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. 2012 May 2;134(17):7423-30.
doi: 10.1021/ja300174v. Epub 2012 Apr 19.

Reductively responsive siRNA-conjugated hydrogel nanoparticles for gene silencing

Stuart S Dunn  1 , Shaomin Tian, Steven Blake, Jin Wang, Ashley L Galloway, Andrew Murphy, Patrick D Pohlhaus, Jason P Rolland, Mary E Napier, Joseph M DeSimone
Affiliations

Reductively responsive siRNA-conjugated hydrogel nanoparticles for gene silencing

Stuart S Dunn et al. J Am Chem Soc. .

Abstract

A critical need still remains for effective delivery of RNA interference (RNAi) therapeutics to target tissues and cells. Self-assembled lipid- and polymer-based systems have been most extensively explored for transfection with small interfering RNA (siRNA) in liver and cancer therapies. Safety and compatibility of materials implemented in delivery systems must be ensured to maximize therapeutic indices. Hydrogel nanoparticles of defined dimensions and compositions, prepared via a particle molding process that is a unique off-shoot of soft lithography known as particle replication in nonwetting templates (PRINT), were explored in these studies as delivery vectors. Initially, siRNA was encapsulated in particles through electrostatic association and physical entrapment. Dose-dependent gene silencing was elicited by PEGylated hydrogels at low siRNA doses without cytotoxicity. To prevent disassociation of cargo from particles after systemic administration or during postfabrication processing for surface functionalization, a polymerizable siRNA pro-drug conjugate with a degradable, disulfide linkage was prepared. Triggered release of siRNA from the pro-drug hydrogels was observed under a reducing environment while cargo retention and integrity were maintained under physiological conditions. Gene silencing efficiency and cytocompatibility were optimized by screening the amine content of the particles. When appropriate control siRNA cargos were loaded into hydrogels, gene knockdown was only encountered for hydrogels containing releasable, target-specific siRNAs, accompanied by minimal cell death. Further investigation into shape, size, and surface decoration of siRNA-conjugated hydrogels should enable efficacious targeted in vivo RNAi therapies.

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Figures

Figure 1
Figure 1
(a) Reaction scheme for PEGylation of hydrogels with succinimidyl succinate monomethoxy PEG2K (SS-mPEG2K), (b) time-dependent release of siRNA from particles incubated at 2 mg/mL and 37 °C in PBS, and (c) Scanning electron micrograph (SEM) of particles illustrating their 200×200 nm cylindrical dimensions (scale bar = 2 μm).
Figure 2
Figure 2
siRNA delivery with PEGylated cationic hydrogels to luciferase-expressing human cervical cancer (HeLa/luc) cells. (a) Cellular uptake. HeLa/luc cells were dosed with particles for 4 h followed by trypan blue treatment and flow cytometry analysis. (b) Luciferase expression. HeLa/luc cells were dosed with particles for 4 h followed by removal of particles and 72 h incubation in media. Data are representative of at least three independent experiments. The error bars represent standard deviation from triplicate wells in one experiment. (c) and (d) Confocal micrographs. HeLa/luc cells were dosed with 50 μg/mL particles containing (c) luciferase or (d) control siRNA cargos for 4 h. Cellular actin cytoskeleton was stained with phalloidin (red) and nuclei with DAPI (blue), while particles (green) were labeled with the fluorescent monomer fluorescein O-acrylate during particle fabrication.
Figure 3
Figure 3
(a) Structures of degradable and non-degradable siRNA macromers as well as native siRNA, (b) Illustration of pro-siRNA hydrogel behavior under physiological and intracellular conditions, and (c) SEM micrograph of pro-siRNA, 200 × 200 nm cylindrical nanoparticles (scale bar = 2 μm).
Figure 4
Figure 4
Release profiles and stability of siRNA in 30% AEM-based hydrogels. All hydrogels were washed with 10x PBS buffer to remove the sol fraction containing unconjugated siRNA before release studies were performed. (a) Time-dependent incubation of pro-siRNA hydrogels (1 mg/mL) in PBS and under reducing conditions (glutathione, 5 mM) at 37 °C. (b) Selective release of the disulfide-coupled siRNA prodrug (PD) from hydrogels under reducing conditions compared to the acrylamide (AA) macromer and native siRNA (NH2). Hydrogels were incubated in 10x PBS with or without 5 mM glutathione for 4 h at 1 mg/mL and 37 °C. (c) Retention of siRNA integrity when conjugated to hydrogels after exposure to 10% fetal bovine serum (FBS) over time. Naked siRNA PD macromer was incubated at 36 ug/mL in 10% FBS for given times, proceeded by storage of solution. pro-siRNA hydrogels were incubated at 1.2 mg/mL in 10% FBS at 37 °C for given times followed by incubation in 10x PBS (5 mM glutathione) for 4 h at 1.2 mg/mL and 37 °C to release siRNA. Differences in siRNA migration observed in gels among the standards and samples which were released from hydrogels incubated in PBS and 10x PBS may arise from the differences in salt concentrations of sample solutions.
Figure 5
Figure 5
(a) Luciferase expression and (b) viability of HeLa/luc cells dosed with cationic pro-siRNA hydrogels fabricated with different amine (AEM) contents. Cells were dosed with particles for 5 h followed by removal of particles and 48 h incubation in media. Data are representative of two independent experiments. The error bars represent standard deviation from triplicate wells in the same experiment. Half maximal effective concentrations (EC50s) of siRNA (nM) for luciferase gene knockdown are listed in the legend. EC50 was not available (NA) for hydrogels prepared with 5 wt% AEM due to the absence of dose-dependent luciferase knockdown.
Figure 6
Figure 6
30% AEM-based hydrogel particles charged with different siRNA cargos for transfection of HeLa cells. (a) Cellular uptake. HeLa/luc cells were dosed with particles for 4 h followed by trypan blue treatment and flow cytometry analysis. (b) Luciferase expression. HeLa/luc cells were dosed with particles for 4 h followed by removal of particles and 48 h incubation in media. Data in (a) and (b) represent one of two independent experiments. The error bars represent standard deviation from triplicate wells in the same experiment. Note that all hydrogels were thoroughly washed after fabrication to remove non-conjugated siRNA in the sol fraction. (c)-(f) Confocal micrographs. HeLa/luc cells were dosed with 50 μg/mL hydrogels containing (c) luc PD (d) luc siRNA-NH2, (e) luc acrylamide, and (f) control PD siRNA cargos for 4 h. Cellular actin cytoskeleton was stained with phalloidin (red) and nuclei with DAPI (blue) while particles (green) were labeled with the fluorescent monomer, DyLight 488 maleimide.
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