Targeted delivery of gossypol acetate prodrug chemically programmable biomimetic nanoparticles and uses thereof

Abstract

The present application relates to gossypol acetic acid, and particularly relates to a kind of targeted delivery gossypol acetic acid prodrug chemical programmable biomimetic nanoparticles and application thereof;The drug delivery carrier has the characteristics of non-toxicity, good biocompatibility and simple preparation, in addition, its particle size is small, and the targeting ability is strong, is conducive to direct targeting to liver stellate cell.In addition, the design of prodrug makes RAET NPs remain inert state under normal physiological conditions, to minimize systemic toxicity.And in the fibrosis microenvironment of high active oxygen level, prodrug is selectively converted into active form, to realize controllable release at lesion site.

Description

Technical Field

[0001] This invention relates to gossypol acetate, and more specifically to a chemically programmable biomimetic nanoparticle for targeted delivery of gossypol acetate prodrugs and its applications. Background Technology

[0002] Chronic liver disease has become a significant challenge in global public health, imposing a heavy burden on society. Liver fibrosis is a pathological repair response of the body to various chronic injuries, primarily caused by viral infections, insulin resistance-related metabolic disorders, cholangiocarcinoma, and autoimmune diseases. It is characterized by activation of hepatic stellate cells (HSCs), excessive deposition and abnormal cross-linking of the extracellular matrix (ECM). The persistent accumulation of ECM disrupts the liver microenvironment, impairs the structural integrity of liver tissue, and affects the maintenance of normal hepatocyte function. Without timely intervention, liver fibrosis can progress to cirrhosis, significantly increasing the risk of chronic liver failure and hepatocellular carcinoma—both diseases with high morbidity and mortality. Therefore, developing safe and effective anti-fibrotic treatment strategies is crucial to halting the progression of chronic liver disease.

[0003] Given the crucial role of HSC activation in the progression of liver fibrosis, inhibiting its activation is considered one of the most promising directions for developing anti-fibrotic treatment strategies. Existing research indicates that key factors driving HSC activation mainly include: (1) soluble signaling molecules, such as platelet-derived growth factor, vascular endothelial growth factor, and TGF-β; and (2) interactions with macrophages, other immune cells, hepatocytes, and endothelial cells through paracrine mechanisms. Therefore, drug development targeting these signaling pathways has received widespread attention, and most candidate drugs have shown significant anti-fibrotic effects in animal models. For example, rosutinol, sulvotetamide, and rosutinol have all shown therapeutic potential. However, due to mechanism-related toxicities or adverse reactions, only a few drugs have entered the clinical trial stage. Therefore, there is an urgent need to develop anti-liver fibrosis drugs with novel mechanisms of action and high selectivity to improve efficacy and safety. However, in the process of developing new anti-liver fibrosis drugs, many candidate drugs have problems such as insufficient target specificity, non-specific accumulation in multiple organs, low bioavailability and high toxicity. These defects seriously restrict their efficacy and make it difficult to effectively reverse or delay the progression of liver fibrosis. Summary of the Invention

[0004] Purpose of the invention Based on transcriptomics, molecular docking, and functional validation, we discovered for the first time that acetate gossypol (ACE) inhibits hepatic stellate cell activation by targeting Per2. Secondly, through chemical reconstruction, ACE was designed as a ROS-responsive prodrug and encapsulated using a macrophage / HSC exosome hybrid membrane to construct RAET NPs, a nanomedicine capable of actively targeting the liver, intelligently releasing drugs at the lesion site, and achieving immune evasion. In vitro and in vivo characterization, targeting validation, and safety evaluation of the system were completed. Both in vitro and in vivo experiments demonstrated that RAET NPs can effectively inhibit hepatic stellate cell activation, improve liver tissue microenvironment homeostasis, and thus alleviate the progression of liver fibrosis.

[0005] Technical solution Application of gossypol in the preparation of drugs for treating liver fibrosis.

[0006] The gossypol mentioned is gossypol acetate.

[0007]

[0008] Gossypol acetate A nanoparticle ACEP NPs, characterized in that it is prepared by the following steps: Preparation of prodrug nanoparticles ACEP NPs: PEG2000-COOH was used as a raw material. After carbonyl activation by EDC / NHS, it was reacted with the oxidation-responsive linker AMBA at room temperature for 24 h. Gossypol acetate (ACE) was then added, and the pH of the system was adjusted with triethylamine. The reaction product was dialyzed through a 1000 Da dialysis bag and then freeze-dried to obtain the triblock amphiphilic polymer ACEP. The prodrug ACEP NPs were prepared by utilizing the amphiphilic self-assembly of ACEP.

[0009] A nanoparticle ACEP NPs, characterized in that it is prepared by the following steps: 1) Dissolve PEG2000-COOH in pure water to prepare a PEG2000-COOH solution with a concentration of 4.5–5.5 mg / mL; add EDC and NHS to the PEG2000-COOH solution, stir and activate for 25–35 min, then add AMBA, and stir and react at room temperature for 22–26 h; wherein, based on 100 mg PEG2000-COOH, the amount of EDC is 6.2–7.3 mg, the amount of NHS is 5.2–6.3 mg, and the amount of AMBA is 8.0–9.3 mg; 2) Add 27.5–30.5 mg of gossypol acetate (ACE) to the reaction system obtained in step 1), adjust the pH of the system to 7.9–8.1 with triethylamine, and stir the reaction at room temperature for 22–26 h. 3) Dialyze the reaction solution obtained in step 2) using a dialysis bag with a molecular weight cutoff of 1000 Da for 22-26 h, and then freeze-dry it to obtain the triblock amphoteric polymer ACEP; the triblock amphoteric polymer ACEP can form prodrug nanoparticles ACEP NPs by its own amphiphilic self-assembly.

[0010] As an optimization method: A nanoparticle ACEP NPs, characterized in that it is prepared by the following steps: 1) Dissolve PEG2000-COOH in pure water to prepare a PEG2000-COOH solution with a concentration of 5 mg / mL; add EDC and NHS to the PEG2000-COOH solution, stir and activate for 30 min, then add AMBA, and stir and react at room temperature for 24 h; wherein, the feed mass of PEG2000-COOH, EDC, NHS and AMBA are 100 mg, 6.762 mg, 5.754 mg and 8.67 mg, respectively; 2) Add 29 mg of gossypol acetate (ACE) to the reaction system obtained in step 1), adjust the pH of the system to 8.0 with triethylamine, and stir the reaction at room temperature for 24 h. 3) The reaction solution obtained in step 2) was dialyzed for 24 h using a dialysis bag with a molecular weight cutoff of 1000 Da, and then freeze-dried to obtain the triblock amphoteric polymer ACEP; the triblock amphoteric polymer ACEP can form prodrug nanoparticles ACEP NPs by its own amphiphilic self-assembly.

