## 4 Discussion  
Extensive research has been conducted on mRNA delivery systems, with LNPs emerging as the primary platform for COVID-19 mRNA vaccines used worldwide [26; 27]. While LNP-based delivery has enabled the rapid development and deployment of mRNA vaccines, challenges related to stability [28], biodistribution [29], and potential inflammatory responses have driven continued efforts to explore alternative delivery methods. In this study, we investigated IM-EP as a non-carrier-based approach for mRNA vaccine delivery, aiming to enhance immune responses without the need for LNP encapsulation. Our results demonstrate that IM-EP successfully delivers naked SARS-CoV-2 spike mRNA into cells, leading to strong humoral and cellular immune responses and providing robust protection against lethal viral challenge in mice. By facilitating direct cytoplasmic entry of mRNA, IM-EP circumvents the limitations associated with LNPs, offering a safer and  
Figure 5: SARS-CoV-2 mRNA vaccine delivered via IM-EP protects mice from lethal SARS-CoV-2 infection. (**A**) Schematic of the immunization and challenge protocols in K18-hACE2 mice, showing three immunizations with EP and subsequent SARS-CoV-2 challenge. (**B**) Post-challenge monitoring of K18-hACE2 female mice (n = 4–5/group) for survival. (**C**) Neutralizing antibody levels measured by performing a PRNT (red line indicates the PRNT\({}_{50}\) threshold). (**D**,**E**) Post-challenge monitoring of K18-hACE2 female mice for body weight (**D**) and body temperature (**E**). more accessible alternative for mRNA vaccine administration. These findings highlight the potential of IM-EP as an effective delivery platform for mRNA vaccines, not only against SARS-CoV-2 but also for other infectious diseases and emerging pathogens requiring rapid vaccine deployment. EP-mediated nucleic acid delivery has been widely explored for both DNA and RNA vaccines. Intramuscular DNA immunization with in vivo EP has been shown to significantly enhance both cellular and humoral immune responses, further highlighting the immune-boosting potential of EP-based vaccine delivery [30]. EP-based DNA vaccines have induced enhanced antigen expression and immune responses in various infectious disease models, including HIV, Zika virus, and influenza, exhibiting promising results in preclinical and clinical trials [31]. However, unlike DNA vaccines, which require nuclear localization for transcription, mRNA vaccines are directly translated in the cytoplasm, allowing for faster antigen expression and immune activation [32]. Furthermore, although the clinical application of RNA vaccines remains limited, electroporation has been proposed as a promising strategy to enhance their delivery. [PERSON] and [PERSON] emphasized that EP significantly improves the intracellular uptake and expression of DNA vaccines, resulting in stronger immune responses in preclinical models [9]. Self-amplifying mRNA vaccines, such as those targeting influenza have demonstrated enhanced immunogenicity [33], while studies using EP-based delivery further support EP as an effective platform for self-amplifying RNA-based vaccine administration [19]. These findings validate the efficacy of IM-EP in delivering naked mRNA vaccines and inducing protective immune responses  
Given the success of EP in vaccine delivery, we sought to evaluate its potential for mRNA vaccine administration as a viable alternative to LNP formulations. Recent studies have reported various adverse effects associated with LNP-mRNA vaccines, including vaccine-induced immune thrombotic thrombocytopenia [34], myocarditis [35], IgA vasculitis [36], and autoimmune disorders [37; 38]. Although the exact mechanisms underlying these adverse effects remain unclear, LNP components, particularly cationic lipids, have been implicated in triggering innate immune activation and systemic inflammation [39]. In contrast, EP allows for the direct cytoplasmic delivery of mRNA without requiring lipid encapsulation, reducing concerns related to carrier-induced immune activation.