Probing the Effects of Plasma-Treated Water Solutions on Healthy and Head and Neck Cancer Cells via FTIR and Raman Spectroscopy

Building on evidence that plasma-treated water solutions (PTWS) exhibit antitumor activity, this study investigated their selective effects on healthy keratinocytes versus head and neck cancer (HNC) cells. PTWS were generated from clinically approved rehydrating solutions supplemented with tyrosine (SIII-Tyr) and treated with oxygen or air plasma at different treatment times. Human keratinocytes (HaCaT) and HNC (FaDu and SAS) cells were exposed to different PTWS formulations for 30 min followed by 24-hours incubation in culture medium. Cells were analysed using Fourier Transform Infrared (FTIR) and Raman spectroscopy, with principal components analysis (PCA) to identify biochemical changes related to cytotoxicity. Results were correlated with cell viability (MTT assay) and intracellular reactive oxygen species (ROS) levels (flow cytometry). HaCaT cells showed minimal sensitivity, while FaDu and SAS cells were significantly affected. Notably, SAS cells exhibited over 90% mortality after exposure to PTWS oxy 20’ sample. ROS levels increased in all cell lines following exposure to PTWS, but for HaCaT cells remained below the baseline ROS of untreated HNC. The highest ROS accumulation was observed in SAS cells treated with PTWS oxy 20’, aligning with cytotoxicity data. PCA of FTIR and Raman spectra revealed distinct biochemical signatures in HNC cells, particularly under PTWS oxy 20’ treatment exposure. Even under milder conditions (air 10’), significant spectral deviations between HNC and HaCaT cells suggested a potential window for selective action. These findings support PTWS selectivity against HNC cells. From a vibrational spectroscopy perspective, this study provides a novel, rapid, label-free tool combining FTIR with Raman to assess selective biochemical responses in HNC models. These results support future preclinical and translational applications.