[0011] A chemically programmable biomimetic nanoparticle for targeted delivery of gossypol acetate prodrug, characterized in that it is prepared by the following steps: S1. Preparation of prodrug nanoparticles ACEP NPs: PEG2000-COOH was activated by EDC / NHS and reacted with AMBA, followed by the addition of gossypol acetate for ACE reaction; the product was dialyzed and freeze-dried to obtain the amphoteric polymer ACEP, which was then self-assembled to obtain prodrug ACEP NPs; S2. Preparation of macrophage membrane stock solution: Prepare a cell membrane extraction reagent containing Tris-HCl, magnesium salt, potassium salt and protease inhibitor and filter it for sterilization; take RAW264.7 cells induced by LPS, wash and resuspend them, lyse the cells with the extraction reagent, collect the supernatant by centrifugation, dry them and reconstitute with PBS to obtain macrophage membrane stock solution. S3. Preparation of exosome membrane stock solution: Take JS-1 cells induced by TGF-β and collect cell supernatant; remove impurities by centrifugation, add exosome extraction reagent to enrich and obtain exosomes; lyse the exosomes with cell membrane extraction reagent, centrifuge to collect supernatant and dry, and reconstitute with PBS to obtain exosome membrane stock solution; S4. Preparation of hybrid membrane RE: Macrophage membrane reserve solution and exosome membrane reserve solution are mixed, ultrasonic treatment is used to promote membrane fusion, and large-sized structures are removed by filtration to obtain hybrid membrane RE; S5. Preparation of functionalized membrane material RAET: DSPE-Apt-CD44 was prepared by EDC / NHS chemical coupling; DSPE-Apt-CD44 was mixed with hybrid membrane RE, and hybrid biomembrane vesicles were formed by membrane extrusion. After centrifugation, the functionalized membrane material RAET was obtained. S6. Package ACEP NPs to obtain RAET NPs. Add 300 μL of ACEP NPs, vortex mix, and then filter through a 0.45 μm filter to remove excessively large particles, keeping the diameter of the remaining particles to around 150 nm.

[0012] As an optimization method: A biomimetic nanoparticle for targeted delivery of gossypol acetate prodrug is characterized by comprising the following steps: S1. Preparation of prodrug nanoparticles ACEP NPs: Using PEG2000-COOH as raw material, after carbonyl activation by EDC / NHS, it was reacted with the oxidation-responsive linker AMBA at room temperature for 24 h; then, gossypol acetate (ACE) was added, and the pH of the system was adjusted to 8.0 with triethylamine, and the reaction was carried out at room temperature for 24 h; the reaction product was dialyzed through a 1000 Da dialysis bag for 24 h and then freeze-dried to obtain the triblock amphiphilic polymer ACEP; the prodrug ACEP NPs were prepared by utilizing the amphiphilic self-assembly of ACEP.

[0013] S2. Preparation of macrophage membrane stock solution: RAW264.7 cells induced with 100 ng / mL LPS for 24 h and grown to 90% confluence were washed with PBS, resuspended, centrifuged and the supernatant was discarded, and then resuspended with macrophage membrane extraction reagent; the cells were then subjected to ice water bath for 15 min, repeated freeze-thaw cycles at -80℃ and 37℃ for 3 rounds, and centrifuged at 4℃ 3000 rpm for 10 min, and the supernatant was collected; after freeze-drying the supernatant, macrophage membrane stock solution was prepared with PBS to a concentration of 5 mg / mL.

[0014] S3. Preparation of exosome membrane stock solution: JS-1 cells grown to 40% confluence were induced with 10 ng / mL TGF-β for 48 h, and the medium was changed. The cell supernatant was collected twice at 24 h intervals and stored at -80℃. The supernatant was centrifuged at 3000xg for 15 min to remove cells and cell debris. The supernatant was collected and 0.5 times the volume of exosome extraction reagent was added. After vortexing and mixing, it was incubated at 4℃ overnight. The sample was centrifuged at 10000xg for 30 min at 4℃. The supernatant was discarded and the precipitate was resuspended in PBS to obtain exosomes. The exosomes were mixed with macrophage membrane extraction reagent and resuspended. The mixture was subjected to ice water bath for 15 min, repeated freeze-thaw cycles at -80℃ and 37℃ for 3 rounds, and centrifuged at 3000 rpm for 10 min at 4℃. The supernatant was collected and freeze-dried. The exosome membrane stock solution was prepared with PBS to a concentration of 5 mg / mL.

[0015] S4. Preparation of hybrid membrane RE: Take equal volumes of 5 mg / mL macrophage membrane stock solution and 5 mg / mL exosome membrane stock solution, respectively, mix them, and sonicate at 30 kHz and 60 W power for 1 min to promote the fusion of the two membrane structures; filter the fusion product through a 0.45 μm filter to remove large-sized vesicles and obtain hybrid membrane RE with uniform particle size.

[0016] S5. Preparation of functionalized membrane material RAET: Using the EDC / NHS chemical method, the 3' end of the CD44 nucleic acid aptamer was coupled with activated DSPE to obtain DSPE-Apt-CD44; the hybrid membrane RE was mixed with DSPE-Apt-CD44 accounting for 0.05–0.2 mol% of the total lipids, and extruded through a 200 nm polycarbonate membrane 21 times to form hybrid biomembrane vesicles. The product was collected by ultracentrifugation to obtain the functionalized membrane material RAET.

[0017] S6. Package ACEP NPs to obtain RAET NPs. Add 300 μL of ACEP NPs, vortex mix, and then filter through a 0.45 μm filter to remove excessively large particles, keeping the diameter of the remaining particles to around 150 nm.

[0018] The nucleic acid sequence of the CD44 aptamer is: 5'-CCAAGGCCTGCAAGGGAACCAAGGACACAGTTTTTTTT-3'.

[0019] Macrophage membrane extraction reagent: The macrophage membrane extraction reagent is prepared by using Tris-HCl, MgCl2·6H2O, KCl, protease inhibitor and distilled water. After adjusting the pH to 7.50 with NaOH, it is filtered through a 0.22μm filter for sterilization before use. Application of the described ACEP NP nanoparticles in the preparation of drugs for treating liver fibrosis.

[0020] The application of the chemically programmable biomimetic nanoparticles for targeted delivery of gossypol acetate prodrug in the preparation of drugs for treating liver fibrosis.

