Anticancer effects of plasma-activated saline (PAS): optimization and stability of reactive species in Ar-PAS/air-PAS combinations

The biological effects of plasma-activated solutions (PAS) depend on the composition and evolution of reactive oxygen and nitrogen species (RONS) in the liquid phase. However, it remains unclear whether PAS generated from different working gases constitute aqueous systems with different dominant RONS, and how these differences shape their anticancer effects after mixing and storage. In this study, PAS was prepared using argon and air dielectric barrier discharge (DBD) plasmas. Their gas-phase characteristics, aqueous RONS profiles, effects on MCF-7 breast cancer cell viability, and the influence of post-mixing ratio and storage conditions on biological activity were systematically compared. H2O2 predominated in argon-derived PAS, whereas NO3− and O3-related oxidation signals were greater in air-derived PAS. Both PAS compositions reduced MCF-7 cell viability and were associated with different dominant aqueous chemical profiles. Post-mixing of argon- and air-derived PAS induced ratio-dependent cytotoxicity, with the 4:6 and 7:3 mixtures showing more pronounced suppression of cell viability and elevated intracellular ROS levels. Storage experiments showed that PAS underwent continuous post‑discharge evolution, and under the conditions tested, storage at 4 °C better preserved the measured long‑lived species than storage at −20 °C. Separate preparation and storage followed by post-mixing immediately before use preserved biological activity better than storage after premixing. These results demonstrate that PAS with different dominant RONS exhibit different anticancer effects, and that the post-mixing ratio and storage conditions further modulate anticancer efficacy. This study provides experimental evidence for optimizing PAS-based strategies in cancer therapy.