Addition of poly (propylene glycol) to multiblock copolymer to optimize siRNA delivery

Dai, Zhi; Arévalo, Maria T; Li, Junwei; Zeng, Mingtao

were the least toxic, while P2 copolymers were the most toxic. When treated with any of the copolymers at about 60 μg/ml, the cells maintained over 80% viability. Cells treated at concentrations of 80 μg/ml maintained over 85% viability when treated with copolymers P1 and P3, but less than 75% when treated with P2. Toxicity has been shown to be related to charge density of the polymer, 28 and may provide an explanation for the different results observed using the different copolymers. Another explanation is that toxicity might be primarily caused by addition of an excess of polymer. 29 Cellular uptake of copolymer-siRNA complexes Cellular uptake is a key step for siRNA delivery. To assess the ability of the various polymers to facilitate siRNA internalization, green fluorescent protein (GFP)-targeted siRNA labeled with a Cy3 fluorescent tag was used to form polyplexes. Fluorescence microscopy was used to monitor uptake of Cy3-tagged siRNA (red) and knockdown of GFP expression (green) in Neuro-2a/ GFP cells (Fig. 8) . The cells shown were transfected with polyplexes composed of the cationic copolymers P1, P2, and P3 with siRNA at N/P ratios of 6, 12, and 24. At the lowest N/P ratio of 6, red fluorescent signal was not detected inside the Neuro 2a cells and GFP expression was still very strong, indicating poor uptake of polyplexes. However, when the N/P ratio was increased to 24, labeled siRNA was detected inside the cells and GFP expression decreased. Furthermore, Cy3 signal increased while GFP expression decreased in cells treated with P2 and P3 complexes as compared with cells treated with P1 complex. Thus, at a high N/P ratio of 24, copolymers P2 and P3 demonstrated higher efficiencies for cellular uptake and gene silencing than P1.