[0021] A biomimetic nanoparticle for targeted delivery of gossypol acetate prodrug, characterized in that it comprises gossypol acetate, Acetate gossypol (ACE), a polyphenolic compound extracted from cottonseed, possesses various biological activities. Its typical effect is the inhibition of spermatogenesis in men, reducing sperm count and motility by damaging the testicular seminiferous epithelium; it has been studied as an oral contraceptive. Pharmacological studies show that this compound can regulate signaling pathways such as PI3K / AKT and MAPK, downregulate anti-apoptotic proteins (e.g., NDRG1), and induce apoptosis, thereby exerting broad-spectrum anti-tumor activity, exhibiting inhibitory effects on uterine fibroids, tongue squamous cell carcinoma, and glioma. Furthermore, it can reverse the methylation of tumor suppressor gene promoters (such as hMLH1), showing potential in epigenetic regulation. However, there are no reports on its use in treating liver fibrosis; this invention is the first to discover that gossypol inhibits the development of liver fibrosis. To improve the targeted delivery efficiency of ACE and address its low solubility and systemic toxicity, we designed a biomimetic nanodelivery system that targets HSCs and is ROS-responsive.

[0022] Key points of the invention Liver fibrosis is a common pathological process in the progression of various chronic liver diseases, characterized by the activation of hepatic stellate cells (HSCs) and excessive deposition of extracellular matrix. Although the underlying mechanisms have been well understood, there is still a lack of drugs clinically capable of effectively reversing fibrosis. The core problem lies in the lack of selectivity in the distribution of existing antifibrotic drugs, resulting in significant off-target toxicity and difficulty in effectively targeting activated HSCs. Therefore, developing delivery systems that can target activated HSCs and achieve controlled drug release in the local microenvironment has become an important research direction in this field.

[0023] This invention presents a biomimetic nanomedicine (RAET NPs) based on modular assembly design, integrating a reactive oxygen species (ROS)-responsive prodrug, a hybrid cell membrane camouflage, and a nucleic acid aptamer for targeting, constructing a multi-level regulated delivery system. Specifically, this invention fuses exosome membranes derived from activated hepatic stellate cells (HSCs) with M1 macrophage membranes to prepare a hybrid membrane shell, achieving co-localization of functional proteins from both membranes, thereby synergistically achieving chemotaxis and homologous targeting. In terms of drug design, this invention designs ACE as a ROS-responsive prodrug. The endogenously elevated ROS levels in activated HSCs can trigger selective drug release, thereby reducing systemic toxicity. Furthermore, a CD44 nucleic acid aptamer is coupled to the surface of the hybrid membrane, enhancing the targeting precision to activated HSCs through dual recognition by the natural membrane protein and the artificial aptamer. Studies have shown that RAETNPs possess good cyclic stability and biocompatibility, can efficiently accumulate in activated HSCs, and undergo prodrug activation and controlled release under intracellular ROS stimulation, thereby effectively inhibiting HSC activation and extracellular matrix deposition, and delaying the progression of liver fibrosis. Mechanistic studies have shown that its therapeutic effect is related to the regulation of the circadian rhythm core gene Per2, which in turn downregulates the expression of matrix metalloproteinases (MMP3, MMP9) and various inflammatory factors.

[0024] 1. Construction Technology of Hybrid Biomimetic Membrane Shell: This invention is the first to fuse exosome membranes derived from activated hepatic stellate cells with M1 macrophage membranes to prepare hybrid biomimetic membrane shells. This hybrid membrane simultaneously retains functional proteins from both membrane sources, possessing the following three functions: Immune Escape Function: The M1 macrophage membrane component endows nanoparticles with the ability to evade clearance by the mononuclear macrophage system, prolonging their circulation time in vivo; Homologous Targeting Function: The activated HSC exosome membrane component achieves preferential binding to activated HSCs through a homologous recognition mechanism; Inflammatory Chemotaxis Function: The M1 macrophage membrane retains inflammatory factor receptors, endowing nanoparticles with the ability to chemotactically attract fibrotic inflammatory lesions. A single membrane source cannot simultaneously achieve the above three functions; this invention achieves functional complementarity and synergistic effects through a hybrid membrane strategy.

[0025] 2. Reactive Oxygen Response Prodrug Design: This invention designs the lead compound ACE as a reactive oxygen species (ROS) responsive prodrug. Under normal physiological conditions, this prodrug remains inert, significantly reducing systemic toxicity. Only in the fibrotic microenvironment (high ROS levels) is the prodrug specifically activated, transforming into an active drug form to achieve on-demand, controllable release at the lesion site. This design forms a closed-loop system of "microenvironment recognition—endogenous trigger-mediated drug release."

[0026] 3. Dual Recognition Mechanism of CD44 Nucleic Acid Aptamer Modification: This invention further couples a CD44-specific nucleic acid aptamer to the hybrid membrane surface. The CD44 receptor is highly expressed on the surface of activated HSCs and is a classic biomarker of liver fibrosis. Through a dual recognition mechanism of natural membrane protein and artificial aptamer, the targeting precision to activated HSCs is significantly enhanced, while non-specific effects on normal liver tissue and resting HSCs are reduced.

[0027] 4. Modular Assembly Strategy with Multi-Level Regulation: This invention modularly assembles the three functional modules (ROS-responsive prodrug, hybrid biomimetic membrane, and aptamer targeting) to construct a RAET NPs multi-level regulated delivery system. Each module is functionally independent yet synergistically complementary, and can be replaced or optimized as needed, exhibiting good programmability and platform potential.

[0028] Beneficial effects This invention constructs a programmable hybrid biomimetic membrane targeted delivery system modified with CD44 nucleic acid aptamer and loads it with a reactive oxygen species-responsive gossypol acetate prodrug, thus forming a multi-level regulated biomimetic nanomedicine RAET NPs. Compared with existing technologies, this invention has the following beneficial effects: The hybrid membrane strategy integrates the exosome membrane of activated hepatic stellate cells and the membrane of M1 macrophages, achieving for the first time the synergistic effect of immune escape, inflammatory chemotaxis, and homologous targeting, overcoming the technical bottleneck of the functional limitations of single membrane sources; CD44 aptamer modification further enhances the active targeting ability of nanomedicines to activated hepatic stellate cells. In vivo distribution and efficacy experiments have confirmed that the accumulation and anti-fibrotic efficacy of the aptamer-modified group in fibrotic liver are significantly better than those of the unmodified group; The reactive oxygen species-responsive prodrug design enables the drug to remain inert under normal physiological conditions to reduce systemic toxicity, and only transforms into an active form in the highly reactive oxygen species microenvironment of fibrosis, achieving controlled release at the lesion site. In vivo comparative experiments of free drug, non-responsive prodrug, and RAET NPs have confirmed that this design is the key to achieving synergistic effect and reduced toxicity; In animal models of liver fibrosis, RAET NPs significantly inhibited hepatic stellate cell activation and extracellular matrix accumulation, demonstrating excellent in vivo anti-fibrotic efficacy. In summary, this invention is significantly superior to existing single biomimetic systems or traditional nanocarriers in terms of targeting precision, controllable drug release, functional integration, and therapeutic safety, providing a safe, efficient, and programmable novel nanoplatform for the precise treatment of liver fibrosis. Attached Figure Description

[0029] Figure 1 High-throughput screening of antifibrotic drugs. (A) High-throughput drug screening platform; (B) Effect of lead compound on COL1 expression in activated hepatic stellate cells; (C) Effect of lead compound on α-SMA expression in activated HSCs.

