In vitro cytotoxicity profile and physicochemical parameters of a hypochlorous acid based wound irrigation solution

Wound irrigation is an essential step to clean the wound bed through cleansing, irrigating, moistening and debriding the wound, which facilitates healing in acute and chronic wound care. To minimize the occurrence of wound infection and biofilm formation, addition of antiseptics, such as polyhexanide (PHMB), povidone-iodine, octenidine dihydrochloride, sodium hypochlorite (NaClO)/hypochlorous acid (HClO) to the solution is an important therapeutic aspect. However, these antiseptics may have negative side effects on the skin cells, which could delay wound healing.  The objective of this study was to compare the cytotoxicity of a HClO based wound irrigation solution with NaClO and PHMB based wound solutions. Cytotoxicity was evaluated on human keratinocytes (HaCaT) and human dermal fibroblasts (HDFa) after 5, 10 and 15 minute exposure to the wound irrigation solutions at 25%, 50%, 75% and 100% with the neat solutions being at a concentration of ~0.033% HClO, 0.125% NaClO, or 0.1% PHMB. Cells were examined microscopically by evaluating the changes in morphology, vacuolization, detachment, cell lysis and membrane integrity. Cell counting was performed using the CyQuant cell proliferation assay (Thermofisher, UK). Additionally, pH and oxidation-reduction potential values (ORP) were measured for all the solutions at 5, 10 and 15 minutes. The cytotoxicity results showed that the HClO based solution was less cytotoxic than the NaClO and PHMB based wound solutions to both HaCaT and HDFa in all the test conditions. The cytotoxic activity on HaCaT was concentration dependant for all three wound solutions. The cytotoxic activity of the HClO based solution on HDFa was pH and concentration dependant due to HClO/hydrochlorite balance. The results indicate that the HClO based solution has a more favourable biocompatibility profile than the NaClO and PHMB based solutions. The study implies HClO/hypochlorite dynamic balance affects in vitro cytotoxicity, which is explained by the scientific evidence available for the HOCl/hypochlorite equilibrium.