2.3. Luciferase assay in vitro

Hep3B cells were grown in EMEM supplemented with 100 mL/L of FBS. The cells were incubated at 37 °C in a 5% CO₂ environment and subcultured by partial digestion with 0.25% trypsin with EDTA.

Hep3B cells were seeded in white 96-well plates at the density of 2 × 10⁴ cells per well, cultured overnight, and then treated with FLuc mRNA loaded LLNs at the concentration of 50 ng mRNA/well. After 18 h, 100 μL luciferase substrate (Bright-Glo reagent, Promega, Madison, WI) was added to each well. After 5 min, the luminescence intensity was measured by the SpectraMax M5 microplate reader (Molecular Devices, LLC., Sunnyvale, CA).