[0030] Figure 2 1. Validation of the preliminarily screened antifibrotic drugs using 3D models. (A) Schematic diagram of the 3D co-culture model creation and drug administration process; (B) 3D cell spheroid image; (C) Expression level of COL1 in liver fibrosis organoids after treatment with different dominant compounds; (D) Expression level of α-SMA in liver fibrosis organoids after treatment with different dominant compounds; (E) Expression level of ROS in liver fibrosis organoids after treatment with different dominant compounds.

[0031] Figure 3 Validation of the antifibrotic efficacy of gossypol acetate. (A) Immunofluorescence distribution of COLⅠ and α-SMA; n=3. Data are presented as mean ± standard deviation. One-way ANOVA was used for analysis, and post-hoc Tukey test and two-tailed Student's t test were performed; (B) Effect of different concentrations of ACE on α-SMA protein expression.

[0032] Figure 4 A schematic diagram of the construction of a biomimetic nano-drug delivery system.

[0033] Figure 5 1H NMR spectra of ACEP NPs and their raw materials.

[0034] Figure 6 Morphological characterization of ACEP NPs. (A) TEM image of ACEP NPs, scale bar at 500 nm; (B) Particle diameter distribution of ACEP NPs.

[0035] Figure 7 Hybrid membrane characterization. (A) SDS-PAGE protein analysis, including ACE NPs, EXM, RCM, REM and RAETNPs; (B) Venn diagram of proteomics.

[0036] Figure 8 Morphological characterization of RAET NPs. (A) TEM image of hybrid RET NPs, scale bar at 500 nm; (B) Particle diameter distribution of hybrid RET NPs; (C) TEM image of RAET NPs, scale bar at 500 nm; (D) Particle diameter distribution of RAET NPs.

[0037] Figure 9Cellular uptake mechanism and in vivo biodistribution. (A) Confocal laser scanning microscopy revealed that RAET NPs accumulated in JS-1 cells in a time-dependent manner; (B, C) Flow cytometry further quantified the distribution at different time points; (DF) The distribution differences of different nanoparticles in JS-1 cells were visualized and quantitatively analyzed using confocal microscopy and flow cytometry; (GI) The uptake mechanism of CHM NPs by JS-1 cells was analyzed using confocal microscopy, and the changes in RAET NP content in JS-1 cells after different inhibition treatments were detected by flow cytometry; (J, K) Regarding in vivo biodistribution, different Cy5-labeled nanoparticles were monitored in CDHFD mice and CCl4 mice; (L, M) Dynamic distribution and accumulation in various tissues over time (n=3 biologically independent samples per group). Data are expressed as mean ± standard deviation and were analyzed using one-way ANOVA combined with Tukey post-hoc test and two-tailed Student t-test.

[0038] Figure 10 RAET NPs inhibited TGF-β-induced hepatic stellate cell activity in vitro. (A) Expression level of transforming growth factor-β after different nanomedicine treatments; (B) Expression level of α-smooth muscle actin after different nanomedicine treatments; (C) Expression level of type I collagen after different nanomedicine treatments; (D) Expression level of matrix metalloproteinase 9 after different nanomedicine treatments; (E) Expression level of matrix metalloproteinase 3 after different nanomedicine treatments; (F) Protein expression level of α-smooth muscle actin after different nanomedicine treatments; (G) Distribution of α-smooth muscle actin and type I collagen expression in activated hepatic stellate cells after different nanomedicine treatments, scale bar: 20 μm; (H) Expression level of type I collagen in liver fibrosis organoids after different nanomedicine treatments; (I) Expression level of α-smooth muscle actin in liver fibrosis organoids after different nanomedicine treatments; (J) Reactive oxygen species level in liver fibrosis organoids after different nanomedicine treatments; n = 3 biologically independent samples. Data are expressed as mean ± standard deviation. Data were analyzed using one-way ANOVA combined with post-hoc Tukey's test and two-tailed Student's t-test.

[0039] Figure 11. RAET nanoparticles may treat CDHFD-induced liver fibrosis. (A) Timeline and treatment regimens of CDHFD-induced NASH mice; (B) Body weight changes in mice of different treatment groups, n = 7 biologically independent samples; (C) Histological anatomy of livers in mice of different treatment groups at the end of the experiment, n = 7 biologically independent samples; (D and E) TG and TC levels, n = 3 biologically independent samples; (F and G) Serum ALT and AST levels; (H) H&E staining, Masson staining, and Sirius Red staining of livers in mice of different treatment groups, scale bar: 200 μm; n = 7 biologically independent samples; (I) Expression level of COL1 in liver tissues of mice of different treatment groups; (J) Expression level of α-SMA in liver tissues of mice of different treatment groups; (K) Expression level of MMP3 in liver tissues of mice of different treatment groups; (L) Expression level of MMP9 in liver tissues of mice of different treatment groups, n = 3 (M) Immune cell infiltration in liver tissue of mice in different treatment groups, scale bar: 200 μm; n = 7 biologically independent samples; (N) Expression level of CXCL2 in liver tissue of mice in different treatment groups; (OP) Serum levels of TNF-α, IL-6, and IL-1β, n = 3 biologically independent samples. Data are expressed as mean ± standard deviation. Data were analyzed by one-way ANOVA combined with post-hoc Tukey's test and two-tailed Student's t-test. Detailed Implementation

[0040] Example 1: High-throughput drug screening and validation of organoid models of liver fibrosis First, JS-1 cells were seeded in 24-well plates, and the inducer TGF-β was added to a final concentration of 10 ng / mL. The cells were incubated for 24 h, followed by washing with PBS. The screening drug and the positive control drug silymarin were then added to a final concentration of 10 μM, and the cells were incubated for another 24 h. The supernatant was collected. The secreted protein content was detected using an α-SMA (Alpha Smooth Muscle Actin, α-SMA) and COLⅠ (Type I Colagen, COLⅠ) ELISA kit, and the OD value was measured using a microplate reader. In two-dimensionally cultured JS-1 cells (activated by TGF-β1), 500 monomeric compounds from traditional Chinese medicine were initially screened. Using α-SMA and COLⅠ as indicators, candidate compounds with inhibitory effects comparable to the positive control drug (silymarin) were selected.

