Silica nanogelling of environment-responsive PEGylated polyplexes for enhanced stability and intracellular delivery of siRNA.

In this study, poly(ethylene glycol) (PEG)-block-polycation/siRNA complexes (PEGylated polyplexes) were wrapped with a hydrated silica, termed "silica nanogelling", in order to enhance their stability and functionality. Silica nanogelling was achieved by polycondensation of soluble silicates onto the surface of PEGylated polyplexes comprising a disulfide cross-linked core. Formation of silica nanogel layer on the PEGylated cross-linked polyplexes was confirmed by particle size increase, surface charge reduction, and elemental analysis of transmission electron micrographs. Silica nanogelling substantially improved polyplex stability against counter polyanion-induced dissociation under non-reductive condition, without compromising the reductive environment-responsive siRNA release triggered by disulfide cleavage. Silica nanogelling significantly enhanced the sequence-specific gene silencing activity of the polyplexes in HeLa cells without associated cytotoxicity, probably due lower endosomal entrapment (or lysosomal degradation) of delivered siRNA. The lower endosomal entrapment of the silica nanogel system could be explained by an accelerated endosomal escape triggered by deprotonated silanol groups in the silica (the proton sponge hypothesis) and/or a modulated intracellular trafficking, possibly via macropinocytosis, as evidenced by the cellular uptake inhibition assay. Henceforth, silica nanogelling of PEGylated siRNA polyplexes is a promising strategy for preparation of stable and functional siRNA delivery vehicles.

Smart multilayered assembly for biocompatible siRNA delivery featuring dissolvable silica, endosome-disrupting polycation, and detachable PEG.

Multifunctional delivery systems of small interfering RNA (siRNA) are needed to overcome the intrinsic biological barriers toward efficient gene silencing in the cell cytoplasm. In this report, a smart multilayered assembly (SMA) was fabricated by a layer-by-layer method with polyionic materials. The SMA was designed to feature a siRNA-loaded core, a transiently core-stabilizing silica interlayer, an endosome-disrupting polycation interlayer, and a biocompatible poly(ethylene glycol) (PEG) shell with reductive environment-responsive detachability. The SMA was confirmed to be approximately 160 nm in size with narrow distribution and spherical morphology by DLS and TEM analyses. The PEG detachability of the SMA based on disulfide cleavage was also confirmed by the increase in both ζ-potential and size due to the exposure of the polycation interlayer and the compromised colloidal stability. The silica interlayer rendered the SMA highly tolerant to dissociation induced by anionic lipids, while after 24 h dialysis siRNA release from the SMA was clearly observed, presumably due to gradual dissolution of the silica interlayer based on the equilibrium shift to silicate ions. The entrapment ratio of siRNA delivered by the SMA within the endosome was significantly lower than that by nondisulfide control (NDC) without PEG detachability, suggesting the improved endosomal escape of SMA with the exposed, endosome-disrupting interlayer after PEG detachment. SMAs induced significantly higher gene silencing efficiency in various cultured cells, compared to NDC, without associated cytotoxicity. The systemic administration of SMAs for subcutaneous tumor-bearing mice achieved significant endogenous gene silencing in tumor tissue without hematological toxicity.

Enhanced transfection with silica-coated polyplexes loading plasmid DNA.

Silica-coating of positively charged polyplexes was demonstrated through silicic acid condensation to improve the polyplexes for enhanced complex stability and transfection efficiency. Silicic acid was efficiently condensed by polycations to form a silica network in the polyplex through electrostatic interaction and hydrogen bonding. The silica-coated (SC) polyplexes had an anionic surface charge of -20 mV and were 10-20 nm larger in size compared to the non-silica-coated control (+33.4 mV, 106 nm). Silica-coating significantly improved the polyplex stability against both dissociations by counter polyanion exchange and aggregation by salt. The silica network was dissolved to form silicic acid by removing free silicic acid based on the equilibrium, SiO(2) + 2H(2)O right arrow over left arrow Si(OH)(4). Indeed, dialysis of the SC polyplex solution against excess silica-free buffer permitted plasmid DNA release from the silica-coated polyplex, indicating the reversible nature of the silica-layer. The SC polyplex achieved significantly higher transfection efficiency without serious cytotoxicity compared to the polyplex without silica-coating. Detailed examinations of transfection using SC polyplexes revealed that the enhanced transfection efficiency was because of facilitated endosomal escape, possibly due to the protonation of the silica in acidic endosomal compartments. These findings demonstrate the utility of the silica-coating technique for polyplex-mediated gene delivery.