Inhaltszusammenfassung

Cationic nanohydrogel particles are interesting vehicles for systemic siRNA delivery. For the first time, optimized anti-procollagen α1(I) siRNA loaded cationic nanohydrogel particles were tested in vivo as an antifibrotic therapy in a mouse model of liver fibrosis, induced by oral gavage of CCl4. After thorough validation of siRNA loaded nanohydrogel particles in vitro for cell toxicity, cellular uptake and knockdown performance, particles qualified for further biological evaluation in vivo...Cationic nanohydrogel particles are interesting vehicles for systemic siRNA delivery. For the first time, optimized anti-procollagen α1(I) siRNA loaded cationic nanohydrogel particles were tested in vivo as an antifibrotic therapy in a mouse model of liver fibrosis, induced by oral gavage of CCl4. After thorough validation of siRNA loaded nanohydrogel particles in vitro for cell toxicity, cellular uptake and knockdown performance, particles qualified for further biological evaluation in vivo. After systemic administration, siRNA loaded particles almost exclusively accumulated in liver, while lungs, spleen and kidneys were only targeted to a minor extend. On the cellular level, complexes addressed primarily activated myofibroblasts, the major collagen producing cells in liver fibrosis, and to a lesser extend endothelial cells and macrophages. Anti-procollagen α1(I) siRNA loaded cationic nanohydrogel sequence specifically and significantly suppressed hepatic procollagen α1(I) transcript levels and total collagen accumulation in liver fibrotic mice. Furthermore, a significant reduction of α-SMA, a surrogate marker of myofibroblast activation, supported the potent antifibrotic effect of the anti-procollagen α1(I) siRNA/nanoparticle treatment. Thus, cationic nanohydrogel particles qualified as promising vehicle platform for siRNA delivery to nonparenchymal liver cells. Since these first generation cationic nanohydrogel particles proved to be an attractive tool for systemic siRNA delivery, we attempted to improve their in vivo degradability and thus tolerability. This improvement is desirable, especially to prevent potential long-term side effects by tissue and cellular accumulation. A second generation of novel nanohydrogel particles was generated that contained a cationic biodegradable acid-labile cross-linker, endowing the next generation of nanohydrogel particles with enhanced biodegradability (bioNP). The first generation (non-bioNP) were compared 1:1 vs. the second generation cationic nanohydrogel particles (bioNP), loaded with anti-procollagen α1(I) siRNA, in another set of liver fibrosis studies. In vitro, acid-labile biodegradable complexes loaded with anti-procollagen α1(I) siRNA could compete with non-biodegradable complexes and showed no cytotoxicity (up to 400 nM siRNA), high cellular uptake (~100% after 24h) and an even improved knockdown efficiency for procollagen 1α(I) transcript mRNA (up to 80% for bioNP or 60% for non-bioNP) in murine fibroblasts. In vivo biodistribution in CCl4 induced liver fibrotic mice, using near infrared (NIR) imaging of NP that were labeled with two different NIR-dyes (one coupled to the siRNA, one coupled to the NP shell) showed an equally efficient siRNA delivery to liver and liver resident myofibroblasts for both NP species. However, in vivo half-lives of NP and their siRNA cargo differed between the first and second generation of cationic nanohydrogel particles. After repetitive iv injection BioNP/siRNA-complexes exhibited, compared to their first generation, an enhanced biodegradability with less tendency to (over)-accumulate in fibrotic and healthy livers. Nevertheless, anti-procollagen α1(I) siRNA loaded bioNP induced a potent procollagen α1(I) mRNA knockdown and prevented fibrosis progression as determined by liver biochemical and morphometrical collagen quantification. Long-term monitoring of the carriers in the body revealed a significantly enhanced clearance for the acid-degradable carrier, especially after multiple dosing. In healthy mice, both species showed a broad therapeutic index in doses escalation studies up to 10mg siRNA/kg body weight. Therefore, the novel acid-degradable cationic bio-NP could be validated as a promising novel platform for siRNA delivery in vivo to treat fibrotic liver diseases. Since these NP also target other nonparenchymal cells, especially macrophages and endothelial cells, further studies to deliver siRNA to these cells are warranted.» weiterlesen» einklappen

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DDC Sachgruppe:
Medizin