Enhanced Anticancer\nEfficacy of Alkaline Plasma-Activated\nWater through Augmented RONS Production

Despite notable advances in anticancer drug development,\ntheir\nmanufacture and use pose environmental and health risks due to toxic\nbyproducts, drug residue contamination, and cytotoxicity to normal\ncells. Therefore, developing cost-effective anticancer treatments\nwith fewer toxic side effects and higher selectivity is essential\nto the advancement of highly effective anticancer therapies. Plasma-activated\nwater (PAW) offers a green alternative to conventional chemical treatments\nas it reverts to water within days. However, the limited duration\nand dose of reactive oxygen and nitrogen species (RONS) in acidified\nPAW restrict its clinical deployment and the full understanding of\ntheir mechanism. In this study, we propose alkaline PAW as an innovative\nenhancement of the RONS technology. The alkaline PAW generated markedly\nsuperior RONS, with about 10 times higher levels of NO<sub>2</sub><sup>–</sup>, H<sub>2</sub>O<sub>2</sub>, and ONOO<sup>–</sup>/O<sub>2</sub><sup>•–</sup> than acidic PAW. The possible\nRONS generation pathways in alkaline PAW are analyzed by scavengers.\nIn conventional acidic PAW, 70% of the H<sub>2</sub>O<sub>2</sub> concentration\nis contributed by <sup>•</sup>OH but only about 20% in alkaline\nPAW. ONOO<sup>–</sup> is mainly formed through the reaction\nof O<sub>2</sub><sup>•–</sup> with NO in alkaline pH,\nwhile in acidic PAW, it mainly forms from NO<sub>2</sub><sup>–</sup> and H<sub>2</sub>O<sub>2</sub>. The results unveiled the synergistic\nand formidable anticancer effects of alkaline PAW against cancer cells,\ntypified by an increase in intracellular ROS/RNS levels. Furthermore,\nalkaline PAW injection also effectively prevented xenograft tumor\ngrowth in mice. We systematically investigated this high-dose anticancer\nsolution without using noble gases, toxic reagents, or extra energy\nconsumption and successfully demonstrated the possibility of alkaline\nPAW being an effective and environmentally friendly therapeutic technology.\nThe activity is closely linked to the RONS dose, and the generation\npathway provides much-needed insight into the fundamental aspects\nof PAW chemistry required for the optimization of the biochemical\nactivity of PAW.