UBC Theses and Dissertations
Immobilization of antimicrobial peptides onto titanium surfaces Lu, Shanshan
Prosthetic-associated infections are one of the most challenging postoperative complications for orthopedic implants. The consequences that infections may lead to include patient pain, high cost, prolonged hospitalization time, and usually the revision of the implant. Current prophylaxis and therapy utilizing antibiotics are facing an emergency of increasing bacterial resistance; the design of a novel anti-infectious implant surface is therefore required. Among the potential antimicrobial alternatives are the antimicrobial peptides (AMP). AMPs are a family of natural defense peptides that has not received enough recognition until recently. The complex killing mechanisms of these cationic peptides make them very unlikely to encounter resistant mutants, and their broad-spectrum activity offers them great opportunity in possible clinical applications. In this study, a novel short AMP Tet213 with prominent bactericidal activity was chosen as the antimicrobial candidate and was covalently attached to titanium surfaces through a short bifunctional linker. This designed routine was confirmed with single cysteine before being applied to the 9-mer AMP candidate. The surface density of the immobilized AMP was determined by detecting its arginine residues after a reaction with 9,10-phenanthrequenon (PHQ). The reaction between arginine and PHQ generates a fluorescent product, by the emission of which the quantity of the arginine-containing peptide can be calculated. The density of the surface-attached Tet213 was measured to be 1.30±0.55 μg/cm². A relatively large proportion of physically adsorbed Tet213 was also observed, with the net adsorbed quantity to be 0.74±0.20 μg/cm². The affinity of the cationic AMP to the bare titanium surface is believed to be a result of electrostatic interactions. Both the covalently immobilized and the physically adsorbed Tet213 showed bactericidal activities of generally > 50% against a Pseudomonas aeruginosa (P. aeruginosa) strain which constitutively expresses luminescence when alive. The inhibition rate was calculated by the luminescence reduction and confirmed by the colony counts of the surviving bacteria. Several parameters were found to be influential to the overall inhibition rate, including the selection of the AMP candidate, the dilution of the bacterial culture and the bacterial incubation time.
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