antibacterial spectrum. Accordingly, nisin bioactivity was also evaluated by in vitro release tests both in physiological and inflammatory pH conditions. Several methods, such as X-ray photoelectron spectroscopy (XPS), and Kelvin Probe atomic force microscopy confirmed the presence of a physisorbed nisin layer on the alloy surface. The functionalization performed at pH 6-7 was found to be especially effective due to the nisin configuration exposing its hydrophobic tail outwards, which is also responsible for its antimicrobial action. In addition, the first evidence of gradual nisin release both in physiological and inflammatory conditions was obtained: the static contact angle becomes half of the starting one after 7 days of soaking on the functionalized sample, while it becomes 0 degrees on the control samples. Finally, the evaluation of the antibacterial performance toward the pathogen Staphylococcus aureus after 24 h of inoculation showed the ability of nisin adsorbed at pH 6 to prevent bacterial microfouling into biofilm-like aggregates in comparison with the uncoated specimens: viable bacterial colonies showed a reduction of about 40% with respect to the un-functionalized surface and the formation of (microcolonies (biofilm-like aggregates) is strongly affected.