P. Melendez Navarro, M. Finkle, S.U. Ahmed, K. Vig
Alabama State University,
Keywords: alginate, Staphylococcus aureus, Ag-PVP, antimicrobial peptides
Summary:Skin tissue engineering has gained attention due to wound healing applications. Often, wounds get infected with bacteria such as Staphylococcus aureus, hence the need to develop a proficient material, with antimicrobial properties to prevent bacterial infection. The present study aims to develop an antimicrobial scaffold using polyvinylpyrrolidone coated silver nanoparticles (Ag-PVP) and the antimicrobial peptides, p753 and p359. Inhibitory action of agents against S. aureus was studied through Minimum Inhibitory Concentration (MIC) Assay, Kirby Disc Diffusion Assay (KB test), Live/dead Assay and Plate Count Assay. To determine toxicity of antimicrobial agents, MTT Assay was performed using HEK293 cells. The MIC of Ag-PVP, p753 and p359 was 100µg/mL, 6.25µg/mL, and 12.5µg/mL, respectively. The KB test displayed a zone of inhibition for Ag-PVP, p753 and p359 of 19mm, 14mm, and 12mm. MTT Assay exhibited that Ag-PVP and the peptides were non-toxic to cells at 100µg/mL and 50µg/mL, respectively. For the 3D model, 2% alginate hydrogels cross-linked with 2% CaCl2 were prepared and incubated with antibacterial agents overnight. Scaffolds were washed and seeded with 20,000 cells. After 24 hours, cells were infected with bacteria at Multiplicity of Infection (MOI) of ¬¬¬¬1:10 for studies. Live/dead analysis and plate count of treated bacteria exhibited bacterial inhibition on 3D cell culture compared to non-treated hydrogels. SEM was performed to analyze the structural changes of bacteria after treatment with antimicrobial agents. Gene expression studies were conducted on bacterial rna from bacteria treated with Ag-PVP and peptides using qRT-PCR. There was a down regulation for gene expression of sodA in S. aureus with Ag-PVP and peptides compared to the control. Results show that the antimicrobial agents are non-toxic to cells but inhibited S. aureus growth which thus can be of aid in the development of antibacterial skin scaffolds.