[0041] Secondly, HSCs, hepatocytes, and sinusoidal endothelial cells were co-cultured in a three-dimensional system using Matrigel (purchased from Jiangsu Kaiji Biotechnology Co., Ltd., product model KGL5101-1) as a scaffold to construct basic liver organoids with key cellular components and functions. To simulate the pathological process of liver fibrosis, the model was continuously stimulated with 10 ng / mL TGF-β1 for 7 days, and the expression of α-SMA and COL1, as well as the collagen deposition stained with Sirius red, were dynamically monitored to rigorously verify the reliability of the model at the molecular and pathological levels.

[0042] result: like Figure 1 As shown in AC, gossypol acetate exhibited excellent anti-liver fibrosis potential in the initial screening and was therefore selected as the lead compound for this study. In a more physiologically relevant organoid model of liver fibrosis, ACE was co-incubated with liver fibrosis organoids to evaluate its anti-fibrotic efficacy and to corroborate the results of the initial screening. As shown in Figures 2A-E, ACE was better than silymarin in inhibiting the expression of COL1, α-SMA and ROS in activated HSCs.

[0043] Example 2: Further Validation of the Anti-Hepatic Fibrosis Efficacy of Gossypol Acetate JS-1 cells were seeded in six-well plates, and TGF-β, the inducer, was added to a final concentration of 10 ng / mL. The cells were incubated for 48 h, then washed with PBS. A control group was set up, while the treatment groups were treated with 20 μM silymarin, 1 μM gossypol acetate, 5 μM gossypol acetate, and 10 μM gossypol acetate, respectively, for 48 h. After treatment, the cells were washed thoroughly with PBS, and protein was extracted. Changes in the expression levels of α-SMA and COLⅠ were detected by Western blotting and immunofluorescence double staining.

[0044] Result: As Figure 3 As shown, the relative expression level in the ACE-treated group was significantly lower than that in the activation group. Furthermore, compared to the 20 μM silymarin positive control group, the 10 μM gossypol acetate group achieved better inhibitory effects with a lower dosage. Therefore, gossypol acetate can be considered a potential targeted drug for the treatment of liver fibrosis in this study.

[0045] Example 3: Optimization and Construction of Bionic Nanoparticle Drug Delivery System To improve the targeted delivery efficiency of ACE and address its low solubility and systemic toxicity, we designed a biomimetic nanodelivery system that targets HSCs and is ROS-responsive. First, we synthesized phenylboronic acid-linked ACE prodrugs (ACEPNPs), which can specifically cleave in the high ROS environment of liver fibrosis lesions, achieving intelligent controlled drug release. Second, to endow the NPs with the dual functions of actively targeting HSCs and immune escape, we innovatively constructed a hybrid biomembrane (RE).

[0046] Then, a CD44-targeting nucleic acid aptamer (5'-CCAAGGCCTGCAAGGGAACCAAGGACACAGTTTTTTTT-3', see SEQ ID NO.1) was modified onto the surface of the hybrid membrane using a lipid anchoring method to obtain the final functionalized membrane material RAET. Finally, the prodrug ACEP NPs were encapsulated in the hybrid membrane RET to prepare the final biomimetic nanodrug RAET NPs.

[0047] The specific operating steps are as follows: A. Construction of prodrug ACEP NPs like Figure 4 As shown, AMBA was used as a linker in response to oxidative environments to synthesize the prodrug ACEP NPs: Specifically: 100 mg of PEG2000-COOH was dissolved in pure water to prepare a 5 mg / mL solution. 6.762 mg of EDC and 5.754 mg of NHS were added and stirred for 30 min to activate the carbonyl groups. Then, 8.67 mg of AMBA was added, and the reaction was carried out at room temperature with stirring for 24 h. After the reaction was complete, 29 mg of gossypol acetate (ACE) was added, and the pH was adjusted to 8.0 using triethylamine. The reaction was carried out at room temperature with stirring for 24 h. The mixture was then dialyzed using a 1000 Da dialysis bag for 24 h, and finally freeze-dried for storage. The resulting product was the triblock amphoteric polymer ACEP, which was utilized to self-assemble into nanoparticles, i.e., the prodrug ACEP NPs, based on its amphiphilic properties.

[0048] B. Macrophage membrane extraction: Preparation of macrophage membrane extraction reagent: Dissolve 1.576 g Tris-HCl in 10 mL of pure water, adjust the pH to 7.50 with NaOH solution, take 2 mL of the solution into a beaker, add 40.64 mg MgCl2·6H2O, 74.5 mg KCl and a protease inhibitor (cocktail) diluted with 1 mL of pure water, add 75 mL of distilled water and mix well, then make up to 100 mL, filter through a 0.22 μm filter and store.

[0049] Macrophage membrane extraction: RAW264.7 cells that had grown to 70% confluence were treated with lipopolysaccharide (LPS) at 100 ng / mL for 24 h, and the cells were observed and collected. Once the RAW264.7 cells reached 90% confluence in the culture dish, they were washed three times with 3 mL PBS each time, the waste liquid was discarded, and then 3 mL PBS was added again to gently resuspend the cells. The cells were then aliquoted into 2 mL centrifuge tubes. The tubes were centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. 1 mL of macrophage membrane extraction reagent was added to each centrifuge tube, the cells were resuspended, wrapped in plastic wrap, and placed in an ice-water bath for 15 min. The cell suspension was then subjected to three freeze-thaw cycles at -80℃ and 37℃. The lysed cell homogenate was placed in a centrifuge and centrifuged at 3000 rpm for 10 min at 4 ℃. The supernatant was collected, frozen, and then dried in a freeze dryer. When needed, it was prepared into a 5 mg / mL solution with PBS.

