Self-assembled nano composite material as well as preparation method and application thereof

Abstract

The invention discloses a self-assembled nano composite material as well as a preparation method and application thereof, and belongs to the technical field of preparation of novel nano materials. The self-assembled nano composite material comprises a copper-based metal organic framework, an exosome wrapped inside the copper-based metal organic framework, polydopamine wrapped outside the copper-based metal organic framework, and an ASGR1 antibody modified on the surface of the polydopamine. According to the invention, catalytic treatment of nano-enzyme, biological regulation and control of exosome and a molecular targeting function of an antibody are ingeniously integrated, and a complex pathological network of MASLD is coped with through a multi-mechanism synergistic effect. The invention not only provides an effective candidate treatment material, but also provides multiple omics perspectives for understanding a combined treatment mechanism based on the exosome and the nano-enzyme, and has important theoretical significance and transformation application potential.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of novel nanomaterial preparation, and particularly relates to a self-assembled nanocomposite material, a preparation method thereof and application thereof. BACKGROUND

[0002] The information disclosed in this Background section is for the purpose of providing an understanding of the overall background of the application, and does not necessarily constitute an admission of prior art by the inventor or any representative of the inventor, and is not specifically admitted to be prior art by this disclosure.

[0003] Non-alcoholic fatty liver disease (NAFLD) and its severe form metabolic-associated fatty liver disease (MAFLD) have become the most common chronic liver disease, with a continuously rising prevalence rate along with the prevalence of obesity and metabolic syndrome. The pathological process of the disease is complex and variable, involving multiple interrelated pathological links such as abnormal accumulation of hepatocyte lipids, insulin resistance, oxidative stress, inflammatory response and apoptosis, forming a vicious cycle that is difficult to break. At present, the treatment means for NAFLD / MAFLD in clinical practice is still limited, mainly relying on lifestyle intervention and complication management, but the long-term effect is not satisfactory. Although liver transplantation provides the last treatment hope for patients with end-stage liver disease, the shortage of donors, surgical risks and postoperative management make its application greatly limited. This treatment dilemma highlights the urgency of developing new and efficient treatment strategies.

[0004] Although nanomaterials show application potential in the field of targeted drug delivery, the existing technical solutions still have obvious deficiencies. Current research attempts to use metal-organic framework materials as drug carriers to achieve liver-specific delivery by loading a single therapeutic agent (such as thyroid hormone) and modifying a targeting molecule (such as ASGR1 antibody). However, this kind of solution generally has the problem of single treatment component, which is difficult to comprehensively cover the complex multi-factor pathogenic network of NAFLD / MAFLD. For example, although thyroid hormone can regulate lipid metabolism, it has limited intervention on key links such as oxidative stress, inflammatory response and fibrosis during the disease process, and long-term use may cause systemic side effects. The existing material systems mostly focus on the simple superposition of functions, and lack the ability to synergistically intervene in multiple pathological links of the disease, resulting in limited treatment effect. In addition, the integration stability between the carrier structure and the functional components is insufficient, which affects the targeting efficiency and controlled release characteristics of the drug, and the long-term biological safety of the material also needs to be further verified. These limitations seriously restrict the clinical translation prospects of existing nanotherapeutic strategies. SUMMARY

[0005] In order to solve the problems in the prior art, the self-assembled nanocomposite, the preparation method and the application thereof are provided, the self-assembled nanocomposite provided by the application can target the liver to reduce the degree of liver steatosis, and the material itself can also scavenge active oxygen and reduce the damage of liver cells.

[0006] In order to achieve the above-mentioned purpose, the technical scheme of the application is as follows:

[0007] In a first aspect, the application provides a self-assembled nanocomposite, comprising a copper-based metal organic framework, an exosome wrapped inside the copper-based metal organic framework, a polydopamine coated outside the copper-based metal organic framework, and an asialoglycoprotein receptor 1 (ASGR1) antibody modified on the surface of the polydopamine.

[0008] Although nanozymes have high catalytic activity of natural enzymes and stability of nanomaterials, their future development depends on the integration with other bioactive modules due to the core limitations of poor targeting and single action mechanism. Although mesenchymal stem cell-derived exosomes have shown great potential in the treatment of liver diseases due to their natural bioactive substance delivery capacity, their stability, targeting and cargo protection capacity are insufficient, which limits their direct application.

