Evaluating the effect of plasma activated water on keratinocytes

Abstract

Rising antimicrobial resistance has highlighted the need for alternative and novel disinfection technologies. Cold atmospheric plasma and its application to solutions such as plasmaactivated water (PAW) fulfil the requirement of a novel technique with antimicrobial characteristics. The suitability of such a product for use as a hand sanitiser would require a balance between antimicrobial efficacy and safety for dermal application. To this end, immortalised keratinocyte cell lines N/TERT1 and N/TERT2G were exposed to PAW for 5 minutes, and cytotoxic markers were evaluated. Specifically, post-exposure cellular viability was assessed using a 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay, and the secretion of immune-response-related cytokines IL-1α, IL4, IL-6, and IL-8 was measured using ELISA. Membrane integrity was evaluated for lipid peroxidation via a TBARS assay, and potential mitochondrial dysregulation was assessed using JC-1 fluorescence. Cleaved caspase-3 was assessed as an apoptotic marker, and potential oxidative stress was evaluated by measuring Glutathione (GSH) concentrations and Superoxide Dismutase (SOD) activity. Exposure to PAW decreased viability similarly to water, to 90% in N/TERT1 and 80% in N/TERT2G; however, this was not statistically significant. No apoptotic induction or cellular dysregulation was indicated as a result of PAW exposure. The GSH concentration increased fivefold upon challenge with PAW in N/TERT2G (p < 0.05), but no such effect was observed in N/TERT1 keratinocytes. The SOD activity remained unaffected following exposure to PAW in both cell lines, indicating that the GSH pathway is sufficient to mitigate oxidative stress. No lipid peroxidation occurred in either N/TERT1 or N/TERT2G keratinocytes following exposure to PAW. Similarly, the mitochondrial membrane potential was also comparable to that of the untreated control cells. Together, these findings demonstrate that transient application of PAW is safe for dermal use. Slight variations in the responses from both cell lines highlight the potential biological variation that can be found in a non-clonal population, emphasising the need for further studies to account for such variation. Moreover, testing must also be carried out on in vitro models to further ensure the safe use of PAW as a topical disinfectant.