Cationic α-cyclodextrin:poly(ethylene glycol) polyrotaxanes for siRNA delivery

Abstract

RNA interference has broad therapeutic potential due to its high specificity and ability to potentially evade drug resistance. Three cationic α-cyclodextrin:poly(ethylene glycol) polyrotaxanes derived from polymer axles of different sizes (MW 2,000, 3,400, and 10,000) have been synthesized for delivering siRNA. These polyrotaxanes are able to condense siRNA into positively charged particles that are <200 nm in diameter, enabling their facile internalization into mammalian cells. The cationic polyrotaxanes display cytotoxicity profiles that are >10(2)-fold lower than the commercial standard bPEI and gene silencing efficiencies that are comparable to those of both Lipofectamine 2000 and bPEI. Our findings suggest that the cationic polyrotaxanes display a size-activity relationship, wherein the higher molecular weight polyrotaxanes (PEG3,400 and 10,000) are able to condense and deliver siRNA better than the lower molecular weight material (PEG2,000).

Figure 1

(A) Conceptual diagram of PR⁺:siRNA complexation and (B) general structure of PR⁺ prepared in this study.

Figure 2

Gel retardation assay showing the complexation properties of the PR materials. M: Marker; R: free siRNA; L: lipofectamine 2000; 2-30: N/P ratios of bPEI, 2k, 3.4k and 10k PR⁺ complexes with siRNA. Arrows indicate uncomplexed siRNA.

Figure 3

Characterization of siRNA complexes formed by 2k, 3.4k and 10k PR⁺. (A) ζ potentials; (B) DLS measurements; and AFM images of (C) 10k PR⁺; (D) 10k PR⁺:siRNA at N/P = 20.

Figure 4

Biological performance of PR⁺:siRNA complexes. (A) Relative cell viabilities PR⁺ and bPEI as a function of amine densities in 10% serum-supplemented media; (B) relative cellular uptakes of PR⁺ and bPEI with L2k as positive control; and (C) in vitro GFP knockdown efficiencies of 2k, 3.4k and 10k PR⁺:siRNA complexes in 10% serum-supplemented media with L2k and bPEI as controls.