Co-delivery of miR-29b and germacrone based on cyclic RGD-modified nanoparticles for liver fibrosis therapy

Abstract

Hepatic stellate cells (HSCs) were activated and secreted excessive amounts of extracellular matrix (ECM) proteins during pathogenetic progress of liver fibrosis. Germacrone (GMO) and miR-29b can play an important role in inhibiting growth of HSCs and production of type I collagen. GMO and miR-29b were co-encapsulated into nanoparticles (NPs) based on poly(ethylene glycol)-block-poly(lactide-co-glycolide) (PEG-PLGA). Then, NPs were modified with cyclic RGD peptides (cRGDfK). cRGDfK is an effective ligand to bind integrin α_vβ₃ and increase the targeting ability for fibrotic liver. GMO- and miR-29b-loaded NPs exhibited great cytotoxicity to activated HSCs and significantly inhibited production of type I collagen. Liver fibrosis model of mice was induced by administration of carbon tetrachloride. Great targeting ability was achieved in liver fibrotic mice treated with cRGD-modified NPs. Significant ant-fibrotic effects have been presented based on hematoxylin and eosin (H&E), Masson and Sirius Red staining results of liver tissues collected from mice treated with drug-loaded NPs. All these results indicate GMO- and miR-29b-loaded cRGD-modified NPs have the potential for clinical use to treat liver fibrosis.

Keywords: Germacrone; Hepatic stellate cells; Liver fibrosis therapy; cRGDfK; miR-29b.

Fig. 1

Size distribution of blank NPs a, G/R-NPs b and G/R-RGD-NPs c measured by DLS; Zeta potential of blank NPs d, G/R-NPs e and G/R-RGD-NPs f measured by DLS; TEM photos of G/R-NPs g and G/R-RGD-NPs h; photo of fluorescent images of blank NPs and ICG-RGD-NPs i using a Maestro imaging system

Fig. 2

Fluorescent images of activated HSCs incubated with ICG-NPs and ICG-RGD-NPs with or without free cRGD pretreatment for 4 h a; fluorescent images of activated HSCs incubated with ICG-RGD-NPs for 2 h, 4 h, 8 h and 12 h b; cell viability of blank NPs (1 mg/mL) and R-RGD-NPs (1 mg/mL) against HSCs and LO2 for 24 h, 48 h and 72 h c; cell viability of free GMO, G-RGD-NPs and G/R-RGD-NPs against HSCs d and LO2 cells e. Scale bar: 20 μm

Fig. 3

Immunofluorescence and immunohistochemistry of activated HSCs treated with different drug formulations a; collagen I expression of HSCs treated with different drug formulations b; quantitative analysis of type I collagen in activated HSCs c. (*p < 0.05, **p < 0.01 and ***p < 0.001 vs Control; ^#p < 0.05 vs G-RGD-NPs)

Fig. 4

Biodistribution of ICG-NPs and ICG-RGD-NPs in mice of liver fibrosis and healthy mice after 24 h a; H&E, Masson and Sirius Red staining of livers collected from healthy and liver fibrotic mice b; collagen I expression of liver tissues from liver fibrotic mice treated with different drug formulations c; quantitative analysis of type I collagen in treated fibrotic livers d. (*p < 0.05, **p < 0.01 and ***p < 0.001 vs Control)