## 4 Discussion  
In recent years, research on therapies using nanoparticles has gained popularity, with the aim of targeting cancer cells more selectively [28]. Strategies have included the combination of antibody and GNP conjugation increase delivery efficiency, diagnostic accuracy, and exert anticancer effects using lasers of specific wavelengths [27; 29; 30; 31; 32]. In the present study, NTP combined with Ab-conjugated GNP were used with an aim of increasing the targeting efficiency and enhancing cell death. As a result, the combination of the PD-L1 antibody to GNP + NTP was found to exert a synergistic effect due to the antibody's targeting ability. According to the result of the SRB assay, this was the most effective when GNP, NTP, and PD-L1 ab were used in combination. Similar to the previous results [33], although the combination of GNP + NTP itself produced anticancer effects, the treatment of SCC-25 cells with PD-L1 ab + GNP + NTP was more effective. Thus, it was possible to implement a targeted treatment more effectively and selectively for cancer cells [23]. This study was designed based on the overexpression of PD-L1 in cancer cells. Although the PD-L1 antibody is widely used in immunotherapy [8], only a few studies deal with a combination of GNP and nonthermal plasma. Thus, this study aimed to establish a new combination approach for cancer treatment using antibodies against PD-L1. To this end, oral squamous cell carcinoma cells expressing high levels of PD-L1 were selected. Comparing the expression of PD-L1 between the SCC cells and HaCaT cells, a marked  
Figure 7: Induction of apoptosis in SCC-25 cells. Upon treatment with PD-L1 ab + GNP + NTP, after 4 h of incubation, immunocytechanistry visualized the redistribution of cyt C and AIF using an anti-cyt C antibody and an anti-AIF antibody, respectively. Translocation of cyt C and AIF are indicated with arrows. Abbreviations: NT, nontreatment; ab, antibody; GNP, gold nanoparticle; NTP, nonthermal plasma; cyt C, cytochrome C; AIF, apoptosis-inducing factor. increase in PD-L1 was observed in SCC cells, with the HaCaT cells weakly expressing PD-L1. According to our recent studies, the PD-L1 overexpression in oral cancer cells is associated with a poor prognosis. As such, SCC-25 cells, which showed high levels of PD-L1 expression, were selected as the primary cells for this experiment (Figure 2). Immunofluorescence staining was performed to confirm the selective binding of the PD-L1 antibodies, which was only weakly observed in HaCaT cells. In contrast, SCC-25 cells treated with PD-L1 ab + GNP showed strong red staining in the cytoplasm around the nucleus (Figure 3). Similar to a previous study [34], higher levels of antibody-GNP uptake were observed in the cancer cells. These results confirmed the selective binding of SCC-25 cells by the PD-L1 antibody + GNP. While the treatment with GNP and NTP had no effect on the HaCaT cells, a decrease in cell viability was observed in the SCC-25 cells. As demonstrated in previous studies [17; 27], both GNP and NTP exert anticancer effects in cancer cells. When only PD-L1 ab was applied to SCC-25 cells, the cells proliferated to 110.1%, unlike normal cells. Further studies are required to determine why the cells proliferated after the application of the PD-L1 antibody alone. The survival of the SCC-25 cells treated with PD-L1 ab + GNP + NTP was markedly reduced compared to the HaCaT cells. This suggests that PD-L1 ab + GNP specifically attaches to the PD-L1 protein and synergizes with the nonthermal plasma (59.96% in SCC-25 cells), strongly inducing cell death. These results suggest that this combination method is effective for the selective treatment of cancer cells (Figure 4). An analysis using a LIVE/DEAD(tm) Viability/Cytotoxicity Kit also showed similar results compared to the SRB assay. The former analysis enabled the differentiation between dead and living cells using different levels of fluorescent staining.