Cellular uptake studies

For uptake analysis, the cells were cultured in six-well plates containing glass coverslips for 24 hours with a density of 3×10⁵ cells/well. The cells were treated with lipoplexes prepared with FAM-labeled siRNA. After 4 hours of incubation, the cells were fixed with 4% paraformaldehyde followed by staining with Hoechst 33258 to stain the nucleus. The cell imaging was performed by a CLSM (FV1000-IX81; Olympus, Tokyo, Japan).

The quantitative measurement of cellular uptake siRNA was determined using a fluorescence-activated cell sorter (FACS; BD FACSCalibur; BD Biosciences, San Jose, CA, USA). MCF-7/ADR cells were seeded in six-well plates with a density of 1×10⁶ cells/well and incubated overnight to allow adhesion of cells. The original culture medium was then replaced by fresh supplemented medium, and then cells were incubated with FAM-labeled siRNA–liposome complexes, LR, PSLR, and EPSLR-loaded 50 nM FAM-siRNA. After 4 hours of incubation, MCF-7/ADR cells were washed twice with cold PBS and dissociated with 0.25% trypsin–EDTA, and then harvested and resuspended in PBS solution. The cell suspension obtained was further analyzed by FACS. The level of cellular uptake was quantified based on FAM-siRNA fluorescence determined in FL1H, in which a total of 10,000 events were analyzed. For free ligand competition study, cells were coincubated with 10 μg/mL of anti-EphA10 antibody with formulations.