Stabilization of polyplexes via polymer crosslinking for efficient siRNA delivery.

Thomas Fr__hlich, Daniel Edinger, Verena Russ, Ernst Wagner

Index: Eur. J. Pharm. Sci. 47(5) , 914-20, (2012)

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Abstract

The pseudodendritic, biodegradable polymer HD-O, consisting of an OEI800 core with several OEI800 molecules attached to it via 1.6-hexanediol diacrylate linkers, has potent pDNA but poor siRNA delivery ability, due to instability of the resulting siRNA polyplexes. Stabilization of such nanoparticles by crosslinking surface amines of HD-O in the polyplexes with dithiobis-(succinimidylpropionate) (DSP) greatly enhanced gene silencing efficiency. Successful crosslinking on the polyplex surface was indicated by a decrease of the positive Zeta potential of the polyplexes. Tuning the polymer/siRNA ratio in combination with adjustment of the linker to a molar ratio of 0.05/1 between linker and polymer amines proved essential for transfection efficiency and prevention of particle aggregation. Gene silencing ability of the crosslinked particles was demonstrated in murine neuroblastoma N2A and human hepatoma HUH-7 cells. Flow cytometry showed efficient cellular uptake already after 1h incubation with the crosslinked but not with unstabilized particles. Downregulation of endogenous AHA1 mRNA (85% knockdown compared to control) by crosslinked HD-O/AHA1-siRNA particles was detected by quantitative real-time PCR.