Pre-clinical Safety Study of Cold Atmospheric Plasma (CAP) Produced by an Inbuilt CAP Device and ROS Mediated Apoptotic Activity in Human Skin Melanoma Cells

Introduction: In recent decades, Cold Atmospheric Plasma (CAP) has become increasingly popular in healthcare for managing diseases, especially skin cancer. This study aimed to assess the preclinical safety of an indigenously developed dielectric barrier discharge-CAP device and its cytotoxic efficacy against melanoma cells while adhering to OECD 402 guidelines for acute dermal toxicity study. The safety evaluation includes ex vivo studies on mouse peritoneal exudates and in vivo acute dermal toxicity tests on Wistar rats. Methods: The ex vivo study of mice peritoneal cells treated for up to 120 seconds, showed a survival rate of over 90% up to 90 seconds of CAP treatment for applied voltage 18.6 kV at 20 kHz with no significant difference with control. In the acute dermal toxicity tests, CAP exposure for up to 30 seconds caused minimal inflammatory cell infiltration and no significant Dermal Inflammation Scoring (DIS) (<1). Results: The efficacy study against G361 human melanoma cells showed reduced cell viability by ~50% (MTT assay) upon 30 seconds of CAP treatment for applied voltage 24 kV at 20 kHz through ROS-mediated apoptosis, confirmed by a 3-fold increase in intracellular reactive oxygen species levels and nuclear fragmentation (4',6-diamidino-2-phenylindole staining). Annexin V/PI (propium iodide) staining further revealed ~30% apoptosis after 24 hours of incubation. These findings establish the developed DBD-CAP device is safe for rat skin exposure durations of up to 30 seconds and effective in inducing apoptosis in melanoma cells. Conclusion: This study supports CAP's optimization for clinical applications and its integration with existing therapies for enhanced outcomes. However, further study is needed to examine the possible risks associated with using CAP devices in the biomedical field. result: Following exposure to CAP, the survival rate of treated mouse peritoneal cells remained safe for up to 90 seconds. The acute dermal toxicity test found that the CAP was safe up to 30 seconds of exposure, with no significant changes in the dermal inflammation score observed. However, Exposure to CAP for a duration of up to 30 seconds resulted in significant cell death as a result of an elevated production of intracellular reactive oxygen species (ROS), leading to apoptosis as confirmed by flow cytometric analysis.