Enhancement in Sustained Release of Antimicrobial Peptide from Dual-Diameter-Structured TiO₂ Nanotubes for Long-Lasting Antibacterial Activity and Cytocompatibility

Novel films on Ti-based orthopedic implants for localized antimicrobial delivery, which comprises dual-diameter TiO₂ nanotubes with the inner layers of compact and fluorine-free oxide tightly bonding to Ti, were formed by voltage-increased anodization with F^- sedimentation procedure. The nanotubes were closely aligned and structured with upper 35 and 70 nm diametric tubes as nanocaps, respectively, and the underlying 140 nm diametric tubes as nanoreservoirs. Followed by loading ponericin G1 (a kind of antimicrobial peptide (AMP)) into the dual-diameter nanotubes with vacuum-assisted physisorption, the resultant films were investigated for loading efficiency and release kinetics of AMP, antibacterial activity against Staphylococcus aureus, and osteoblastic compatibility, together with the AMP-loaded single-diameter (140 nm) nanotube film. The loaded films had no statistical difference in the loading efficiency of AMP and revealed burst release within 6 h followed by steady release of AMP in phosphate-buffered solution. At day 42, almost all of AMP was released from the single-diameter nanotube film. However, the dual-diameter nanotube films loaded with AMP still showed sustained release at least up to 60 days, and the sustained efficacy was enhanced with decreasing diameter of nanocaps. In the case of nominal AMP loading amount of 125 μg, the resultant 35 nm capped dual-diameter nanotube film exhibited significant short- and long-term (even for 49 days) antibacterial activity not only against planktonic bacteria, which is ascribed to the release-killing efficacy of AMP, but also against adhered bacteria, which is ascribed to the AMP-derived killing efficacy and the nanocaps-derived adhesion resistance. Moreover, this loaded film presented cytocompatibility comparative to that of Ti but higher than that of the other AMP-loaded films. Increasing the nominal loading amount of AMP to 200 μg improved antibacterial activity but gave rise to obvious cytotoxicity of the loaded films.