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. 2021 Dec;28(1):2534-2547.
doi: 10.1080/10717544.2021.2008054.

Liver-targeted delivery of asiatic acid nanostructured lipid carrier for the treatment of liver fibrosis

Ya-Wen Zhang  1 , Ling-Lan Tu  2 , Yi Zhang  1 , Jie-Chao Pan  3 , Gao-Li Zheng  4 , Li-Na Yin  1
Affiliations

Liver-targeted delivery of asiatic acid nanostructured lipid carrier for the treatment of liver fibrosis

Ya-Wen Zhang et al. Drug Deliv. 2021 Dec.

Abstract

Liver fibrosis is a major global health concern. Management of chronic liver disease is severely restricted in clinics due to ineffective treatment approaches. However, a lack of targeted therapy may aggravate this condition. Asiatic acid (AA), a pentacyclic triterpenoid acid, can effectively protect the liver from hepatic disorders. However, the pharmaceutical application of AA is limited by low oral bioavailability and poor targeting efficiency. This study synthesized a novel liver-targeting material from PEG-SA, chemically linked to ursodeoxycholic acid (UA), and utilized it to modify AA nanostructured lipid carriers (UP-AA-NLC) with enhanced targeting and improved efficacy. The formulation of UP-AA-NLC was optimized via the Box-Behnken Experimental Design (BBD) and characterized by size, zeta potential, TEM, DSC, and XRD. Furthermore, in vitro antifibrotic activity and proliferation of AA and NLCs were assessed in LX-2 cells. The addition of UP-AA-NLC significantly stimulated the TGF-beta1-induced expression of α-SMA, FN1, and Col I α1. In vivo near-infrared fluorescence imaging and distribution trials in rats demonstrated that UP-AA-NLC could significantly improve oral absorption and liver-targeting efficiency. Oral UP-AA-NLC greatly alleviated carbon tetrachloride-induced liver injury and fibrosis in rats in a dosage-dependent manner, as reflected by serum biochemical parameters (AST, ALT, and ALB), histopathological features (H&E and Masson staining), and antioxidant activity parameters (SOD and MDA). Also, treatment with UP-AA-NLC lowered liver hydroxyproline levels, demonstrating a reduction of collagen accumulation in the fibrotic liver. Collectively, optimized UP-AA-NLC has potential application prospects in liver-targeted therapy and holds great promise as a drug delivery system for treating liver diseases.

Keywords: Box–Behnken design; Liver-targeted therapy; asiatic acid; liver fibrosis; nanostructured lipid carrier.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1.
Figure 1.
Synthesis scheme (A) and 1H-NMR spectra (B) of UA-PEG-SA conjugate. (a) Acetylated ursodeoxycholic acid; (b) UA-PEG-SA conjugate.
Figure 2.
Figure 2.
Characterization of UP-AA-NLC. The size (A) and zeta potential (B) distribution of UP-AA-NLC. (C) TEM photograph of UP-AA-NLC.
Figure 3.
Figure 3.
DSC image of (A) AA powder, (B) Ingredients mixtures, (C) Physical mixtures, (D) UP-AA-NLC, (E) P-AA-NLC.
Figure 4.
Figure 4.
X-ray Diffraction Pattern of (A) AA powder, (B) Ingredients mixtures, (C) Physical mixtures, (D) UP-AA-NLC, (E) P-AA-NLC.
Figure 5.
Figure 5.
3D Response surface plots showing the effect of independent variables on response parameters. (A) Particle size (Y1), (B) Zeta potential (Y2), (C) Encapsulation efficiency (Y3), (D) Drug loadings (Y4).
Figure 6.
Figure 6.
Stability of UP-AA-NLC at 4? and 37? (pH7.4, containing 10% fetal bovine serum) (mean ± SD, n = 3).
Figure 7.
Figure 7.
In vitro release profiles of AA, P-AA-NLC and UP-AA-NLC in PBS (pH7.4, containing 1% SDS) (mean ± SD, n = 5).
Figure 8.
Figure 8.
(A) Cytotoxicity of AA and UP-AA-NLC in LX-2 cells. (B) Quantitative real-time PCR analysis of ?-SMA, FN1 and Col I ?1 expression in LX-2 cells. Each column represents the mean ± SD from three independent experiments. ##p < 0.01 vs. Control group, ###p < 0.001 vs. Control group, *p < 0.05 vs. TGF-β1 group, **p < 0.01 vs. TGF- β1 group, ***p < 0.001 vs. TGF- β1 group.
Figure 9.
Figure 9.
In vivo imaging of AA, P-AA-NLC and UP-AA-NLC biodistribution in rats. (A) Whole body imaging from 0 to 48 h. (B) The fluorescence intensity of the liver sites. (C) The imaging of the dissected major organs. *p? 0.05, **p? 0.01, ***p? 0.001.
Figure 10.
Figure 10.
10 Mean concentration of AA in various tissues at 24 h after oral administration at dose of 64 mg/kg (n = 3). *p? 0.05, **p? 0.01, ***p? 0.001.
Figure 11.
Figure 11.
Body weight curve (A) and organ coefficient (B).
Figure 12.
Figure 12.
Histopathological analysis of anti-fibrosis efficacy. The hematoxylin-eosin (H&E) staining (A) and Masson staining (B) examination of the dissected livers at the treatment endpoint (scale bar: 200 μm). (a) Normal group; (b) Liver fibrosis group; (c) AA (32 mg/kg); (d) Colchicine; (e) UP-AA-NLC (16 mg/kg); (f) UP-AA-NLC (32 mg/kg); (g) UP-AA-NLC (64 mg/kg). (C) Statistical analysis of liver tissue collagen fiber area after treated with AA, colchicine and UP-AA-NLC. (D) Liver fibrosis grading for all rats according to histopathological results. Grade S0 was defined as healthy liver and from S1 (slight fibrosis) to S4 (severe fibrosis) was represented the severity of liver fibrosis.
Figure 13.
Figure 13.
Biochemical analysis of anti-liver fibrosis efficacy. The serum biochemical parameters of AST (A), ALT (B), and ALB (C), and the liver tissue biochemical parameters of SOD (D), MDA (E), and HYP (F).
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