Tailoring the RNAi efficiency of polyplexes

Abstract

RNA interference (RNAi) is a cellular pathway preserved in eukaryotic cells of animals, plants and fungi. In the RNAi process, double-stranded small interfering RNA (siRNA) is recognized by the RNA-induced silencing complex (RISC). The antisense strand of siRNA binds to the mRNA that are homologous to the siRNA sequence and induces the degradation of the target mRNA with the help of argonaute 2 (Ago2) protein in RISC, ultimately resulting in the inhibition of the mRNA translation.1 The principle of RNAi was first employed to downregulate the target gene in mammalian cells by Tuschl et al. in 2001.2 Since then employing synthetic siRNAs to silence targeted genes has been intensively investigated for uses as a powerful tool in laboratory studies3,4 as well as in RNAi-based therapies.5-7 Delivery vehicles for siRNA are crucial to guarantee an effective RNAi, which prevent siRNAs from degradation and deliver them into the cell as well. However, taken up by cells is far from enough for the effective RNAi if delivered siRNAs fail to be localized at the cytoplasm where RISC is staying.3 Viral vector systems include adeno-associated viruses and lentiviruses. There are now many examples of the use of viral vector-mediated RNAi to inhibit gene expression in animal models of disease, and in many cases proof-of-principle has been demonstrated.8 However, a number of concerns have raised questions regarding the clinical application of this technology, including off-target effects, risks of immunogenicity and potential carcinogenicity.9 Hence nonviral vectors, particularly synthetic cationic polymers, have been attracting ever-growing interests due to facile synthesis, controllable modification and convenient manipulation,10 only a simple mixing of the cationic polymers and siRNAs allow the formation of polyplexes. However, two crucial issues are raising major challenges to the cationic polymeric carriers: (1) Although polyplexes can be steadily internalized by various kinds of cells, the efficiency of lysosomal escape for the siRNA is limited. (2) The cytotoxicity of polyplexes is high, which is attributable to positive charges of the cationic polymers. To overcome these barriers, efforts are mainly placed in three aspects (Fig. 1). Figure 1. Schematic illustration to strategies of optimizing cationic polymer-mediated RNAi: (1) regulating the interaction between polyplexes and the serum in the biological environment to modulate the particles size for efficient internalization ... Regulating Interactions between Polyplexes and the Serum Zeng’s group proposed a novel cationic copolymer platform for siRNA delivery in a safe and efficient way,11 they reported three block copolymers including PEG-PLL, PLL-PEG-PLL, and PLL-PPG-PEG-PPG-PLL. The PEG and PPG are used for improving the biocompatibility and modulating the size of the polyplexes, which allows them efficient cellular internalization as well as low cytotoxicity. In the serum-containing culture medium the gene silence mediated by PLL-PPG-PEG-PPG-PLL is comparable to that mediated by Lipofectamine 2000. This is attractive because serum can significantly compromise the RNAi efficiency mediated by cationic polymers due to the nonspecific adsorption of proteins, while serum-free condition is not only toxic to the cells, but also not available in biological environment, particularly in blood. The high RNAi efficiency should be attributed to the PEG and PPG segments, for they are able to repel the protein adherence. We speculate that the PEG and PPG chain segments may partly locate on the surface and partly in the core of the polyplexes particles. Moreover, the PEGylation strategy can hinder the clearance by RES system in vivo and increase the circulation time of polyplexes in blood.6 Our previous work found that serum free is not always necessary for cationic polymer carriers in the RNAi. The serum at a proper concentration in the culture medium not only modulates the size of polyplexes particles to allow the efficient cellular uptake and lysosomal escape, but also reduces the cytotoxicity of the polycomplex.12