[0009] To solve the challenges faced by nanozymes and exosomes, the application provides a new "cooperative integration" design concept, and constructs a self-assembled nanocomposite integrating "active targeting", "catalytic scavenging" and "gene regulation" functions, named CMEPA. Through precise nanoengineering technology, the three functional modules are organically combined: 1) catalytic module: a copper-based metal Prussian blue analogue nanozyme (i.e. copper-based metal organic framework) with clear superoxide dismutase / hydrogen peroxidase mimetic activity is selected as a "first-line defense barrier" for direct neutralization of active oxygen; 2) regulation module: human umbilical cord mesenchymal stem cell-derived exosomes are used as a natural and biocompatible "signal molecule library", especially their rich microRNA components are used to systemically regulate multiple pathological pathways in liver cells; 3) targeted navigation module: by coupling ASGR1 antibody, the nanocomposite is endowed with the ability to specifically recognize and enrich in liver cells.

[0010] The CMEPA provided by the application can realize cascade synergistic treatment: first, the active targeting effect of the ASGR1 antibody is used to ensure efficient and specific enrichment in liver tissue; then, the nanoscale enzyme core (i.e., a copper-based metal organic framework) can quickly remove excess active oxygen in the local area, break the vicious cycle of oxidative stress, and create a microenvironment conducive to cell repair; at the same time, the exosomes carried are delivered into liver cells, and the microRNA and other regulatory molecules in the exosomes can deeply intervene in key processes such as lipid synthesis, fatty acid oxidation, inflammatory signaling and cell apoptosis at the post-transcriptional level, thereby achieving the multi-level treatment goal from rapid symptom relief to long-term recovery of homeostasis. The CMEPA regulates the molecular mechanism of the sialic acid glycoprotein receptor 1-breast cancer susceptibility gene 1 / breast cancer susceptibility gene-related protein 1-liver X receptor alpha (ASGR1-BRCA1 / BARD1-LXRa) signal axis and inhibits the interleukin 6-tyrosine kinase 2-signal transduction and transcriptional activator 3 (IL-6 / JAK / STAT3) pathway through the synergistic effect of the three of "nanoscale enzyme-exosome-targeting antibody".

[0011] The application not only provides a nano therapeutic candidate drug with great transformation potential for the treatment of NAFLD / MAFLD, but more importantly, through the construction and verification of a multi-module integrated nano therapeutic platform, a new design paradigm and theoretical basis are provided for overcoming the limitations of single therapy for complex metabolic diseases. The multi-omics integration strategy adopted by the application also provides a methodological reference for in-depth analysis of the complex interaction between nanomaterials and biological systems.

[0012] In some embodiments of the application, the copper-based metal organic framework is formed by self-assembly of ferrous ions, copper ions, cyanide ions and citric acid (CA), and divalent copper ions and monovalent copper ions coexist.

[0013] In the application, citric acid is not only a simple dispersant, but also a structure directing agent and a modulator. Citric acid is a multidentate organic ligand, and the carboxyl and hydroxyl groups on the molecule can coordinate with Fe 2+ and Cu 2+ ions. This coordination can control the release rate of metal ions, avoid their rapid reaction with cyanide ions to generate a large amount of disordered precipitate, and thus facilitate the formation of a more ordered nanostructure. This coordination also participates in the final skeleton construction. In the reaction, citric acid coordinates with metal ions and cooperates with [Fe(CN)6] 4- units to guide the formation of metal organic frameworks with specific channels and structures. Citric acid has a certain reducing property, which helps to maintain the Fe 2+ state and adjust the redox environment of the system. Moreover, through its coordination and steric hindrance effect, citric acid can effectively prevent the excessive aggregation of nanoparticles and help form nanoscale products with uniform size and good dispersity.

[0014] In some embodiments of the present invention, the chemical formula of the copper-based metal-organic framework is Cu2[Fe2(CN)2].

