Don’t Stop Me Now: Investigating the Influence of Novel Antimicrobial EPC-373K on Keratinocyte Migration In-Vitro

Introduction: Public health concerns over antibiotic-resistant pathogens has spurred the development of non-traditional antimicrobials. These non-traditional antimicrobials offer advantages over conventional antibiotics; however, their cytotoxicity is often called into question.

This study explores the effect of a novel, antimicrobial on epidermal keratinocyte cell viability, migration, and signaling pathways.

Method: The cytotoxicity of EPC-373K on human epithelial keratinocytes (HaCaT) cells was evaluated in-vitro using an MTT cell viability assay. Confluent HaCaT cells treated with a solution of DMEM cell media containing EPC-373K over a concentration range of 1 µM to 50 µM for a period of 2-hours and 24-hours at 37˚C in triplicate and subsequently evaluated for cell viability using an MTT assay kit versus untreated control. Keratinocyte gap wound closure in response EPC-373K was evaluated against an untreated control. HaCaT cells were exposed to a solution of DMEM and EPC-373 and imaged over a 24-hours using a VivaView microscope and quantified with TScratch software. Cytokine & chemokine quantification was performed at 4-hour gap migration time point via a multi-plex human assay on the cell media supernatant for EPC-373K versus untreated control.

Results: In the present work, epidermal keratinocyte HaCaT cells maintained a minimum viability of 74% following a 24-hour exposure to concentrations up to 50 µM of EPC-373K. Keratinocyte gap assays shows that EPC-373K treated cells exhibit accelerated wound migration (gap closure at 12 hrs) in comparison to untreated control trials (gap closure at 20-hrs). Decreased abundance IL-1β (-1.6 fold) and increased abundance of VEGF-A (1.8 fold), EGF (2.8 fold), and PDGF-AA/BB (0.42 fold) were observed for EPC-373K versus untreated control.

Discussion: Within in-vitro keratinocyte migration assays, EPC-373K promotes wound migration versus untreated controls. IL-1b is well established as an immune system modulator and lymphocyte chemoattractant. IL-1b depression may be indicative of an anti-inflammatory response. EGF enhances migratory responses of keratinocytes, contributing to wound remodeling. VEGF-A is integral early in the wound healing process, promoting angiogenesis through endothelial cell migration. Upregulation of these cytokines may have implications in pro-healing and angiogenesis. Further in-vivo and paracrine signaling studies are required to verify these findings.