Hydrogen Peroxide-Mediated Inhibition of Membrane Resealing Drives Synergistic Cytotoxicity of Combined Cold Atmospheric Plasma and Pulsed Electric Field Treatment

Cold atmospheric plasma (CAP) combined with pulsed electric fields (PEF) demonstrates synergistic cytotoxicity against HeLa cells; however, the differential contributions of short-lived versus long-lived reactive oxygen and nitrogen species (RONS) remain unclear. This study compared direct CAP treatment with indirect CAP-treated liquid treatment, both followed by PEF, to elucidate underlying mechanisms. Direct CAP + PEF treatment resulted in significantly greater cell death than indirect CAP + PEF, with both showing synergistic effects relative to single treatments. Analysis of intracellular RONS and membrane integrity revealed that direct CAP treatment enhanced intracellular RONS levels and PEF-induced membrane permeabilization immediately after treatment. Time-course analysis demonstrated that hydrogen peroxide specifically inhibits membrane resealing following PEF-induced electroporation, as evidenced by progressive calcein leakage over 20 min, while immediate pore formation remained unaffected. Catalase rescue experiments confirmed that hydrogen peroxide removal prevented progressive membrane damage without affecting immediate pore formation, thereby restoring cell viability. These findings identify hydrogen peroxide-mediated inhibition of membrane resealing as a novel mechanism underlying synergistic cytotoxicity, distinct from immediate membrane damage. This two-phase mechanism provides new insights for optimizing plasma-based cancer therapies.