*2.5. Preparation of RNA-Lipoplexes (RNA-LPX)*

A lipid film was formed by the evaporation of an ethanolic solution containing DOTMA and DOPE at a molar ratio of 1:1 (10 mM, 400 µL). PBS(-) (800 µL) was then added to the lipid film, followed by incubation for 7 min at room temperature to hydrate the lipids. To form empty liposomes, the lipid film was then sonicated for approximately 30 s in a bath-type sonicator. After dilution with PBS(-) 5.92 times (final lipid concentration of 0.845 mM), equal volumes of the liposome suspension and the mRNA solution (0.2 mg/mL in PBS(-)) were mixed to form RNA-LPX with a nitrogen per phosphate ratio of 1.3/2. The resulting sample was used in in vivo experiments without further purification.
