Enhanced Anticancer Efficacy of Alkaline Plasma-Activated Water through Augmented RONS Production

Abstract

Despite notable advances in anticancer drug development, their manufacture and use pose environmental and health risks due to toxic byproducts, drug residue contamination, and cytotoxicity to normal cells. Therefore, developing cost-effective anticancer treatments with fewer toxic side effects and higher selectivity is essential to the advancement of highly effective anticancer therapies. Plasma-activated water (PAW) offers a green alternative to conventional chemical treatments as it reverts to water within days. However, the limited duration and dose of reactive oxygen and nitrogen species (RONS) in acidified PAW restrict its clinical deployment and the full understanding of their mechanism. In this study, we propose alkaline PAW as an innovative enhancement of the RONS technology. The alkaline PAW generated markedly superior RONS, with about 10 times higher levels of NO₂^–, H₂O₂, and ONOO^–/O₂^•– than acidic PAW. The possible RONS generation pathways in alkaline PAW are analyzed by scavengers. In conventional acidic PAW, 70% of the H₂O₂ concentration is contributed by ^•OH but only about 20% in alkaline PAW. ONOO^– is mainly formed through the reaction of O₂^•– with NO in alkaline pH, while in acidic PAW, it mainly forms from NO₂^– and H₂O₂. The results unveiled the synergistic and formidable anticancer effects of alkaline PAW against cancer cells, typified by an increase in intracellular ROS/RNS levels. Furthermore, alkaline PAW injection also effectively prevented xenograft tumor growth in mice. We systematically investigated this high-dose anticancer solution without using noble gases, toxic reagents, or extra energy consumption and successfully demonstrated the possibility of alkaline PAW being an effective and environmentally friendly therapeutic technology. The activity is closely linked to the RONS dose, and the generation pathway provides much-needed insight into the fundamental aspects of PAW chemistry required for the optimization of the biochemical activity of PAW.