Broad-spectrum antibacterial activities of activities of proteolytically stable self-assembled peptide and peptide/chitosan gels for biomaterial-related infections

Abstract

Biomaterial-related infections pose significant challenge on account of increasing antibiotic resistance. Pseudomonas aeruginosa and Staphylococcus aureus, resistant pathogens, are majorly responsible for such infections. To circumvent this problem, several strategies have been explored, which involves the use of silver ions, silver and gold nanoparticles, carbon nanotubes, and graphene but these strategies are associated with side effects like cytotoxicity, genotoxicity, inflammation, and oxidative DNA damage1,2. Therefore, the current focus of antibacterial research has shifted towards self-assembled gels fabricated from ultra-short peptides, as they often possess inherent antibacterial activities and mimic extracellular matrix but their use in biomedical applications is limited by their poor proteolytic stability3. The main focus of the present study was to develop self-assembled antibacterial peptide gels with enhanced proteolytic stability. In this context, we report the fabrication and characterization of self-assembled peptide gels, Boc-D-Phe-γ4-L-Phe-PEA (NH007) and Boc-L-Phe-γ4-L-Phe-PEA (NH009) in aqueous DMSO4. The surface morphology, secondary structures, rheological properties, self-healing characteristics, and swelling and degradation properties were investigated. Proteolytic stability studies showed that NH007 and NH009 undergo 23 and 77% degradation after 24 h of incubation with proteolytic enzymes, which is a significant improvement when compared to similar peptides reported in literature. These gels exhibited broad-spectrum antibacterial activities against Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, and Staphylococcus aureus at high inoculum of 107-108 cfu/mL. The gel fabricated from NH009 showed a maximum of 78% inhibition against Pseudomonas aeruginosa. Upon complexation with chitosan, the gels exhibited an improved and sustained antibacterial activity for a longer duration5. NH009/chitosan gels showed two times better proteolytic stability than NH009 gels, whereas NH007/chitosan gels showed four times enhanced stability than NH007 gels, which might be due to the complexation with chitosan, in addition to α/γ hybrid linkage. The gels fabricated from NH009/chitosan showed maximum inhibition of 81% against Pseudomonas aeruginosa, which is known for its multi-drug resistance properties. The SEM and HR-TEM studies revealed the entrapment of bacteria within the porous gel networks, followed by interaction with cell membrane components, and lysis. The bacterial live-dead cell assay indicated abundance of dead cells upon incubation with gels and the mechanism was further confirmed by investigating antibacterial activities of gels against mutant strains of E. coli. Cell viability and toxicity studies revealed that gels were not toxic towards mammalian cells. Thus, the gels developed in the current work can potentially be used to prevent biomaterial-related infections.