## 5 Conclusions  
Our findings show that IM-EP-mediated delivery of naked mRNA encoding the SARS-CoV-2 spike protein induces high levels of neutralizing antibodies and strong cellular immunity, effectively preventing lethal viral infections in mice. These results demonstrate that delivering mRNA directly into the cytoplasm via IM-EP, without the need for lipid carriers, not only simplifies the processes of vaccine production and distribution but also reduces adverse effects associated with LNPs. Given the rising interest in optimizing mRNA vaccine platforms, our study supports IM-EP as a promising alternative for enhancing vaccine efficacy while improving safety and accessibility. Furthermore, given the ongoing advancements in mRNA-based cancer vaccines, EP-mediated delivery could also be considered as a potential platform for tumor mRNA vaccine delivery. Further studies are necessary to optimize and expand the application of this approach in both infectious disease and cancer vaccine development. The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/app15084428/s1](https://www.mdpi.com/article/10.3390/app15084428/s1)]([https://www.mdpi.com/article/10.3390/app15084428/s1](https://www.mdpi.com/article/10.3390/app15084428/s1)), Figure S1. Gating strategy for flow cytometry analysis of muscle and lymph node samples. A: Lymphocytes were initially gated based on forward scatter area (FSC-A) vs. side scatter area (SSC-A) characteristics. Live cells were then selected using a Live/Dead fixable violet dead cell stain. Single cells were further gated based on forward scatter area (FSC-A) vs. forward scatter height (FSC-H) to exclude doublets, leaving only single cells. CD45\({}^{+}\) immune cells were then gated for further analysis. B-E: Representative flow cytometry plots from muscle samples are shown for CD45\({}^{+}\) immune cell subsets, including neutrophils (CD11b\({}^{+}\)Ly6G\({}^{+}\)), dendritic cells (DCs, CD11c\({}^{+}\)MHCII\({}^{+}\)CD11b\({}^{+}\)), and monocytes/macrophages (CD169\({}^{+}\)CD115\({}^{+}\)CD11b\({}^{+}\)). Figure S2: Immune response induced by IM-EP immunization. A: Immunication schedule for C57 BL/6 mice receiving three doses of the SARS-CoV-2 mRNA vaccine via IM-EP. B: RBD-specific total IgG serum measured by RBD-specific ELISA after the third dose and one week post-immunization serum. C: IFN-\(\gamma\) concentration in splenocytes stimulated with a SARS-CoV-2 spike peptide library for 48 h, measured by IFN-gamma ELISA. (* \(p<0.05\) Control vs. EP group). Conceptualization, Y.J.L. and S.-ML. ; methodology, S.-H.P., Y.K., M.K. and Y.S. ; investigation, S.-H.P., Y.K., M.K. and Y.S. ; data curation, S.-H.P., Y.K., M.K. and Y.S. ; writing--original draft preparation, S.-H.P., M.K., Y.S. and H.J. ; writing--review and editing, S.-M.L. and H.J. ; project administration, Y.J.L. and S.-M.L. All authors have read and agreed to the published version of the manuscript. GenAI tools were utilized to assist with English editing and proofreading. The authors take full responsibility for the final content of the manuscript. This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health and Welfare, Republic of Korea (Grant No: RS-2022-KH128441). It was also supported by the Technology Innovation Program (20019452, Development of LNP-free mRNA vaccine using new concept vaccination equipment based on Korean technology), funded by the Ministry of Trade, Industry and Energy (MOTIE, Korea). Additional support was provided by the Ministry of Food and Drug Safety through a grant from the Korean government (Grant No: 22213 MFDS421). The animal studies were performed with the approval of the Institutional Animal Care Committee (IACUC) of CBNUA-2046-22-01 (23 December 2022). Not applicable. Data Availability Statement: The original contributions presented in this study are included in the article/Supplementary Material.