[0015] In some embodiments of the present invention, the core framework of the copper-based metal-organic framework provided by the present invention is formed by the self-assembly of ferrous ions, copper ions, and cyanide ions, which is a Prussian blue analogue (PBAs) structure. Citric acid is a key ligand guiding synthesis and regulating structure and performance. The copper-based metal-organic framework is a multifunctional metal-organic framework material based on a cyanide-bridged copper-iron framework, while integrating citric acid ligands. The introduction of citric acid greatly alters the electronic structure, surface properties, and catalytic activity (i.e., nanozyme performance) of the material.

[0016] In some embodiments of the present invention, the exosomes are umbilical cord mesenchymal stem cell exosomes (UC-MSC-Exos).

[0017] In some embodiments of the present invention, the particle size of the self-assembled nanocarrier is 95~130 nm.

[0018] In some embodiments of the present invention, the total mass of exosomes and polydopamine is 15% to 20% of the mass of the self-assembled nanocarrier.

[0019] In some embodiments of the present invention, the self-assembled nanocarrier has an exosome loading of 12-17% of the mass of the copper-based metal-organic framework, a polydopamine loading of 2%-5% of the mass of the copper-based metal-organic framework, and an ASGR1 antibody loading of 0.1%-1% of the mass of the copper-based metal-organic framework.

[0020] A second aspect of the present invention provides a method for preparing the self-assembled nanocarrier described in the first aspect, comprising: Potassium ferrocyanide and citric acid were dissolved in water, and exosome suspension and copper salt aqueous solution were added sequentially under stirring to obtain a reaction mixture. The reaction mixture was stirred at room temperature to obtain a copper-based metal-organic framework-exosome precipitate. Copper-based metal-organic framework-exosome precipitate was dispersed in water, and dopamine solution and Tris-HCl buffer were added sequentially under stirring. The reaction was stirred to obtain copper-based metal-organic framework-exosome-polydopamine complex. The copper-based metal-organic framework-exosome-polydopamine complex was resuspended in phosphate buffer, and an activator and ASGR1 antibody were added. The mixture was then shaken to react. Bovine serum albumin was added to the system to block unreacted binding sites. After centrifugation, the self-assembled nanocomposite material was obtained.

[0021] In some embodiments of the present invention, the copper salt includes any one or more of copper chloride or its hydrate, copper sulfate or its hydrate.

[0022] In some embodiments of the present invention, the copper salt is copper sulfate pentahydrate, and the mass ratio of potassium ferrocyanide, citric acid, exosomes and copper sulfate pentahydrate is (3~4):(28~32):(0.1~0.5):(3~7), preferably (3.5~4):30:(0.3~0.5):(4~6), and more preferably 3.7:30:0.4:5.

[0023] In some embodiments of the present invention, the reaction mixture is stirred at room temperature for 15-20 h, washed, and copper-based metal-organic framework-exosome precipitate is obtained.

[0024] In some embodiments of the present invention, the mass ratio of copper-based metal-organic framework-exosomes to dopamine is (9~11):(0.3~0.4), preferably (9~11):(0.35~0.4), and more preferably 10:0.375.

[0025] In some embodiments of the present invention, a copper-based metal-organic framework-exosome precipitate is dispersed in water, a dopamine solution is added while stirring, the mixture is stirred, and then a Tris-HCl buffer solution is added while stirring. The pH of the Tris-HCl buffer solution is 8-9, preferably 8.5.

[0026] In some embodiments of the present invention, dopamine solution and Tris-HCl buffer are added sequentially under stirring. After stirring and reacting, the product is collected by centrifugation, washed, and resuspended in phosphate buffer to obtain a copper-based metal-organic framework-exosome-polydopamine complex.

[0027] In some embodiments of the present invention, the activator is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and the mass ratio of the copper-based metal-organic framework-exosome-polydopamine complex, the activator and the ASGR1 antibody is 100:(6000~6500):(1~5), preferably 100:(6200~6300):(2~4), and more preferably 100:6300:3.

[0028] A third aspect of the present invention provides the application of the self-assembled nanocarrier described in the first aspect in the preparation of a drug for treating fatty liver.

[0029] In some embodiments of the present invention, the fatty liver includes non-alcoholic fatty liver disease.

