Figure 4. Generating siRNA from RNAi-microsponges by the RNAi pathway, and condensing RNAi-microsponges for transfection.

a, Schematic of the generation of siRNA from RNAi-microsponges by Dicer in the RNAi pathway. b, Gel-electrophoresis result after Dicer reaction. On the left, lanes 1 and 2 indicate double-stranded RNA ladder and RNAi-microsponges (MS) after treatment with Dicer (1 unit) for 36 h, respectively. On the right, lanes 1 and 2 indicate double-stranded RNA ladder and RNAi-microsponges without Dicer treatment. Lanes 3–8 correspond to 12 h, 24 h, 36 h and 48 h reaction with 1 unit of Dicer, and 36 h reaction with 1.25 and 1.5 units of Dicer, respectively. Increasing the amount of Dicer did not help to generate more siRNA (lane 7 and 8 of b). The amount of generated siRNA from RNAi-microsponges was quantified relative to double-stranded RNA standards. 21% of the cleavable double-stranded RNA was actually diced to siRNA because Dicer also produced the two or three repeat RNA units. The results suggest the possibility that in a more close-packed self-assembled structure, some portion of the RNA is not as readily accessed by Dicer. c, Particle size and zeta potential before and after condensing RNAi-microsponges with PEI. d, SEM image of further condensed RNAi-microsponges with PEI. The size of the RNAi-microsponges was significantly reduced by linear PEI because the RNAi-microsponges with high charge density would be more readily complexed with oppositely charged polycations. The porous structure of the RNAi-microsponges disappeared following condensation.