Design, characterization, and evaluation of antibacterial gels, Boc-D-Phe-γ4-L-Phe-PEA/chitosan and Boc-L-Phe-γ4-L-Phe-PEA/chitosan, for biomaterial-related infections.

Self-assembled peptide gels have generated interest as antibacterial materials to prevent biomaterial-related infections but these peptides are often associated with poor proteolytic stability. Efforts have been made to stabilize peptides by incorporating non-natural amino acids and/or linkages but complexation with polymers have not been explored. Therefore, we developed self-assembled peptide/chitosan gels, Boc-D-Phe-γ4-L-Phe-PEA (NH007)/chitosan and Boc-L-Phe-γ4-L-Phe-PEA (NH009)/chitosan, by complexing dipeptide NH007 or NH009 with chitosan in DMSO:acetic acid. The gels were characterized using SEM, FTIR, contact angle, and rheology data and found to exhibit excellent viscoelastic and self-healing characteristics. Complexation with chitosan led to an increase in stability against proteolytic degradation. Peptide/chitosan gels showed broad spectrum antibacterial activities against Gram-negative and Gram-positive bacteria, such as Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis at a high inoculum of 107-108 cfu/mL. NH007/chitosan gels showed 70-75% inhibition, whereas NH009/chitosan showed 78-81% inhibition and NH009/chitosan gels, in particular, showed strong antibacterial activity against pathogenic strain of P. aeruginosa. A unique feature of these gels is that the antibacterial activities did not decrease gradually but were sustained for up to 48 h. The mechanistic studies using SEM and HR-TEM indicated interaction of gels with bacterial membrane components, leading to cell lysis. The MTT and LDH assays indicated >90% cell viability and only 8-10% toxicity towards NIH 3T3 fibroblast cells. Thus, peptide/chitosan gels developed in the present work showed improved proteolytic stability and sustained antibacterial activities and, therefore, may be used for preventing biomaterial-related infections.