[0030] The beneficial effects of this invention are as follows: This invention provides a self-assembled nanocarrier that synergistically integrates three core functional components: a copper-iron-based nanozyme with dual superoxide dismutase (SOD) and catalase (CAT) mimicry activities; UC-MSC-Exos loaded with regulatory microRNA; and a hepatocyte-specific ASGR1 antibody. Western blot analysis of exosome marker proteins such as CD9, CD81, TSG101, and Alix demonstrated that the self-assembled nanocarrier exhibits a high exosome encapsulation efficiency; and compared to free exosomes, its protein retention rate after repeated freeze-thaw cycles is significantly improved, indicating that this platform possesses excellent carrier protection capabilities. In vitro experiments showed that the self-assembled nanocarrier can effectively scavenge reactive oxygen species (ROS), significantly reduce lipid droplet accumulation in palmitic acid-treated hepatocytes, and inhibit hepatocyte apoptosis. In vivo experiments showed that the self-assembled nanocarrier can selectively accumulate in liver tissue with good safety, and no systemic toxicity was detected. In a diet-induced mouse model of MASLD, administration of self-assembled nanocarriers significantly improved serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglycerides, and cholesterol. Histological results confirmed that it reduced the degree of hepatic steatosis and simultaneously increased the activity of endogenous superoxide dismutase (SOD) and catalase (CAT). Transcriptome sequencing analysis showed that the self-assembled nanocarriers significantly downregulated key signaling pathways related to inflammatory responses and lipid synthesis. Therefore, the self-assembled nanocarriers provided by this invention are highly efficient multi-target nanotherapeutic agents that integrate antioxidant catalysis, exosome-mediated gene regulation, and active hepatocyte targeting, offering a novel strategy with great translational potential for the treatment of metabolic-related fatty liver disease. Attached Figure Description

[0031] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0032] Figure 1 This diagram illustrates the synthesis process and mechanism of action of the self-assembled nanocomposite material of this invention. A represents the one-pot preparation process of CMEPA. B illustrates the mechanism of action of CMEPA in treating metabolic-related fatty liver disease.

[0033] Figure 2 The structure and physicochemical properties of CMEPA obtained in Example 1 of this invention are characterized. A is a schematic diagram of the CMEPA preparation process; B is a high-angle annular dark-field scanning transmission electron microscope image of CMEPA; C is the X-ray diffraction pattern of CuMOF and CMEPA; D is the Zeta potential distribution of CuMOF, ASGR1 antibody, exosomes, and CMEPA. n= 3); E is the Fourier transform infrared spectrum of CuMOF and CMEPA; F is the nitrogen adsorption-desorption isotherm of CuMOF and CMEPA; G is the thermogravimetric analysis curve of CuMOF and CMEPA; H is the full X-ray photoelectron spectrum of CMEPA; I is the copper 2p orbital spectrum; J is the copper LMM Auger spectrum; K is the sulfur 1s orbital spectrum.

[0034] Figure 3 This document describes the multi-enzyme simulated antioxidant activity of CMEPA obtained in Example 1 of this invention. A is a schematic diagram of the simulated catalytic mechanism of CMEPA involving catalase and superoxide dismutase; B shows the change in absorbance (A-A0) over time in the nitroblue tetrazolium reduction experiment after the addition of CuMOF and CMEPA; C shows the UV-Vis absorption spectra of the nitroblue tetrazolium-xanthine-xanthine oxidase system after the addition of CuMOF and CMEPA; D shows the electron paramagnetic resonance spectra characterizing the scavenging of superoxide anion radicals by CuMOF and CMEPA; E shows the concentration-dependent UV-Vis absorption changes corresponding to the scavenging of superoxide anion radicals by CMEPA; F shows the kinetic curves of oxygen production from hydrogen peroxide catalyzed by CuMOF and CMEPA; G shows the oxygen production kinetics of CMEPA at different hydrogen peroxide concentrations. n = 3); H represents the net oxygen production from the decomposition of hydrogen peroxide catalyzed by CMEPA at different concentrations; I represents the effect of temperature and pH on the simulated activity of CMEPA catalase.

