Targeted Delivery of Anti-TGF-β1-siRNA Using PDGFR-β Peptide-Modified Chitosan Nanoparticles for the Treatment of Liver Fibrosis.

Activated hepatic stellate cells (aHSCs) are key players in the fibrotic cascade in inflamed livers, with transforming growth factor-beta 1 (TGF-β₁) being the most potent pro-fibrotic cytokine. Therefore, TGF-β₁ downregulation in aHSCs may serve as an interesting therapeutic approach for the treatment of liver fibrosis. However, excessive collagen deposition in the extracellular matrix (ECM) hinders drug delivery to aHSCs. Chitosan nanoparticles (CS-NPs) show intrinsic affinity for collagen, holding potential for drug delivery to fibrotic livers. In this study, CS-NPs were used for the in vivo delivery of anti-TGF-β₁ siRNA. To promote delivery into aHSCs, CS-NPs were modified with different densities of platelet-derived growth factor receptor-β (PDGF-β) binding peptides as the targeting moiety. The CS-NPs showed an average hydrodynamic diameter of 103 ± 7 nm, a zeta potential of 24 ± 1 mV, and an siRNA encapsulation efficiency of 92.39 ± 6.4%. In healthy mice, biweekly treatment of siRNA-free unmodified CS-NPs up to a concentration of 120 mg/kg for 4 weeks was well tolerated and caused no organ-specific toxicity. The biodistribution of the unmodified and peptide-modified CS-NPs was carried out to identify potential sites of accumulation in healthy and fibrotic mice, and histopathological studies confirmed the safety of the formulations in nontarget sites in the case of fibrotic animals. Cell culture experiments confirmed the affinity of unmodified CS-NPs to the collagenated ECM, whereas collagen density reduction with collagenase-loaded CS-NPs (Coll-NPs) was necessary to promote cellular uptake of the peptide-modified CS-NPs. When loaded with anti-TGF-β₁ siRNA and used for the treatment of the CCl₄ liver fibrosis model in mice, CS-NPs decorated with a high density of PDGF-β binding peptide could significantly reduce the hepatic TGF-β₁ (by approximately 65%) and fibronectin (by approximately 63%) levels. Pretreatment with Coll-NPs could contribute to a further reduction of both markers by around 10%. Histopathological evaluations with Masson Trichrome staining revealed a reduction in the aggregation of the portal inflammatory cells, an absence in the proliferation of the fibroblastic cells in between hepatocytes, and a decrease in collagen deposition in the liver following treatment with anti-TGF-β₁ siRNA-loaded peptide-modified CS-NPs, both with and without Coll-NP pretreatment. Accordingly, the results demonstrate the plausibility of anti-TGF-β₁-siRNA delivery to aHSCs using PDGF-β binding peptide-modified CS-NPs for the treatment of liver fibrosis.