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Efficient Sirna Delivery with Acetylated Polyethylenimine

Lily Wong and Daniel W. Pack. Chemical and Biomolecular Engineering, University of Illinois, Box C-3, 600 S. Mathews Ave, Urbana, IL 61801

Small-interfering RNA (siRNA) – 21 nucleotide, double-stranded oligomers – can shut down expression of essentially any gene in a cell in a sequence specific manner in a process termed RNA interference (RNAi). As a result, RNAi holds great potential as a therapeutic against many classes of disease, especially cancer and infectious diseases. RNAi has been touted as the greatest advance in therapeutics in two decades. However, safe and efficient delivery of siRNA remains the critical barrier to successful implementation of siRNA therapies. Delivery methods for siRNA have largely mirrored those for plasmid DNA, with many of the same liposomal and polymeric gene delivery materials being “recycled” for siRNA delivery. The requirements for siRNA delivery are likely to be different, but there is a general lack of mechanistic studies of siRNA delivery in the literature. Thus, a goal of our research is to quantitatively investigate the structure-activity relationships of siRNA-polymer complexes and their trafficking in target cells in order to build a mechanistic understanding upon which new siRNA delivery agents can be designed. In particular, we hypothesize that the primary barriers to siRNA delivery are endolysosome escape and polymer-siRNA complex dissociation. Further, we hypothesize that the optimal polymer chemistry will balance protection of siRNA, endocytic escape, and efficient unpackaging in the cytosol. Therefore, we synthesized a series of “capped” polymers based on acetylation of polyethylenimine (PEI) to convert primary and secondary amines into secondary and tertiary amides, respectively. We have previously reported that these polymers are more efficient plasmid DNA delivery agents compared to unmodified PEI because they bind to DNA less strongly and, as a result, unpackage inside the cell more efficiently. Further, we found an optimal amount of capping that strikes a balance between buffering capacity needed for escape from endocytic vesicles and efficient unpackaging. In this paper, we report the effect of PEI capping density on siRNA-mediated gene knockdown. As with DNA, we find an optimal capping percentage, but at much lower ratios than for plasmid DNA delivery.