SiRNA Crosslinked Nanoparticles for the Treatment of Inflammation-induced Liver Injury

Abstract

RNA interference mediated by small interfering RNA (siRNA) provides a powerful tool for gene regulation, and has a broad potential as a promising therapeutic strategy. However, therapeutics based on siRNA have had limited clinical success due to their undesirable pharmacokinetic properties. This study presents pH-sensitive nanoparticles-based siRNA delivery systems (PNSDS), which are positive-charge-free nanocarriers, composed of siRNA chemically crosslinked with multi-armed poly(ethylene glycol) carriers via acid-labile acetal linkers. The unique siRNA crosslinked structure of PNSDS allows it to have minimal cytotoxicity, high siRNA loading efficiency, and a stimulus-responsive property that enables the selective intracellular release of siRNA in response to pH conditions. This study demonstrates that PNSDS can deliver tumor necrosis factor alpha (TNF-α) siRNA into macrophages and induce the efficient down regulation of the targeted gene in complete cell culture media. Moreover, PNSDS with mannose targeting moieties can selectively accumulate in mice liver, induce specific inhibition of macrophage TNF-α expression in vivo, and consequently protect mice from inflammation-induced liver damages. Therefore, this novel siRNA delivering platform would greatly improve the therapeutic potential of RNAi based therapies.

Keywords: acid labile; copper free click; positive‐charge‐free; siRNA crosslinking; targeting.

Figure 1

Schematic representation of siRNA cross‐linked nanoparticles (PNSDS).

Figure 2

Synthetic routes of AAPEG. i) azidopropanol, PTSA, 5Å molecular sieves; ii) acrylate chloride, Et₃N; ii) eight‐armed PEG amine, Et₃N.

Figure 3

a) Synthetic route of cyclooctyne modified siRNA. b) PAGE gel verification of successful modification of siRNA. Lane 1: free siRNA; Lane 2: cyclooctyne modified siRNA. c) Agarose gel electrophoresis assay for the release of siRNA by acid induced hydrolysis. Lane 2: cyclooctyne modified siRNA duplex; Lane 3: PNSDS; Lane 4: acid hydrolysis of PNSDS. d) SEM image of PNSDS. Scale bar is 1 μm. e) Particle size of PNSDS. f) The hydrolysis of AAPEG at pH 7.4 and 5.0.

Figure 4

a) Fluorescence images showing internalization of PNSDS‐FAM‐siRNA‐in RAW 264.7 cells. b) Cell viability results after incubation of macrophage cells with various concentrations of AAPEG and PNSDS.

Figure 5

Knock down of TNF‐α produced by LPS in the presence of serum.

Figure 6

Mannose conjugated siRNA cross‐linked nanoparticles protected mice from LPS/d‐GalN‐induced acute hepatic injury. a) Serum TNF‐α level of mice administered with siRNA at 50 μg kg⁻¹. b) Relative TNF‐α mRNA levels in mouse liver. c) ALT levels in mice 6 h after LPS/d‐GalN stimulation. d) H&E stained liver sections from mice 6 h post LPS/d‐GalN stimulation. n = 4.