Cellular uptake assay

Both fluorescence microscopy and flow cytometry measurement (FCM) were used to measure the cellular uptake level of different formulations. For fluorescence microscopy, cells were seeded into six-well plate with 1×10⁵ cells per well to allow attachment. The medium was replaced with serum-free RPMI 1640, and different FAM-siRNA-loaded formulations were added into each well and incubated. At scheduled time points, medium was removed and the cells were washed with PBS. The cells were fixed with formalin, stained with Hoechst 33258, and finally visualized with fluorescence microscopy.

Flow cytometry was also used to quantitatively analyze the samples. Briefly, cells were seeded into six-well plate with 1×10⁶ cells per well to allow attachment. Then the culture medium was replaced with RPMI 1640, and different FAM siRNA-loaded formulations were added into each well. The cells were collected, washed with PBS, and finally analyzed using FCM.