[0035] Figure 4 This image illustrates the ASGR1-mediated targeting and in vitro therapeutic effects of CMEPA. A shows a confocal laser scanning microscope image: demonstrating the specific binding of DiL-labeled CMEPA (orange) to the DiO-PLUS-stained MIHA cell membrane; cell nuclei are counterstained with DAPI (blue), and the yellow fluorescence in the combined image represents the co-localization signal of both. Scale bar: 40 μm. B shows the flow cytometry quantitative analysis of the binding levels of DiR-labeled CuMOF, CuMOF@ASGR1, exosomes, and CMEPA to normal human hepatocytes (MIHA) (first row) and hepatocellular carcinoma cells (Huh7) (second row). C shows the live / dead staining experiment verifying the cytoprotective effect of CuMOF against hydrogen peroxide-induced oxidative damage in HeLa cells. D shows a representative Oil Red O staining image: showing lipid accumulation in Huh7 cells under different treatment conditions (red). E shows the quantitative analysis of the Oil Red O positive area corresponding to Figure D; data are expressed as mean ± standard deviation. n = 6). F represents representative fluorescence images of intracellular reactive oxygen species levels detected by the dichlorofluorescein diacetate (DCFH-DA) probe in different treatment groups. G represents the fluorescence images of the dichlorofluorescein diacetate (DCFH-DA) probe in different treatment groups. Figure 4 Quantitative analysis of the relative fluorescence intensity of DCFH-DA in F, data are expressed as mean ± standard deviation.n = 6). H represents the quantitative detection results of apoptotic cells in different treatment groups based on flow cytometry.

[0036] Figure 5 This section presents the in vivo liver targeting and induced transcriptome remodeling of CMEPA. A shows representative in vivo whole-body fluorescence images collected at specified time points after intravenous injection of DiR-labeled CMEPA in mice, demonstrating preferential accumulation and subsequent clearance of CMEPA in the liver. B shows the flow cytometry quantitative analysis results of DiR-positive cells in single-cell suspensions of mouse liver 24 hours after intravenous injection of different nanocomposite formulations. C shows principal component analysis of transcriptome sequencing data from liver tissues of mice in the control and CMEPA-treated groups, revealing significant differences in transcriptome clustering characteristics between the two groups. D shows a volcano plot of differentially expressed genes in liver tissues of mice in the CMEPA-treated and control groups. E shows a heatmap of the relative abundance of mouse liver immune cell subsets derived from transcriptome deconvolution analysis. F shows the gene set enrichment analysis of representative Kyoto genes and genome encyclopedia pathways that showed significant changes after CMEPA treatment. G shows the gene set enrichment analysis of selected landmark pathways that were significantly regulated after CMEPA administration.

[0037] Figure 6 The study aimed to improve liver injury, restore lipid homeostasis, and regulate key metabolic and inflammation-related proteins in a diet-induced metabolic-associated fatty liver disease mouse model using CMEPA. In this study, A represents the serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride, and total cholesterol levels in mice from different treatment groups. n = 6). B represents the activities of superoxide dismutase and catalase in mouse liver ( n = 6). C shows representative images of liver tissue stained with hematoxylin-eosin and Oil Red O, scale bars: 100 μm, 50 μm. D shows the quantitative analysis of the relative area of ​​lipid droplets under Oil Red O staining ( n = 3). E represents the quantitative analysis of BRCA1, BARD1, LXRα, ASGR1, interleukin-1β, and interleukin-6 protein expression in liver tissue using Western blot. F represents the quantitative statistical results of protein expression levels. n = 3). Data are expressed as mean ± standard deviation. P≤0.0001. Detailed Implementation

[0038] This invention provides a self-assembled nanocomposite material, its preparation method, and its application, which addresses the technical deficiencies faced by nanozymes and exosomes in the prior art.

[0039] The technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0040] In the following examples, all reagents or drugs used are commercially available or self-made.

[0041] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0042] Example 1 A method for preparing a self-assembled nanocomposite material (CMEPA) includes the following steps: 1. Isolation and identification of human umbilical cord mesenchymal stem cells and extraction and identification of their exosomes Human umbilical cord mesenchymal stem cells (UC-MSCs) were isolated from umbilical cord tissue of healthy women undergoing full-term cesarean section, cultured in a specialized culture medium, and passaged to passages 3-5 for future use. Flow cytometry was used to identify stem cell surface markers.

