An integrin-based quercetin 7-rhamnoside liver-targeted delivery liposomes for intrahepatic cholestasis in pregnancy

Abstract

Intrahepatic cholestasis in pregnancy (ICP) is a characteristic disease during the perinatal period; however, its therapy remains unsatisfactory, and the pathogenesis remains unclear. The ameliorative effect of naturally occurring quercetin 7-rhamnoside (Q7R) in cholestasis has been established. In this study, we aimed to establish a nanoparticle-based peptide, A20FMDV2-modified liposome (t-QL), to encapsulate and deliver Q7R. Q7R bioavailability improved significantly when liposomes were used as carriers. This peptide A20FMDV2-modified nanosystem targeted integrin αvβ6 on biliary epithelial cells and improved stillbirth rates and liver function indicators better than free Q7R without a carrier. Q7R improved ICP by regulating mitochondrial function and bile metabolism. Our nanosystem provides a promising nanotherapeutic strategy for applying Q7R in ICP. We also elucidated a therapeutic mechanism underlying the action of ICP by simultaneously targeting mitochondrial structure and function, as well as bile acid metabolism.

Keywords: Bile acid metabolism; Intrahepatic cholestasis in pregnancy; Mitochondrial function; Nanodrug delivery system; Peptide A20FMDV2; Quercetin 7-rhamnoside.

Graphical abstract

Fig. 1

Preparation and characterization of t-QL. (A) Response surface plots (three-dimensional) showing the effect of the SPC:HSPC (X1; w/w), TPC:SCS (X2; w/w), and TPC:Q7R (X3, % w/w) on the Stability (%) and EE (%) in the QL; (B) Representative transmission electron microscopy (TEM) images of QL and t-QL; scale bar = 50 nm; (C) Particle sizes of QL and t-QL; (D) Stability of QL and t-QL; (E) Drug release profiles of QL and t-QL in PBS; (F) Safety evaluation about haemolysis.

Fig. 2

Evaluation of hepatocyte and biliary epithelial cells targeting characteristics in vitro. (A) LSCM fluorescence images of Heap1-6 cell line after incubation with Free-C6, C6-Lip, and t-C6-Lip for 1, 2, and 4 h; (B) LSCM fluorescence images of HCCC-9810 cell line after incubation with Free-C6, C6-Lip, and t-C6-Lip for 1, 2, and 4 h; (C) Semi-quantitative fluorescence intensity analysis of the Heap1-6 and HCCC-9810 cell line uptake of Free-C6, C6-Lip, and t-C6-Lip for 1, 2, and 4 h (n = 3); ∗, p < 0.05; ∗∗, p < 0.01; and ∗∗∗, p < 0.001 compared to the Free-C6 group.

Fig. 3

Targeting and the biodistribution of C6- t-QL in a mouse. (A) Distribution of Free-DiR, DiR-Lip, and t-DiR-Lip at 6, 12, and 24 h in mice; (B) Fluorescence images after administration of Free-DiR, DiR-Lip, and t-DiR-Lip in different tissues in mice; (C) Semi-quantitative fluorescence intensity analysis of the liver uptake of Free-DiR, DiR-Lip, and t-DiR-Lip (n = 3); ∗, p < 0.05; ∗∗, p < 0.01; and ∗∗∗, p < 0.001 compared to the DiR group.

Fig. 4

Establishment of an ICP rat model induced by EB (n = 8). (A) Schematic diagram of the experimental design and t-QL for treatment of ICP; (B) Representative image of livers and feces; (C) Liver index, number of stillbirths and stillbirth rate of ICP rat; ∗, p < 0.05; ∗∗, p < 0.01; and ∗∗∗, p < 0.001 compared to the model group. ^#, p<0.05; ^##, p<0.01;^###, p<0.001 compared to the control; ▽, p < 0.05; ▽▽, p < 0.01; and ▽▽▽, p < 0.001 compared to the Q7R; ^ψ, p < 0.05; ^ψψ, p < 0.01; and ^ψψψ, p < 0.001 compared to the QL.

Fig. 5

Effect of t-QL treatment on ICP. (A–H) Serum ALT, AST, and ALP levels of ICP rat after treatment with different concentrations of t-QL (n = 8); ∗, p < 0.05; ∗∗, p < 0.01; and ∗∗∗, p < 0.001 compared to the model group. ^#, p<0.05; ^##, p<0.01;^###, p<0.001 compared to the control; ▽, p < 0.05; ▽▽, p < 0.01; and ▽▽▽, p < 0.001 compared to the Q7R; ^ψ, p < 0.05; ^ψψ, p < 0.01; and ^ψψψ, p < 0.001 compared to the QL; (I) Representative images of H&E stained liver tissue of ICP rat after treatment with different concentrations of t-QL, scalebar = 50 μm; (J) Representative images of Masson-stained liver tissue of ICP rat after treatment with different concentrations of t-QL, scalebar = 50 μm.

Fig. 6

Effect of t-QL on mitochondrial structure and function of ICP rat. (A) TEM images of hepatocyte mitochondrial, scalebar = 0.5 μm; (B) Liver NAD⁺/NADH ratio; (C) ATP levels and (D) GDF-15 levels in CTR and ICP rat were measured after treatment (n = 8). ∗, p < 0.05; ∗∗, p < 0.01; and ∗∗∗, p < 0.001 compared to the model group; ^#p<0.05;^###, p<0.001 compared to the control; ▽, p < 0.05; ▽▽▽, p < 0.001 compared to the Q7R; ^ψ, p < 0.05; ^ψψ, p < 0.01; ^ψψψ, p < 0.001 compared to the QL.

Fig. 7

Effects of t-QL on mRNA and protein expression in the liver of ICP rat. (A) Analysis of mRNA level are related to bile acid metabolism and mitochondria metabolism in the liver; (B) Analysis of proteins are related to bile acid metabolism and mitochondria metabolism in the liver, (n = 3), ∗, p < 0.05; ∗∗, p < 0.01; and ∗∗∗, p < 0.001 compared to the model group; ^#p<0.05;^###, p<0.001 compared to the controlgroup; (C) The brief diagram of bile acid metabolism and mitochondria metabolism pathway.

Fig. 8

Effect of t-QL on bile acid metabolism in ICP rat (n = 6). (A) Representative UPLC-MS/MS chromatogram of bile acid detection; (B) PCA-X and OPLS-DA score plot of ICP model rat; (C) Heatmap analysis of ICP model rat.

Fig. 9

Bile acid metabolite correlations with stillbirth rate scores (n = 6). Scatter plot correlations between (A) serum GHDCA and stillbirth rate scores; (B) serumTMCA and stillbirth rate scores; (C) serum TCDCA and stillbirth rate scores; (D) serum TDCA and stillbirth rate scores; (E) serum THDCA and stillbirth rate scores; (F) serum TUDCA and stillbirth rate scores; dashed lines indicate the mean value confidence interval for the line of best fit.