[0050] C. Exosome isolation and exosome membrane extraction Exosome extraction: JS-1 cells confluent to 40% were added to a culture dish and induced with 10 ng / mL TGF-β inducer. After 48 h, the medium was changed, and the supernatant was collected after 24 h. This was repeated after another 24 h, and the supernatant was stored at -80 °C. The supernatant was then centrifuged at 3000 xg for 15 min to remove cells and cell debris. The supernatant was transferred to a new, clean EP tube, taking care not to disturb any clumps of precipitate. 0.5 times the volume of exosome extraction reagent (Exosome Extraction Kit (General Type), KGI: KGA3201-10) was added to the cell-free cell culture supernatant. The mixture of cell culture supernatant and exosome extraction reagent was vortexed until homogeneous and incubated overnight at 4 °C. After incubation, the sample was centrifuged at 10000 xg for 30 min at 4 °C. Discard the supernatant. The beige or white precipitate at the bottom of the EP tube contains exosomes (usually invisible to the naked eye). Resuspend the precipitate with an appropriate amount of PBS buffer. The resuspended precipitate can be stored at -20°C for long-term storage. Exosome membrane extraction: Collected exosomes were placed in 1.5 ml centrifuge tubes, 1 ml of macrophage membrane extraction reagent was added, and the cells were resuspended by pipetting. The tubes were then wrapped in plastic wrap and placed in an ice-water bath for 15 min. The cell suspension was then subjected to three freeze-thaw cycles at -80°C and 37°C. The lysed cell homogenate was centrifuged at 3000 rpm for 10 min at 4°C. The supernatant was collected, frozen, and then freeze-dried. A 5 mg / mL solution was prepared with PBS before use. D. Membrane fusion to obtain hybrid membrane RE A mixture of macrophage and exosome membranes was pretreated using ultrasound (60 W, 1 min) to form heterologous membrane vesicles. This step disrupts the rigid structure of the membranes, allowing the two cell membrane fragments to fuse during ultrasound treatment, utilizing the fluidity of the cell membranes. Because phospholipid molecules have hydrophobic segments and hydrophilic heads, the hydrophobic segments and hydrophilic ends attract each other, causing the two membranes to fuse together. Due to the "self-sealing" behavior of their lipid bilayers, when the cell membrane breaks down, approximately spherical nanovesicles with internal cavities are spontaneously formed. However, the obtained derived vesicles are not homogeneous. They are then filtered through a 0.45 μm filter to eliminate large vesicles, controlling the final product diameter to around 150 nm.

[0051] Specific operations: Exosome membranes and macrophage membranes were dissolved in PBS to a concentration of 5 mg / mL, and 500 μL of each were mixed and sonicated (30 kHz, 60 W) for 1 min to promote membrane fusion. D. Targeting CD44 aptamer modification on the surface of hybrid membrane RE to obtain functionalized membrane material RAET. The 3' end of the CD44-targeting aptamer was coupled to activated DSPE via an NHS / EDC chemical method to obtain DSPE-Apt-CD44. The prepared hybrid membrane was mixed with DSPE-aptamer (0.05–0.2 mol% of total lipids) and then extruded 21 times through a 200 nm polycarbonate membrane to form hybrid biomembrane vesicles, which were then collected by ultracentrifugation.

[0052] D. Load ACEP NPs to obtain RAET NPs Add 300 μL of ACEP NPs, vortex to mix, and then filter through a 0.45 μm filter to remove larger particles, keeping the diameter of the remaining particles to around 150 nm.

[0053] Result: As Figure 5 As shown, the characteristic CH2 peak of PEG2000 appeared in the 1H NMR spectrum of ACEP. Simultaneously, the characteristic CH peaks of AMBA and the benzene ring of ACE also appeared at their corresponding positions in ACEP. These results indicate that PEG2000 and ACE were successfully coupled to form ACEP via AMBA.

[0054] Example 3: Characterization and in vitro functional evaluation of RAET NPs We will perform systematic physicochemical characterization of RAET NPs. Particle size and PDI will be determined using a Malvern laser particle size analyzer. Transmission electron microscopy will be used to observe whether the morphology exhibits a regular core-shell structure and dispersion. Cellular uptake assays: RhB-labeled RAET NPs will be co-incubated with activated JS-1 cells, and uptake efficiency will be quantitatively and qualitatively assessed using flow cytometry and confocal microscopy. An unmodified RE NPs group will be used as a negative control, and a CD44 aptamer pre-blockade assay will be employed to fully demonstrate its targeting specificity. In vivo targeting capability verification: Cy5-labeled RAET NPs will be injected into a liver fibrosis model mouse via tail vein, and their real-time distribution in vivo will be observed using an in vivo imaging system. After 24 hours, major tissues (heart, liver, spleen, lung, kidney, etc.) will be collected, and the fluorescence intensity ratio of the liver relative to other organs will be quantitatively analyzed to objectively evaluate its liver enrichment capacity and off-target effects.

[0055] result: like Figure 6 As shown in Figure A, ACEP NPs are uniform in size and spherical in shape; Figure 6 As shown in B, the average particle size is 143.3 nm and the dispersion index (PDI) is 0.133, exhibiting good dispersibility.

[0056] like Figure 7 As shown in Figure A, Coomassie blue staining results indicate that, compared to exosome membranes and macrophage membranes, hybrid membranes (REs) possess the protein profiles of both exosome and macrophage membranes. Furthermore, through proteomics Venn diagrams, such as... Figure 7 B shows that the hybrid membrane contains protein components of both exosome membrane and macrophage membrane.

[0057] like Figure 8 As shown in A and B, the morphological characterization of RAET NPs demonstrates that, as observed by TEM, RAET NPs possess a uniformly sized core-shell structure; Figure 8 The particle size distributions shown in C and D are somewhat increased compared to the hybrid film, with an overall distribution of around 157.4 nm, which further illustrates the successful encapsulation by the hybrid film.

[0058] like Figure 9As shown, the cellular uptake mechanism and in vivo biodistribution of RAET NPs were investigated. This study systematically evaluated the cellular uptake mechanism and in vivo distribution of RAET NPs. Using Rhodamine B as a model drug, it was found that the uptake of RAET NPs by hepatic stellate cells (HSCs) was time-dependent. After modification with CD44 aptamer, the targeting of NPs to activated HSCs was significantly enhanced. Mechanistic studies showed that NPs mainly enter cells via the caveolin-mediated endocytosis pathway. In in vivo distribution experiments, in non-alcoholic steatohepatitis and liver fibrosis models, Cy5-labeled RAET NPs showed strong and persistent fluorescence signals in the fibrotic liver region and were mainly enriched in the liver, without significant accumulation in major organs such as the heart, lungs, and kidneys, confirming their excellent active targeting ability.

[0059] 4. RAET NPs were used to further evaluate the in vitro anti-liver fibrosis efficacy.

