## 4 Conclusions  
This study conducted a comprehensive lipidomics analysis of microplasma-irradiated cells, utilizing highly sensitive LC/MS techniques. Rat intestinal epithelial cells were  
Figure 6: Analysis of cell death in microplasma-irradiated cells using flow cytometry. (**A**) Representative dot plots illustrating the relative counts of live, early apoptotic, apoptotic, and necrotic cells in both control and microplasma-irradiated cell populations. (**B**) Bar charts show the proportions of live, early apoptotic, apoptotic, and necrotic cells in microplasma-irradiated cells and control cells. The data are presented as mean \(\pm\) standard deviation (n = 3). Statistical analysis compared the C4K0H group (n = 3), C4K12H group (n = 3), and C4.5K0H groups with the control group (n = 3). UL: necrotic cells; UR: late apoptotic cells; LL: viable cells; LR: early apoptotic cells.  
subjected to microplasma irradiation at 4.0 kV, a condition previously identified as optimal for facilitating the absorption of high-molecular-weight drugs (up to 2000 kDa) into cells. Additionally, cells were irradiated at 4.5 kV in order to investigate lipid changes under extreme conditions.  
The results demonstrate that irradiation at 4.0 kV induces a significant increase in cell membrane lipids within 10 min post-irradiation. This lipid augmentation potentially enhances cell membrane permeability, facilitating the penetration of high-molecular-weight drugs. Notably, the levels of cell membrane lipids return to baseline levels 12 h after irradiation, indicating a transient effect of microplasma on the cell membrane.  
Conversely, irradiation at 4.5 kV did not elicit significant changes in cell membrane lipids. Consistent with our previous findings where drug absorption was not observed under this condition, our current study unequivocally demonstrates that microplasma promotes drug absorption by modulating cell membrane lipids.  
Flow cytometry analysis revealed no significant differences in live cell and apoptotic cell percentages between the control and 12 h post-irradiated groups (at 4.0 kV), aligning with the observations from the LC/MS analysis. This consistency between flow cytometry and LC/MS data underscores the reliability of our findings.  
In summary, our study elucidates the transient modulation of cell membrane lipids by microplasma irradiation, thereby potentially facilitating drug absorption. These insights contribute to our understanding of the mechanisms underlying microplasma-mediated cellular responses and hold potential implications for drug delivery strategies as well as other microplasma-based biomedical technologies.  
The supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/app14103978/s1](https://www.mdpi.com/article/10.3390/app14103978/s1)]([https://www.mdpi.com/article/10.3390/app14103978/s1](https://www.mdpi.com/article/10.3390/app14103978/s1)), Table S1: LipidSearch (version 4.2.13) software parameters.  
Figure S1: Changes in PUFA-PC after microplasma irradiation at 4.0 kV condition.  
Conceptualization: S.A.R., M.A.M. and K.S.; methodology: S.A.R., M.J.A., J.K., A.H.S., M.H. and M.A.M.; software, formal analysis, and investigation: S.A.R., M.A.M., J.K., M.S. and K.S.; validation and resources: K.S.; data curation: S.A.R., M.A.M. and K.S.; original draft writing: S.A.R. and M.A.M.; review and editing: S.A.R., M.J.A., J.K., A.H.S., M.H., M.A.M., M.S. and K.S.; supervision and funding: K.S. All authors have read and agreed to the published version of the manuscript.  
This work was supported by JSPS KAKENHI (Grant Numbers JP23K17473, and JP24H00794).  
Not applicable.  
Data Availability Statement: Data supporting this study are included in the article and the Supplementary Information.  
The authors declare no conflict of interest.