Endocytic Pathways and Endosomal Escape

For endocytic pathways study, 2 × 10⁴ cells per well of Hep 3B cells were seeded in 24-well plates and allowed to attach to the bottom of plates for overnight. Then, a cellular uptake assay was carried out by treating cells with enhanced green fluorescent protein mRNA-encapsulated FTT5 LLNs with the addition of different endocytic inhibitors, EIPA, MβCD, and CPZ, respectively. We used BD LSR II flow cytometry analyzer (BD Biosciences, San Jose, CA) for quantifying the cellular uptake of cells in the presence of different inhibitors. For the endosomal escape assay, 2 × 10⁴ cells per well of Hep 3B cells were seeded in an ibidi μ-Dish 35-mm Quad dish (ibidi GmbH, Gräfelfing, Germany) and incubated overnight. Then, cells were treated with Alexa Fluor 647–labeled RNA containing FTT5 LLNs for 6 hours followed by the addition of calcein (150 μg/ml) in each well as previously reported (38 (link)). After washing with phosphate-buffered saline, images of cells were captured by a Nikon A1R Live Cell Imaging Confocal Microscope (Melville, NY) and analyzed by NIS-Elements.