###### Abstract  
Combination therapies for the treatment of oral squamous cell carcinoma have been studied extensively and represent a synergistic approach with better outcomes than monotherapy. In this study, a novel combination therapy was investigated using gold nanoparticles (GNP) conjugated to programmed cell death protein ligand 1 (PD-L1) antibodies and nonthermal plasma (NTP). The present study describes the effectiveness of NTP using PD-L1 antibody conjugated to GNP in PD-L1 expressing SCC-25 cells, an oral squamous cell carcinoma line. Immunocytochemistry revealed higher levels of PD-L1 expression and an increase in the selective uptake of PD-L1 antibody + GNP on SCC-25 cells compared to HaCaT cells. In addition, cell viability analyses confirmed higher levels of cell death of SCC-25 cells after treatment with PD-L1 antibody, GNP, and NTP compared to HaCaT cells. Among the experimental groups, the highest cell death was observed upon treatment with PD-L1 antibody + GNP + NTP. Following the Western blot analysis and immunofluorescence staining, the expression of apoptosis-related proteins was found to increase after treatment with PD-L1 antibody + GNP + NTP among the other experimental groups. In conclusion, the treatment of SCC-25 cells with PD-L1 antibody + GNP + NTP significantly increased the number of dead cells compared to other experimental groups. The results of this in vitro study confirmed the therapeutic effects of PD-L1 antibody + GNP + NTP treatment on oral squamous cell carcinoma. gold nanoparticle; nonthermal plasma; oral cancer; PD-L1; combination therapy +
Footnote †: journal:Among these, a recent study investigated the use of monoclonal antibodies (mAb) to selectively attack cancer cells. In fact, mAb against cancer cell-specific antigens have been proposed as alternatives to cancer-selective treatment. However, most mAb acting alone do not show a sufficiently strong therapeutic effect. Nevertheless, cancer cell-specific antibodies continue to show beneficial effects when used in cancer treatments. Since these antibodies specifically bind to the surfaces of cancer cells, they show clinical potentials of cancer cell selectivity that are differentiated from conventional chemotherapy. To find antigens that selectively target cancer cells, these antigens must be specifically expressed on cancer cells. In a previous study, PD-L1 was found to be highly expressed in patients with oral cancer and was associated with a poor prognosis [4]. PD-L1 was recently proposed as an immune checkpoint because of its association with the immune evasion of cancer. Moreover, the inhibition of PD-L1 has been shown to promote T cells with anticancer activity [5]. In fact, the achievement of the immune checkpoint block treatment in patients with various malignant tumors is changing the paradigm of the treatment [3; 6]. Advances in immunotherapy improved the survival rates of cancer patients. In many clinical trials, monoclonal antibodies that target immune checkpoint proteins have demonstrated significant responses in the treatment of lung cancer and terminal stage melanoma patients [7]. The United States Food and Drug Administration (FDA) has accepted the use of three PD-L1 inhibitors and two PD-1 inhibitors for the cure of melanoma, lymphoma, and lung cancers [7; 8]. However, a large number of patients do not react to immune checkpoint inhibitors or develop a resistance to treatment [9]. Therefore, the complete elimination of cancer and the reduction of its recurrence rate are difficult to achieve using only one method, such as immunotherapy [10]. Thus, many clinical trials involving immune checkpoint inhibitors are currently being conducted in combination with other treatment methods, including radiation therapy or chemotherapy, to improve the cancer survival rate and the synergistic effects on the suppression of the growth of cancer cells [8; 10; 11; 12]. However, this approach has limitations in clinical practice due to its potential toxicity, resulting in dose limitations and low responses in patients.