Briefly, in 24-well plates, trypsinized cells were seeded (n = 3) at 5 × 104 cells per well and incubated overnight until they reached 60 to 80% confluence, after which they were transfected with various formulations. FAM-siRNA uptake by the cells was assessed after 6 hours using an inverted fluorescence microscope (Olympus, IX71, Japan). In addition, cells were trypsinized, centrifuged (2000g, 3 min), resuspended in PBS, and analyzed using flow cytometry (Beckman Coulter).

Next, T24 cells were seeded in a 35-mm glass-bottom culture dish (MatTek Corp.) at 5 × 104 cells per well and incubated at 37°C in 5% CO2 for 24 hours. The cell culture medium was then substituted by HNTs/FAM-siRIPK4 complexes in 500 μl of serum-free DMEM in each well. At various time points, the cell culture medium was removed, and the cells were washed three times with PBS. DAPI (Sigma-Aldrich) was then used to stain the nuclei for 5 min. The cells were directly observed using confocal laser scanning microscopy with an Olympus FLUOVIEW confocal microscope and analyzed using the FV10-ASW viewer software (Olympus, Tokyo, Japan).

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