Intracellular Trafficking of RGP Nanoparticles

The HeLa cells were inoculated in a 6-well plate with a sterilized coverslip at an initial density of 2.5×10⁵ cells/well and cultured at 37°C overnight. Subsequently, the cells were incubated with 1 mL FBS-free DMEM containing RGP nanoparticles (RNase A concentration of 4 μg/mL) for 2 and 6 hrs, respectively. Afterward, the cells were stained with LysoTracker Green DND-26 (ThermoFisher, Eugene, OR) for 5 mins as described in previous reports.31 (link)–33 (link) After washing with PBS three times, the cells were fixed with 4% paraformaldehyde solution for 15 mins and stained with DAPI solution (1 μg/mL) for 5 mins. Finally, the coverslip was subjected to the analysis on an LSM 710 CLSM (Carl Zeiss Microscopy LLC to detect the endosomal escape of RGP nanoparticles.