Rationale design of polymeric siRNA delivery systems

Regulation of gene expression using small interfering RNA (siRNA) is a promising strategy for research and treatment of numerous diseases. However, siRNA cannot easily cross the cell membrane due to its inherent instability, large molecular weight and anionic nature. For this reason, a carrier that protects, delivers and unloads siRNA is required for successful gene silencing. The goal of this research was to develop a potential siRNA delivery system for in vitro and in vivo applications using cationic polymers, chitosan and polyethylenimine (PEI), poly(ethylene glycol) (PEG), mannose, and poly(D,L-lactic-co-glycolic acid) (PLGA). Furthermore, the delivery system was constructed in two different ways to explore the effect of mannose location in the structure. In the first approach, mannose and PEG were directly conjugated to the chitosan/PEI backbone, while mannose was connected to the chitosan/PEI backbone through PEG spacer in the second approach. First, the ability of modified chitosan polymers to complex and deliver siRNA for gene silencing was investigated. Despite the modified chitosan polymers successfully formed nanoplexes with siRNA, entered target cells and reduced cytotoxicity of unmodified chitosan, they showed limited gene silencing efficiency. For this reason, modified PEIs were examined to improve in vitro gene knockdown. The modified PEI polymers also complexed with siRNA and facilitated endocytosis of the nanoplexes. In addition, the modifications reduced inherent cytotoxicity of unmodified PEI without compromising the gene silencing efficiency on both mRNA and protein levels. Interestingly, we found that complexation of siRNA with PEI-PEG-mannose resulted in higher cell uptake and gene silencing than complexes made with mannose-PEI-PEG. Finally, the effect of sustained release of the mannosylated pegylated PEI/siRNA nanoplexes on gene silencing was tested by encapsulating the nanoplexes within PLGA microparticles. The modified PEIs enhanced the entrapment efficiency of siRNA into the particles and resulted in reduced initial burst