Intracellular Delivery of Q-Starch based RNAi Therapeutics for Reducing Drug Resistance in Ovarian Cancer Cells

RNA interference (RNAi) therapeutics targeting the multi-drug efflux pump P-glycoprotein (P-gp), over expressed in ovarian cancer cells, will render the cells susceptible to anti-cancer drugs. A safe and efficient carrier for RNAi was recently developed, based on modified potato starch with quaternary amine groups (Q-starch). Q-starch and siRNA, are co-assembled to form of Q-starch/siRNA nanocomplexes, which effectively reduces the expression of P-gp (50%). Here we studied the delivery pathways of Q-starch/siRNA complexes to define intracellular delivery barriers.

We show here, that siRNA/Q-starch complexes readily adhere to the cell membrane and accumulate in the cells within minutes. FRET analysis of fluorescently labeled siRNA /Q-starch complexes decreased following 24hrs, indicating de-complexation and si-RNA release. Internalization of the complexes was blocked by dynasore (80mM), a dynamin inhibitor, leading to recovery of P-gp expression. Inhibition of clathrin mediated endocytosis using pitstop2 partially inhibited complexes uptake (42±2% of the transfected cells not treated with pitstop2), suggesting a combination of clathrin dependent and independent entry mechanisms. In agreement, complexes co-localize with early endosomes (rab5a positive) up to 4 hr, and co-localize to late endosomes (rab7a positive) and lysosomes (using lysotracker) following 24 hr. Interestingly, internalization of fluorescently labeled complexes applied for a 4hr pulse, continued to accumulate in the cells, up to a maximum at 24hrs. Fluorescence of the cells was sustained for the next 48hrs. Therefore, we suggest that endosomal escape and decomplexation, and not exocytosis, are the major barriers in delivering RNAi to the cell cytoplasm using our carrier. Thus, increasing endosomal escape by further manipulations of Q-starch will promote efficiency of gene silencing and enhancement of drug sensitivity of the cells.

Acknowledgement: This work was supported by the Focal Technological Area Program of the Israeli National Nanotechnology Initiative (INNI).