Endosomal escape of siRNA-ONV

To visualize the co-localization of internalized siRNA-ONV with endosomal compartments, the aptamer-bound CEM cells were incubated with Cy3-siRNA-ONV and Cy3-siRNA-ONV without rigid structure (Cy3-siRNA-ONV-NR) at siRNA concentration of 200 nM for 2 or 6 h at 37 °C. Then, the cells were washed with PBS and stained with 100 nM of LysoTracker Green and 5 μg ml⁻¹ of DAPI for 15 min for imaging. LysoTracker Green was excited at 488 nm with an argon ion laser and the emission was collected from 505 to 535 nm.

The endosomal escape was further demonstrated with calcein release assay by incubating the aptamer-bound CEM cells with the mixture of 25 μM calcein and Cy3-siRNA-ONV or Cy3-siRNA-ONV-NR for 6 h at 37 °C and then washing these cells with PBS. Calcein was excited at 488 nm with an argon ion laser and the emission was collected from 500 to 530 nm.