Acidic plasma-activated povidone-iodine induces copper-dependent oxidative death in oral squamous cell carcinoma cells

Oral squamous cell carcinoma (OSCC) requires therapeutic strategies that preserve oral function while providing effective tumor control. Here we demonstrate that non-thermal plasma (NTP) irradiation of a clinically established povidone-iodine solution generates a potent cytotoxic formulation termed plasma-activated povidone-iodine (PAI). PAI selectively reduced viability of OSCC cell lines (SAS, HSC-2, HSC-4) compared with normal fibroblasts. Mechanistically, PAI activity strictly depended on its acidic pH (3.0 or below) and hydrogen peroxide (H₂O₂) generated during NTP irradiation. Neutralization or freeze-drying abolished both H₂O₂ content and cytotoxicity, indicating that Reactive Oxygen Species are essential mediators. Unlike plasma-activated media, whose effects are predominantly iron-dependent, PAI-induced cytotoxicity required intracellular monovalent copper (Cu⁺). The Cu⁺ chelator tetrathiomolybdate suppressed ROS accumulation, glutathione depletion, lipid peroxidation, and cell death, whereas classical Ferroptosis or Apoptosis inhibitors failed to rescue viability. PAI increased intracellular Cu⁺ levels and triggered oxidative damage including γH2AX induction; however, the resulting death program was mechanistically distinct from Apoptosis, Ferroptosis, and Cuproptosis. Our findings reveal a previously unrecognized Cu⁺-dependent oxidative pathway initiated by PAI. This study establishes an accessible iodine-based platform that harnesses metal-driven redox chemistry for selective OSCC elimination.