**4. Conclusions**

In the present study, the DOE-based in vivo optimization of mRNA-loaded LNPs for mRNA vaccines was studied. The screening included a wide range of particle sizes that were controlled by the addition of NaCl in addition to the lipid type used and its composition. The results showed a clear correlation between particle size and uptake and gene expression activity in splenic DCs and indicated that a size range from 200 to 500 nm is appropriate for use in conjunction with splenic DCs. It was also found that transgene expression activity in the whole spleen, which can be easily evaluated, was not a valid predictor of transgene expression activity in splenic DCs and potency as mRNA vaccines. The A-11-LNP, which was found to have the optimal formulation, induced better transgene expression activity and maturation in DCs compared to two clinically relevant LNP formulations and induced clear therapeutic antitumor effects in an E.G7-OVA tumor model. The findings reported herein are expected to contribute to the development of future mRNA vaccines.
