To study the escape of the siRNAs from lysosomes, T24 cells (1 × 105 cells per well) were seeded into a confocal dish and incubated for 24 hours. LysoTracker Red (75 nM, 1 hour, Invitrogen) was then used to stain the T24 cells. Thereafter, HNTs/FAM-siRNA and free FAM-siRNA (200 nM FAM-siRNA equivalent) were incubated with the T24 cells at 37°C for 1 hour. The cells were then rinsed and incubated in fresh medium for 1 to 6 hours. Samples were taken at different time points, the cells were fixed using 4% paraformaldehyde (PFA), and observed using confocal microscopy. LysoTracker Red and FAM-siRNA were detected at excitation wavelengths of 550 and 480 nm, respectively.

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