[0060] like Figure 10 As shown, the results indicate that this invention first induces hepatic stellate cells (HSCs) activation using TGF-β1, followed by intervention with free ACE, ACE NPs, RAE NPs, and RAET NPs, respectively. The anti-fibrotic effect was systematically evaluated by detecting changes in the protein and mRNA expression of α-SMA and Col1a1 in activated HSCs. Immunofluorescence analysis showed that RAET NPs significantly inhibited the expression of α-SMA and Col1a1 compared to the free ACE, ACE NPs, and RAE NPs groups. Western blotting results also confirmed that RAET NPs more effectively reduced the expression levels of these two proteins. RT-qPCR analysis also showed that the α-SMA and Col1a1 mRNA expression levels in the RAET NPs group were significantly lower than those in other treatment groups. Furthermore, the study found that RAET NPs significantly reduced reactive oxygen species accumulation and promoted the conversion of HSCs from an activated state to a resting state. In conclusion, RAET NPs significantly enhanced the anti-hepatic fibrotic effect of ACE. To further validate its efficacy in models with higher pathological complexity, this study constructed a three-dimensional spherical model of HSCs. Quantitative morphological analysis showed that after treatment with RAET NPs, the volume and density of the spherical cells were significantly reduced, suggesting that RAET NPs have the ability to effectively penetrate and depolymerize the dense fibrotic structure formed by HSCs. Simultaneously, RAET NPs exhibited strong antioxidant capacity, significantly alleviating the abnormal accumulation of reactive oxygen species within the spherical cells. Furthermore, RAET NPs effectively downregulated the expression of key molecules in the downstream TGF-β1 signaling pathway and significantly inhibited the secretion of pro-fibrotic inflammatory mediators (such as TNF-α and IL-6), suggesting that it may block the vicious cycle in the process of liver fibrosis through a multi-target synergistic mechanism.

[0061] 5. Pharmacodynamic evaluation of RAET NPs in vivo Investigating whether RAET NPs have a therapeutic effect on NASH and its associated fibrosis. A mouse model based on a methionine-choline deficient diet combined with a 45% high-fat diet (CDHFD model) was established. After 4 weeks of modeling, CDHFD model mice were injected via tail vein with PBS buffer, free ACE inhibitors, or RAETNPs, respectively.

[0062] Result: As Figure 11 As shown, the CDHFD diet led to a significant decrease in body weight in mice, while free ACE and RAET NPs could alleviate this weight loss to some extent. Compared with the normal diet group, the livers of CDHFD mice treated with saline were smaller and yellowish; however, after intervention with RAET NPs, both liver weight and appearance were improved. Biochemical analysis showed that RAET NPs could significantly regulate the abnormal serum total cholesterol (TC) and triglyceride (TG) levels induced by the CDHFD diet in mice, and effectively reduce serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, suggesting that they have a positive effect on improving lipid metabolism disorders and inhibiting hepatocyte inflammatory response. Histopathological staining results (H&E staining, Sirius red staining, and Masson staining) showed that compared with the PBS control group, the liver histopathological damage in the RAET NPs treatment group was significantly improved. At the molecular level, RAET NPs significantly downregulated the expression of pro-fibrotic genes (Acta2, Col1a1) and pro-inflammatory cytokine genes (Ccl2, Ccl5) ​​in liver tissue, reduced the Timp1 / Mmp2 ratio, and decreased the hydroxyproline content in liver homogenate. At the protein expression level, the levels of type I collagen (COL1) and α-smooth muscle actin (α-SMA) were significantly downregulated, indicating that RAET NPs help improve liver function and regulate extracellular matrix deposition. These results suggest that RAET NPs have therapeutic potential for NASH and its associated liver fibrosis. Given the central role of hepatic macrophages in the pathogenesis of NASH, the researchers further examined the expression of the macrophage marker CD68. Immunofluorescence assays showed that CD68 expression was significantly increased in the liver of mice treated with PBS, while it was significantly decreased in the RAET NPs treatment group. Meanwhile, RAET NPs treatment significantly inhibited the mRNA expression levels of pro-inflammatory cytokines TNF-α, IL-1β and IL-6 in the liver, and the concentrations of the corresponding inflammatory factors in the serum were also downregulated.

[0063] In conclusion, RAET NPs show promise as a potential strategy for treating NASH and related liver fibrosis.

Claims

1. Application of gossypol in the preparation of drugs for treating liver fibrosis.

2. The application according to claim 1, characterized in that, The gossypol mentioned is gossypol acetate. Gossypol acetate.

3. A type of nanoparticle ACEP NPs, characterized in that, Prepared according to the following steps: Using PEG2000-COOH as raw material, the carbonyl group was activated by EDC / NHS and then reacted with the oxidation-responsive linker AMBA at room temperature for 24 h. Gossypol acetate (ACE) was then added, and the pH of the system was adjusted with triethylamine, and the reaction was carried out at room temperature. The reaction product was dialyzed through a 1000 Da dialysis bag and then freeze-dried to obtain the triblock amphoteric polymer ACEP. The amphiphilicity of ACEP was used for self-assembly to prepare prodrug nanoparticles ACEP NPs.

4. The nanoparticle ACEP NPs according to claim 3, characterized in that, Prepared according to the following steps: 1) Dissolve PEG2000-COOH in pure water to prepare a PEG2000-COOH solution with a concentration of 4.5–5.5 mg / mL; add EDC and NHS to the PEG2000-COOH solution, stir and activate for 25–35 min, then add AMBA, and stir and react at room temperature for 22–26 h; wherein, based on 100 mg PEG2000-COOH, the amount of EDC is 6.2–7.3 mg, the amount of NHS is 5.2–6.3 mg, and the amount of AMBA is 8.0–9.3 mg; 2) Add 27.5–30.5 mg of gossypol acetate (ACE) to the reaction system obtained in step 1), adjust the pH of the system to 7.9–8.1 with triethylamine, and stir the reaction at room temperature for 22–26 h. 3) Dialyze the reaction solution obtained in step 2) using a dialysis bag with a molecular weight cutoff of 1000 Da for 22-26 h, and then freeze-dry it to obtain the triblock amphoteric polymer ACEP; the triblock amphoteric polymer ACEP can form prodrug nanoparticles ACEP NPs by its own amphiphilic self-assembly.

5. The nanoparticle ACEP NPs according to claim 4, characterized in that, Prepared according to the following steps: 1) Dissolve PEG2000-COOH in pure water to prepare a PEG2000-COOH solution with a concentration of 5 mg / mL; add EDC and NHS to the PEG2000-COOH solution, stir and activate for 30 min, then add AMBA, and stir and react at room temperature for 24 h; wherein, the feed mass of PEG2000-COOH, EDC, NHS and AMBA are 100 mg, 6.762 mg, 5.754 mg and 8.67 mg, respectively; 2) Add 29 mg of gossypol acetate (ACE) to the reaction system obtained in step 1), adjust the pH of the system to 8.0 with triethylamine, and stir the reaction at room temperature for 24 h. 3) The reaction solution obtained in step 2) was dialyzed for 24 h using a dialysis bag with a molecular weight cutoff of 1000 Da, and then freeze-dried to obtain the triblock amphoteric polymer ACEP; the triblock amphoteric polymer ACEP can form prodrug nanoparticles ACEP NPs by its own amphiphilic self-assembly.