[0043] Exosomes derived from umbilical cord mesenchymal stem cells (UC-MSC-Exos) were extracted from serum-free conditioned medium using differential ultracentrifugation. The specific steps were as follows: the supernatant of the culture medium was centrifuged sequentially at 300×g, 2000×g, and 10000×g to remove cells and cell debris; subsequently, it was ultracentrifuged at 110000×g for 70 minutes, and the exosome precipitate was collected. The morphology of the exosomes was observed using transmission electron microscopy, and their particle size distribution (approximately 100 nm) was detected using nanoparticle tracking analysis. All human-related experiments strictly followed the relevant regulations of the Ethics Committee of Weifang Maternal and Child Health Hospital (Ethics Approval No.: KY-2025-07).

[0044] 2. Preparation of copper-based metal-organic framework-exosome-polydopamine complex (CuMOF@EX / PDA) 3.7 mg potassium ferrocyanide and 30 mg citric acid were dissolved in 1.5 mL of water by sonication. 0.5 mL of exosome suspension (containing 0.4 mg of exosomes) was added while stirring, and the mixture was stirred for 30 min. Then, 1 mL of an aqueous solution containing 5 mg copper sulfate pentahydrate was added while stirring. The reaction mixture was stirred at room temperature for 18 h, and washed three times with deionized water to obtain a copper-based metal-organic framework-exosome precipitate, named CuMOF@EX.

[0045] 1 mg of copper-based metal-organic framework-exosome precipitate was ultrasonically dispersed in 10 mL of deionized water. 0.25 mL of dopamine solution with a concentration of 1.5 mg / mL was added while stirring continuously for 5 min. Subsequently, 8 mL of tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffer with a concentration of 10 mmol / L and a pH of 8.5 was added to the mixture and stirred for 2 h. Finally, the mixture was centrifuged at 13400 rpm for 15 min, the product was collected, washed twice with water, and resuspended in 1 mL of phosphate-buffered saline (PBS) to obtain the copper-based metal-organic framework-exosome-polydopamine complex, named CuMOF@EX / PDA.

[0046] 3. Preparation of self-assembled nanomaterials (CMEPA) One mg of CuMOF@EX / PDA was resuspended in 1 mL of phosphate buffer, and 63 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and 30 μg of ASGR1 antibody were added. The mixture was shaken for 2 h. Then, 50 μL of 10% bovine serum albumin (BSA) solution was added to the system, and the reaction was continued for another 2 h to block unreacted binding sites. Finally, the self-assembled nanocomposite material was collected by centrifugation at 13400 rpm for 15 min, named CuMOF@EX / PDA / ASGR1 (CMEPA), and resuspended in 1 mL of aqueous solution.

[0047] Comparative Example 1 A copper-based metal-organic framework (CuMOF) is prepared as follows: 0.1 mmol potassium ferrocyanide K4[Fe(CN)6] and 0.3 g citric acid were dispersed in 20 mL of water. Under vigorous magnetic stirring, 10 mL of an aqueous solution of 0.2 mmol copper sulfate pentahydrate was added dropwise to the mixture. Immediately after addition, a wine-red turbidity was observed. The reaction mixture was then stirred continuously at room temperature for 18 h. The resulting copper-based metal-organic framework precipitate, named CuMOF, was collected by centrifugation and washed three times thoroughly with deionized water for subsequent characterization.

[0048] Comparative Example 2 A self-assembled nanomaterial (CuMOF@ASGR1) is prepared by the following method: 1 mg of CuMOF obtained from Comparative Example 1 was ultrasonically dispersed in 10 mL of deionized water. 0.25 mL of dopamine solution with a concentration of 1.5 mg / mL was added while stirring continuously for 5 min. Subsequently, 8 mL of Tris-HCl buffer with a concentration of 10 mmol / L and a pH of 8.5 was added to the mixture and stirred for 2 h. Finally, the mixture was centrifuged at 13400 rpm for 15 min, the product was collected, washed twice with water, and resuspended in 1 mL of phosphate buffer (PBS) to obtain the copper-based metal-organic framework-polydopamine complex, named CuMOF@PDA.

