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. 2010 Jun 23;132(24):8241-3.
doi: 10.1021/ja102132e.

Effect of nanoparticle conjugation on gene silencing by RNA interference

Neetu Singh  1 , Amit Agrawal, Anthony K L Leung, Phillip A Sharp, Sangeeta N Bhatia
Affiliations
Free PMC article

Effect of nanoparticle conjugation on gene silencing by RNA interference

Neetu Singh et al. J Am Chem Soc. .
Free PMC article

Abstract

RNA interference (RNAi) is a cellular process whereby the silencing of a particular gene is mediated by short RNAs (siRNAs). Although siRNAs have great therapeutic potential, cellular delivery has been a challenge. Nanoparticle-siRNA conjugates have emerged as potential delivery vehicles; however, reports describing the effects of nanoparticle conjugation on RISC incorporation and subsequent gene silencing have been mixed. In this report, we have systematically evaluated the effect of siRNA coupling strategies using a model nanoparticle system with varying conjugation schemes. We show that the accessibility of the siRNA linked to the nanoparticle and the lability of the cross-linker are critical for efficient gene knockdown.

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Figures

Figure 1
Figure 1
Probing the effect of conjugation strategy on gene silencing by QD-siRNA conjugates. (a) Scheme for probe synthesis. (b) Characterization of the probes. (Left) Gel electrophoresis of QD-siRNA conjugates. Conjugation with labile cross-linkers (SPDP and SMPT) releases the conjugated siRNA upon treatment with glutathione. Arrow indicates free siRNA. (Middle) Gel electrophoresis of QD-siRNA with nonlabile maleimide cross-linker indicating the absence of unbound siRNA. (Right) Intracellular delivery of QD-siRNA conjugates by electroporation in modified HeLa (GFP-Ago2/Luc-CXCR4) cells. QD-siRNA conjugates are in red, green is Ago2-GFP, and the nuclei are stained with DAPI (blue). Scale bar is 30 μm.
Figure 2
Figure 2
KD of luciferase by QD-siRNA conjugates with antisense (As) or sense (S) strand of siLuc and siCXCR4 conjugated (n = 3). KD by QD-siRNA conjugates with labile (a) SPDP, (b) SMPT, and short nonlabile (c) PEO-12-Mal. KD by nonlabile cross-linker of varying length (d) PEO-4-Mal, (e) PEO-12-Mal, (f) PEO-24-Mal.
Figure 3
Figure 3
Performance of QD-siRNA conjugates with different conjugation chemistries after incubation in 10% FBS at 37 °C. (a) Gel electrophoresis of QD-siRNA showing the loss of siRNA after 8 h when conjugated with a labile cross-linker. (b) Comparison of the luciferase KD efficiency by QD-siRNA conjugates after incubation in 10% FBS.
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