Cellular Uptake and Endosomal Escape Assay

RAW264.7 cells were seeded in a 24-well plate at 10⁵ cells/well and cultured for 24 h. Then, the cells were treated by FLuc mRNA and Alexa-Fluor 647-labeled RNA (1:1, weight ratio) using Lipofectamine 3000, VcLNPs, or electroporation. After 3 h incubation, cellular uptake was quantified by a flow cytometer (LSRII, BD). To study endocytic pathways of VcLNPs, cellular uptake assay was performed in the presence of different endocytotic inhibitors, 5-(N-methyl-N-isopropyl)amiloride (EIPA), methyl-beta-cyclodextrin (MβCD), and chlorpromazine hydrochloride (CPZ), which inhibit macropinocytosis-, caveolae-, and clathrin-mediated endocytosis, respectively. For the endosomal escape assay, 2 × 10⁴ cells were plated in an imaging dish (Ibidi) for 24 h, and then 150 μg/mL of calcein was added to the cells with or without VcLNPs containing Alexa-Fluor 647-labeled RNA for 6 h at 37 °C^{27 (link)}. After washing with PBS to remove extracellular calcein and VcLNPs, cells were lively imaged under the Nikon A1R Live Cell Imaging Confocal Microscope. The images were analysed by NIS-Elements AR 5.20.00.