Microplasma-Generated Plasma-Activated Water: A Coalescence of Ions and Free Radicals Facilitating Cancer Cell Death─Impact of Plasma-Forming Gases

Plasma-activated solutions (PAS), a cocktail of ions and free radicals, have emerged as a therapeutic approach for cancer treatment in which reactive radicals in PAS have marked anticarcinogenic effects on cancer cells. In this context, the anticancer effects of plasma-activated water (PAW) generated using a microplasma device were evaluated with air, argon, and nitrogen as plasma-forming gases at treatment durations of 5-25 min (5 min intervals). Optical characterization revealed that air and nitrogen plasma harnessed nitrogen species, whereas argon and air plasma indicated OH^• and oxygen species. UV-vis spectra of PAW demonstrated a linear increase with the treatment time. Quantitative analysis showed an increase in OH^• concentration around 15 min, and further declined with treatment time due to its short half-life. Hydrogen peroxide (H₂O₂) was highest in argon > air > nitrogen (1667.6 > 794.9 > 627.8 μM) at 25 min plasma treatment. NO₃^- ions concentration increased with treatment time and was highest (250 ppm) in air and nitrogen PAW. The pH of PAW decreased, while total dissolved solids, electrical conductivity, and salt concentration increased with treatment time. Cytotoxicity of 100 and 200 μL PAW was studied in MDA-MB-231 human breast adenocarcinoma cells using MTT assay, incubated for 24 and 48 h. PAW treated for 25 min with argon and nitrogen caused 95% cell death, while air plasma exhibited 81% at 48 h. Confocal microscopic studies revealed substantial cell damage caused by PAW via cell shrinkage and disintegration, nuclear fragmentation, and accumulation of cell debris. This study explicates the cytotoxic potential of PAW and correlates the role of reactive oxygen and nitrogen species (RONS) in cancer cell inhibition.