## 3 Results and Discussion
### The Induction of Apoptosis by CDES  
We hypothesized that treatment with a higher voltage ES would promote the delivery of cisplatin. However, CDES was performed with a voltage of 6 kV or less since ES alone at more than 6 kV impacted cells. To determine the optimal voltage, CDES was performed at various voltages. One day after treatment, cell proliferation was investigated by live/dead analysis (Figure S3A,B). Living cells (green fluorescence) were classified according to intracellular esterase activity, which involved the release of red dye (ethidium homodimer-1). In contrast, the dead cells (red fluorescence) degraded esterase activity and did not release red dye. As a result, there was no significant difference between the cisplatin-only, 2 kV CDES, and 4 kV CDES treatment groups. The fewest live cells were found in the 6 kV CDES treatment group. The analysis showed that the ratio of dead to live cells was almost twice that of the group treated with cisplatin alone. In the WST-1 analysis, it was found that the cell viability was the lowest in the 6 kV CDES treatment group, similar to the live and dead results (Figure S3C). Based on these results, we confirmed that the optimal voltage of CDES for inducing apoptosis in cancer cells is 6 kV. Figure 2 shows the cell viability of the 6 kV CDES and cisplatin treatment groups 3 days after treatment. Interestingly, in the group treated with only 6 kV ES, most of the cells were alive, similar to the control group. However, it was confirmed that the 6 kV CDES treatment group had significantly lower proliferation than the group treated with cisplatin alone. After the fluorescence image analysis for the live and dead assays, it could be seen that the ratio of dead/live cells in the 6 kV CDES treatment group was approximately 7 times higher than that of the cisplatin-treated group (Figure 2B). Cell viability using the WST-1 assay also showed similar results to the live and dead assays (Figure 2C). Based on these results, we expected that cisplatin would be delivered to the cells with higher efficiency through the CDES method, and accordingly, apoptosis of cells was better induced. To determine accurately whether apoptosis was caused by CDES, FACS analysis was performed using the Annexin V assay (Figure 3). Apoptotic cells were detected by staining with phosphatidylserine with green fluorescence (FITC), and necrotic cells were detected by PI through red fluorescence. Early apoptotic cells showed only green fluorescence, while late apoptotic cells showed both green and red fluorescence. Surprisingly, it was confirmed that more than 85% of the cells were in the process of apoptosis in the 6 kV CDES treatment group. In the group treated with cisplatin alone, approximately 40% of the cells were undergoing apoptosis. Few cells were apoptotic in the 6 kV ES treatment group. This means that the ES method promoted the delivery of cisplatin, dramatically increasing the induction of apoptosis, even though it did not cause much damage to the cells by itself.  
Figure 3: **(A)** The results of fluorescence-activated cell sorting (FACS) for Annexin V staining after 6 kV CDES (Control, 6 kV ES, Cisplatin, and 6 kV CDES). **(B)** FACS analysis showed more than 80% cell death in the 6 kV CDES group. Approximately 80% of apoptotic cells were shown in the 6 kV CDES treatment group (n = 3, \(p\) < 0.05. Columns with different letters are significantly different according to one-way ANOVA and Duncan’s test).  
Figure 2: Comparison of cell viability of 6 kV CDES and cisplatin, **(A)** live and dead assay of 6 kV electrospray with cisplatin (Control, 6 kV ES, Cisplatin and 6 kV CDES), **(B)** the bar graph of the dead/live ratio, **(C)** water-soluble tetrazolium salt (WST) assay of 6 kV CDES (scale bar: 1 mm, n = 3, \(p\) < 0.05. Columns with different letters are significantly different according to the Duncan test). The ratio of dead/live cells in the CDES-treated group was approximately 7 times larger than that in the cisplatin-treated group 3 days after treatment.