6. A chemically programmable biomimetic nanoparticle for targeted delivery of gossypol acetate prodrug, characterized in that, Prepared according to the following steps: S1. Prodrug nanoparticles ACEP NPs are obtained according to claim 4 or 5; S2. Preparation of macrophage membrane stock solution: Prepare a cell membrane extraction reagent containing Tris-HCl, magnesium salt, potassium salt and protease inhibitor and filter it for sterilization; take RAW264.7 cells induced by LPS, wash and resuspend them, lyse the cells with the extraction reagent, collect the supernatant by centrifugation, dry them and reconstitute with PBS to obtain macrophage membrane stock solution. S3. Preparation of exosome membrane stock solution: Take JS-1 cells induced by TGF-β and collect cell supernatant; remove impurities by centrifugation, add exosome extraction reagent to enrich and obtain exosomes; lyse the exosomes with cell membrane extraction reagent, centrifuge to collect supernatant and dry, and reconstitute with PBS to obtain exosome membrane stock solution; S4. Preparation of hybrid membrane RE: Macrophage membrane reserve solution and exosome membrane reserve solution are mixed, ultrasonic treatment is used to promote membrane fusion, and large-sized structures are removed by filtration to obtain hybrid membrane RE; S5. Preparation of functionalized membrane material RAET: DSPE-Apt-CD44 was prepared via EDC / NHS chemical coupling; DSPE-Apt-CD44 was mixed with hybrid membrane RE, and hybrid biomembrane vesicles were formed by membrane extrusion. After centrifugation, the functionalized membrane material RAET was obtained; the nucleic acid sequence of the CD44 aptamer is: 5'-CCAAGGCCTGCAAGGGAACCAAGGACACAGTTTTTTTT-3'; S6. Package ACEP NPs to obtain RAET NPs. Add 300 μL of ACEP NPs, vortex to mix, and then filter through a 0.45 μm filter to remove excessively large particles, keeping the diameter of the remaining particles to around 150 nm.

7. A chemically programmable biomimetic nanoparticle for targeted delivery of gossypol acetate prodrug, characterized in that, Includes the following steps: S1. Preparation of prodrug nanoparticles ACEP NPs: Using PEG2000-COOH as raw material, after carbonyl activation by EDC / NHS, it was reacted with the oxidation-responsive linker AMBA at room temperature for 24 h; then, gossypol acetate (ACE) was added, and the pH of the system was adjusted to 8.0 with triethylamine, and the reaction was carried out at room temperature for 24 h; the reaction product was dialyzed through a 1000 Da dialysis bag for 24 h and then freeze-dried to obtain the triblock amphiphilic polymer ACEP; the prodrug ACEP NPs were prepared by utilizing the amphiphilic self-assembly of ACEP. S2. Preparation of macrophage membrane stock solution: RAW264.7 cells induced with 100 ng / mL LPS for 24 h and grown to 90% confluence were washed with PBS, resuspended, centrifuged and the supernatant was discarded. The cells were then resuspended in macrophage membrane extraction reagent. The cells were subjected to three freeze-thaw cycles at -80℃ and 37℃, and centrifuged at 3000 rpm for 10 min at 4℃. The supernatant was collected. After freeze-drying, the supernatant was prepared into a 5 mg / mL macrophage membrane stock solution using PBS. The macrophage membrane extraction reagent consisted of Tris-HCl, MgCl2·6H2O, KCl, protease inhibitor, and distilled water. The pH was adjusted to 7.50 with NaOH, and the solution was filtered through a 0.22 μm filter for sterilization. S3. Preparation of exosome membrane stock solution: JS-1 cells grown to 40% confluence were induced with 10 ng / mL TGF-β for 48 h, and the medium was changed. The cell supernatant was collected twice at 24 h intervals and stored at -80℃. The supernatant was centrifuged at 3000xg for 15 min to remove cells and cell debris. The supernatant was collected and 0.5 times the volume of exosome extraction reagent was added. After vortexing and mixing, the mixture was incubated at 4℃ overnight. The sample was centrifuged at 10000xg for 30 min at 4℃. The supernatant was discarded and the precipitate was resuspended in PBS to obtain exosomes. The exosomes were mixed with macrophage membrane extraction reagent and resuspended. The mixture was subjected to ice-water bath for 15 min, repeated freeze-thaw cycles at -80℃ and 37℃ for 3 rounds, and centrifuged at 3000 rpm for 10 min at 4℃. The supernatant was collected and freeze-dried. The exosome membrane stock solution was prepared with PBS to a concentration of 5 mg / mL. S4. Preparation of hybrid membrane RE: Equal volumes of 5 mg / mL macrophage membrane stock solution and 5 mg / mL exosome membrane stock solution were mixed and ultrasonicated at 30 kHz and 60 W for 1 min to promote the fusion of the two membrane structures; the fusion product was filtered through a 0.45 μm filter to remove large vesicles and obtain hybrid membrane RE with uniform particle size. S5. Preparation of functionalized membrane material RAET: Using the EDC / NHS chemical method, the 3' end of the CD44-targeting nucleic acid aptamer was coupled with activated DSPE to obtain DSPE-Apt-CD44; the hybrid membrane RE was mixed with 0.05–0.2 mol% of the total lipids of DSPE-Apt-CD44, and the mixture was extruded 21 times through a 200 nm polycarbonate membrane to form hybrid biomembrane vesicles. The product was collected by ultracentrifugation to obtain the functionalized membrane material RAET. The nucleic acid sequence of the CD44-targeting nucleic acid aptamer is: 5'-CCAAGGCCTGCAAGGGAACCAAGGACACAGTTTTTTTT-3'; S6. Package ACEP NPs to obtain RAET NPs. Add 300 μL of ACEP NPs, vortex to mix, and then filter through a 0.45 μm filter to remove excessively large particles, keeping the diameter of the remaining particles to around 150 nm.

8. The use of the ACEP NPs nanoparticles according to any one of claims 3-5 in the preparation of drugs for treating liver fibrosis.

9. The use of the chemically programmable biomimetic nanoparticles for targeted delivery of gossypol acetate prodrug according to claim 6 or 7 in the preparation of drugs for treating liver fibrosis.

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