## 3 Results
### Naked mRNA Vaccination via IM-EP Effectively Induces Antigen-Specific Immune Responses in Mice  
To evaluate the immune response induced by IM-EP-mediated mRNA vaccine delivery, we first examined cytokine expression in muscle tissue and draining lymph nodes following the administration of SARS-CoV-2 mRNA vaccine. EP-mediated delivery led to a dose-dependent increase in cytokine mRNA expression levels, with IFN-\(\beta\), ISG56, IL-6, CXCL10, and CCL5 levels significantly elevated in muscle tissue, indicating strong local innate immune activation (Figure 2A). Similarly, dLNs from vaccinated mice exhibited a dose-dependent increase in cytokine expression levels, suggesting the induction of innate immune responses (Figure 2B). Figure 1: Optimization and evaluation of mRNA delivery via IM-EP in mice. (**A**):Administration of 10 \(\upmu\)g firefly luciferase (Fluc) mRNA into the biceps femoris muscle, followed by IM-EP. Control: without IM-EP. Bioluminescence imaging at 6, 24, and 48 h (each group, n = 8). (**B**) (**Left**) Quantification of bioluminescence signals up to 37 days post-IM-EP. (**Right**) Analysis on day 2 post-IM-EP under different voltages (control, 20 V, 40 V, 60 V, and 80 V; duration: 10 ms; pulse: 12, two repetitions) (**\(p<\) 0.01, ***\(p<\) 0.001, and ***\(p<\) 0.0001; control vs. EP). (**C**) Mice were intramuscularly injected with 5 or 25 \(\upmu\)g of the SARS-CoV-2 mRNA vaccine, followed by IM-EP. Levels of SARS-CoV-2 spike protein were evaluated by performing ELISA using muscle tissue lysates (***\(p<\) 0.001; 0 \(\upmu\)g vs. 5 or 25 \(\upmu\)g mRNA vaccine group). We next assessed systemic humoral and cellular immune responses (Figure 3A). Serum analysis conducted 2 weeks after the second immunization revealed substantial elevations in RBD-specific IgG, IgG1, IgG2a, and IgG2b levels, indicating a strong antigen-specific humoral response (Figure 3B). In addition to humoral immunity, IM-EP induced a potent cellular immune response. Immunized mice exhibited enhanced IFN-\(\gamma\) production by CD8+ T cells, indicating robust cytotoxic T lymphocyte (CTL) activation (Figure 3C). Further analysis of splenocytes stimulated with spike protein peptides confirmed a significant increase in IFN-\(\gamma\), IL-2, and tumor necrosis factor-alpha (TNF-\(\alpha\)) secretion (Figure 3D), reinforcing the ability of IM-EP to induce a significant cell-mediated immune response. Figure 2: Expression of innate immune response-related genes in muscle and draining lymph nodes. (**A**) Muscle tissues were harvested from mice to measure expression levels of IFN-\(\beta\), IgG56, IL-6, OAS1, CXCL10, and CCL5 genes using real-time PCR (* \(p\) + 0.05, ** \(p\) + 0.01, and *** \(p\) + 0.0001; compared to the 0 \(\upmu\)g IM-EP group at each time point). (**B**) Draining lymph nodes were harvested from mice to measure expression levels of IFN-\(\beta\), IgG56, IL-6, OAS1, CXCL10, and CCL5 genes using real-time PCR (* \(p\) + 0.05, ** \(p\) + 0.01, and *** \(p\) + 0.001; compared to the 0 \(\upmu\)g IM-EP group at each time point). Interestingly, we also found that IM-EP alone, without mRNA (PBS control), triggered mild immune activation. To further assess the potential immunomodulatory effects of EP, we analyzed immune cell infiltration in muscle tissue by flow cytometry at different time points following IM-EP without mRNA administration. We observed an initial increase in total immune cell infiltration on day 1 and a further increase on day 2, followed by a decline on day 3. Neutroph exhibits a similar trend; however, the differences were not statistically significant because of high individual variability. In contrast, monocytes/macrophages increased on day 1, peaked on day 2, and remained elevated on day 3, suggesting their role in prolonged local inflammation and immune modulation.