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

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

Polyplexes were further characterized by size and Zetapotential measurements. Because an N/P ratio of at least 4 was needed to achieve complete siRNA condensation by all of our copolymers, all experiments discussed hereafter were performed using greater N/P ratios of 6, 12, and 24. Size and surface charge measurements are presented in Figure 4 and Figure 5 , respectively. The diameters of P1 complexes were 128 nm, 130 nm, and 150 nm for respective N/P ratios of 6, 12, and 24. The diameters of P2 complexes were 190 nm, 200 nm, and 210 nm for respective N/P ratios of 6, 12, and 24. The diameters of P3 complexes were 160 nm, 152 nm, and 130 nm for respective N/P ratios of 6, 12, and 24 (Fig. 4) . The diameters of P2 complexes were larger than those of P1 and P3.The two sides of PLL may contribute to making P2 complexes less compact by entrapping many water molecules in the complexes and thus, contributing to the larger Figure 1 . synthesis of copolymers P1, P2, and P3.synthesis of the copolymers began with preparation of LL(Z)-Nca by intramolecular ring closure of LL(Z). The hybrid copolymers were then synthesized using successive ring opening polymerization. Finally, Z-groups on P1-Z, P2-Z, and P3-Z were removed using hBr/hac, and target copolymersP1, P2, and P3 were obtained. Reaction temperatures (°c) and times are indicated at each step shown in the figure. complex sizes measured. It was interesting to find that increasing the N/P ratio of P1 or P2 polymer complexes resulted in larger particles, while increasing the N/P ratio P3 polymer complexes resulted in smaller particles. Because the PPG segments are chemically attached to the PLL segments, formation of a PPG core causes dense packaging of P3-siRNA complexes. The Zeta potential analyses are provided in Figure 5 . The Zeta-potentials of P1 complexes were 28 mv, 29 mv, and 31 mv for respective N/P ratios of 6, 12, and 24. The Zeta-potentials of P2 complexes range were 44 mv, 46 mv, and 52 mv for respective N/P ratios of 6, 12, and 24. Finally, the Zeta-potentials of P3 complexes were 19 mv, 20 mv, and 22 mv for respective N/P ratios of 6, 12, and 24. The Zeta-potentials of P2 complexes were greater than those of P1 and P3. It was interesting that P2 complex has greater potential but lower siRNA binding ability. A possible reason is that the larger surface areas coupled with lower siRNA binding efficiency would induce more charge leave on their surfaces. Taken together, the size and Zeta potential measurements have provided useful information on the architecture of the P1, P2, and P3 complexes: (1) P1 and P2 complexes were less dense, although they still appeared to form micelles, (2) a well-defined boundary between PLL/siRNA complexes and PEG segments was not apparent, (3) PLL segments increasingly localized to the PEG corona, and (4) P3 complex is small in size and has a relatively defined layer structure because of the PPG core. It is possible that the small Zeta potential of P3 (~20 mV) may improve its affinity to the cells and internalization by the cells as compared with the other copolymers tested. The possible micelle architecture of complexes of copolymer with siRNA is shown in Figure 6 . Evaluation of cytotoxicity by P1, P2, and P3 copolymers The cytotoxicity of cationic copolymers P1, P2, and P3 was evaluated by MTT viability assay in Neuro-2a cells. The cells were incubated with various concentrations of copolymers for 48 h. The results in Figure 7 show that P1 copolymers