*2.10. Prophylactic and Therapeutic Antitumor Effect of mRNA-Loaded LNPs on Mice*

For the prophylactic antitumor experiment, C57BL6/N mice (6 weeks old) were intravenously injected with OVA mRNA-loaded A-11-LNPs at the indicated doses on day <sup>−</sup>14 and <sup>−</sup>7. On day 0, E.G7-OVA cells (8 <sup>×</sup> <sup>10</sup><sup>6</sup> cells/50 µL/mouse) were inoculated to the immunized mice subcutaneously in the right flank under isoflurane anesthesia. Tumor volumes were calculated using the equation shown below (tumor volume = major axis <sup>×</sup> minor axis<sup>2</sup> <sup>×</sup> 0.52) from day 6 to 21.

For therapeutic antitumor experiments, C57BL6/N mice (6 weeks old) were anesthetized with isoflurane. E.G7-OVA cells (8 <sup>×</sup> <sup>10</sup><sup>6</sup> cells/40 µL/mouse) were inoculated subcutaneously in the right flank. The tumor-bearing mice were intravenously injected with EGFP mRNA or OVA mRNA-loaded A-11-LNPs, OVA mRNA-loaded MC3-LNPs or OVA mRNA-loaded RNA-LPX at doses of 0.03 mg mRNA/kg on days 8 and 11. Tumor volumes were calculated by the above method from day 6 to 21.
