Pathological Microenvironment-Remodeling Nanoparticles to Alleviate Liver Fibrosis: Reversing Hepatocytes-Hepatic Stellate Cells Malignant Crosstalk

Abstract

During the onset and malignant development of liver fibrosis, the pernicious interplay between damaged hepatocytes and activated hepatic stellate cells (HSCs) induce a self-perpetuating vicious cycle, deteriorating fibrosis progression and posing a grave threat to public health. The secretions released by damaged hepatocytes and activated HSCs interact through autocrine or paracrine mechanisms, involving multiple signaling pathways. This interaction creates a harsh microenvironment and weakens the therapeutic efficacy of single-cell-centric drugs. Herein, a malignant crosstalk-blocking strategy is prompted to remodel vicious cellular interplay and reverse pathological microenvironment to put an end to liver fibrosis. Collagenases modified, bardoxolone and siTGF-β co-delivered nanoparticles (C-NPs/BT) are designed to penetrate the deposited collagen barriers and further regulate the cellular interactions through upregulating anti-oxidative stress capacity and eliminating the pro-fibrogenic effects of TGF-β. The C-NPs/BT shows successful remodeling of vicious cellular crosstalk and significant disease regression in animal models. This study presents an innovative strategy to modulate cellular interactions for enhanced anti-fibrotic therapy and suggests a promising approach for treating other chronic liver diseases.

Keywords: cellular crosstalk; chemogene therapy; hepatic stellate cell; hepatocyte; liver fibrosis.

Scheme 1

The preparation and in vivo mechanism of C‐NPs/BT. Briefly, C‐NPs/BT relieve hepatocytes damage and inhibit HSC activation by eliminating oxidative stress and silencing TGF‐β signaling pathway synergistically, thereby interrupting the malignant cellular interactions. C‐NPs/BT would significantly remodel the pathological microenvironment and promote the resolution of liver fibrosis.

Figure 1

The vicious crosstalk between hepatocytes and HSCs in human fibrotic liver. A) Bulk‐sequence analysis of cellular response to oxidative stress and TGFβ1 expression. B) UMAP plot showing the distribution of five major cell types. C) Signaling pathway enriched in hepatocyte. D) Signaling pathway enriched in HSC. E) Cell chat analysis of interactions among the five major cell types. F) KEGG analysis of gene enrichment related to these pathways. G) Representative images of tissue sections of patients's liver, showing H&E, Masson, Van Gieson, and α‐SMA staining. Data was expressed as mean ± SD. ^*** p < 0.001, ^**** p < 0.0001.

Figure 2

Characterization and in vivo anti‐fibrotic effects of C‐NPs/BT. A) Size distribution and TEM images of C‐NPs/BT. Scale bar = 100 nm B) Size variation in 5% glucose and 10% FBS 1640 culture medium over 72 h. C) Agarose gel electrophoresis of ALC‐0315/siRNA at various weight ratios. D) Lysosomal escape assay on HSC‐T6 cells. Scale bar = 10 nm E) Visualized collagen degradation experiments and quantification (n = 3). F) Representative images of Sirius Red staining. Scale bar = 50 µm G) Representative images of Masson staining. Scale bar = 50 µm H) Semi‐quantification results of Sirius Red positive area (n = 3). I) Semi‐quantification results of Masson positive area (n = 3). J) Serum ALT activity (n = 3). K) Serum AST activity (n = 3). L) Tissue hydroxyproline content (n = 3). C‐NPs/BT: nanoparticles modified with collagenases and loaded with bardoxolone and siTGF‐β; C‐NPs/B: nanoparticles modified with collagenases and loaded with bardoxolone; C‐NPs/T: nanoparticles modified with collagenases and loaded with siTGF‐β; B+T: free bardoxolone and siTGF‐β. Data was expressed as mean ± SD. No significant difference (ns): p > 0.05, ^** p < 0.01, and ^**** p < 0.0001.

Figure 3

The effect of C‐NPs/BT on hepatocytes in vitro and in vivo. A) Representative CLSM images of L02 cells after 2 h treatment. Scale bar = 10 µm B) Mean fluorescence intensity measured by flow cytometry (n = 3). C) Representative images using DCFH‐DA probe to test ROS levels. Scale bar = 100 µm D) Representative images of EdU staining. Scale bar = 100 µm E) Representative images of albumin immunofluorescent‐staining (blue: nucleus; green: albumin; red: DiI). Scale bar = 50 µm F) Representative images of ki67 staining (blue: nucleus; green: albumin; red: ki67). Scale bar = 50 µm G) Western blot and semi‐quantification for p‐Nrf2 and Smad7 (n = 3). H) Representative images of H&E staining. Scale bar = 50 µm Data was expressed as mean ± SD. No significant difference (ns): P > 0.05 and ^**** p < 0.0001.

Figure 4

The effect of C‐NPs/BT on HSCs in vitro and in vivo. A) Representative CLSM images of HSC‐T6 cells after 2 h. Scale bar = 10 µm B) Mean fluorescence intensity measured by flow cytometry (n = 3). C) Representative images of α‐SMA immunofluorescent staining on HSC‐T6 cells. Scale bar = 20 µm D) Representative images of α‐SMA immunofluorescent staining in vivo. Scale bar = 50 µm E) Representative images of α‐SMA‐stained liver sections. Scale bar = 50 µm F) Representative images of α‐SMA staining in a coculture model. Scale bar = 10 µm Data was expressed as mean ± SD. ^**** p < 0.0001.