## 3 Results and Discussion
### Changes in Lipids Associated with Cell Membranes
Statistical analysis compared the B4 KOH group (n = 5) and B4K12H group (n = 5) with the control group (n = 5). (**b**) Relative changes (expressed in percentages) in total cell membrane-associated lipids in the B4 KOH and B4K12H groups compared to the control. (**c**) Relative changes in phosphatidylinositol(PC), phosphatidylethanolamine (PE), phosphatidyserine (PS), and phosphatidylinositol (PI) species in the B4 KOH and B4K12H groups compared to the control. (**d**) Relative changes in sphingomyelin (SM), ganglioside (GM3), monohexosylceamide (Hex1 Cer), and dihexosylceamide (Hex2 Cer) species in the B4 KOH and B4K12H groups compared to the control. The trends observed among the GP classes were remarkably uniform: they exhibited a consistent elevation in B4K0H cells, followed by a restoration to baseline levels (i.e., almost similar to the control cells) in B4K12H cells (Figure 3c). On the other hand, SP classes showed variability in their changes (Figure 3d). The absorption of drugs into cells was markedly enhanced during or immediately following microplasma irradiation, as suggested by previous research [11]. Notably, there was a significant increase in cell membrane lipids within 10 min post-irradiation, suggesting a direct correlation between lipid concentrations and drug uptake. It has been reported that lipids play a crucial role in determining the permeability of cell membranes [16], which supports the hypothesis that lipid augmentation may facilitate drug absorption by altering membrane characteristics. It would be more interesting if the lipid changes were studied immediately after the irradiation. However, the inherent nature of the LC/MS sample preparation renders such timely studies challenging, limiting our understanding of these immediate biochemical changes. The increase in lipids observed in A4K0H or B4K0H cells cannot be attributed to a potential increase in cell numbers after irradiation. Rat epithelial cells typically undergo a cell cycle spanning a day or longer [23], rendering substantial proliferation unlikely within the short timeframe of our experimental protocol. Moreover, the A4K0H and B4K0H cells were washed and snap-frozen within 10 min of irradiation, which excludes the possibility that increased lipid levels originated from cellular proliferation. Hence, it is reasonable to hypothesize that microplasma irradiation stimulated lipid synthesis within the cells, leading to an augmentation in lipid content--a phenomenon often associated with lipogenesis. It was expected that we would observe oxidized lipid species in microplasma-irradiated cells, since ROS species are known to cause lipid peroxidation [24]. However, we could not find any oxidized lipid species in the irradiated cells. This could be due to the analytical challenges, such as the low abundance, poor stability, the limited ionization efficiency of oxidized lipids [25; 26], and the limitations of lipid annotation tools. Our findings are consistent with a previous study, wherein the oxidative modification of skin lipids by CAP was investigated by MS/MS, but oxidized lipid species was rarely detected [27]. Polyunsaturated fatty acids (PUFAs) are highly susceptible to peroxidation due to the presence of multiple carbon-carbon double bonds [28]. Therefore, we analyzed PUFA-containing PC species in order to investigate the lipid peroxidation indirectly. However, our analysis revealed a significant increase in total PUFA-containing PC species in B4K0H cells (Figure S1), indicating that peroxidation was not a major phenomenon in the irradiated cells. These findings resonate with a recent study by [PERSON] et al., who observed a notable increase in PUFA chains within kidney PC, PE, and TG species within 5 min of whole-body irradiation with 10 Gy and 20 Gy in mice [29]. One of the limitations of this study is that the lipids were extracted from whole cells, meaning that the cell membrane-associated lipids mentioned above did not exclusively reflect the composition of the cell membranes.