[0049] 1 mg CuMOF@PDA was resuspended in 1 mL of phosphate buffer, and 63 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and 30 μg of ASGR1 antibody were added. The mixture was shaken for 2 h. Then, 50 μL of 10% bovine serum albumin (BSA) solution was added to the system, and the reaction was continued for 2 h to block unreacted binding sites. Finally, the self-assembled nanocomposite material was collected by centrifugation at 13400 rpm for 15 min, named CuMOF@ASGR1, and resuspended in 1 mL of aqueous solution.

[0050] Experimental Example 1 I. Experimental Methods 1. Detection of superoxide dismutase (SOD)-like activity in CMEPA The superoxide dismutase-like catalytic activity of CMEPA was evaluated based on its inhibition of nitroblue tetrazolium (NBT) reduction. The standard experimental setup was as follows: a Tris-HCl buffer (0.1 mol / L, pH 7.2) containing xanthine oxidase (XO, 1 U / mL), xanthine (X, 0.5 mmol / L), and nitroblue tetrazolium (NBT, 0.4 mmol / L) was prepared. Different concentrations of CMEPA were added, and the absorbance of the reaction solution was continuously monitored at 560 nm for 10 minutes. Simultaneously, the superoxide dismutase-like activity of CMEPA was determined using electron paramagnetic resonance (EPR) technology. The typical experimental procedure is as follows: CMEPA aqueous solution is added to Tris-HCl buffer (0.1 mol / L, pH 7.2) containing xanthine (X) and 5,5-dimethyl-1-pyrrolline-N-oxide (DMPO) to achieve final concentrations of CMEPA, X, and DMPO of 10 μg / mL, 0.5 mmol / L, and 100 mmol / L, respectively; xanthine oxidase (1 U / mL) is added to initiate the reaction, and electron paramagnetic resonance spectra are recorded to evaluate the superoxide dismutase-like activity of CMEPA.

[0051] 2. Detection of catalase (CAT)-like activity in CMEPA The catalase-like activity of CMEPA was analyzed by detecting the amount of oxygen generated in a hydrogen peroxide reaction system with a concentration of 5 mmol / L. Different concentrations of CMEPA were mixed with hydrogen peroxide in a Tris-HCl buffer solution with a concentration of 0.1 mol / L and a pH of 7.2. The dissolved oxygen concentration in the system was continuously monitored over 10 min using a dissolved oxygen meter.

[0052] 3. Cell Culture and Model Construction Human hepatocellular carcinoma cell line Huh-7 (purchased from Yaji Biotechnology Co., Ltd.) was treated with palmitic acid-bovine serum albumin complex for 24 h to induce intracellular lipid accumulation and establish a steatosis hepatocyte model. Human normal hepatocyte line MIHA (C712) and human cervical cancer cell line HELA (catalog number ml086960, RRID: CVCL_1922) were both purchased from Shanghai Enzyme-Linked Biotechnology Co., Ltd.

[0053] C57BL / 6 mice (purchased from Beijing SPF Biotechnology Co., Ltd.) were used and fed a high-fat, high-cholesterol diet for 14 weeks to establish a mouse model of metabolic-associated fatty liver disease (MASLD). All animal experiments were strictly conducted in accordance with the relevant regulations of the Animal Ethics Committee of Qingdao University (ethics approval number: 20220910C576520241001110).

[0054] In a study of the therapeutic effects of diet-induced MASLD in mice, mice were administered either a CMEPA nanoparticle formulation or an equivalent control solution (physiological saline) via tail vein injection. The dosing regimen was 200 μL of the 10 μg / mL formulation per mouse, administered once every 3 days for a total of 10 doses.

[0055] 4. Detection of cell viability, cytotoxicity, and apoptosis Cell viability and cytotoxicity assays: Dual fluorescence staining was performed using a calcein-AM / PI cell viability and cytotoxicity assay kit. Live cells showed green fluorescence, while dead cells showed red fluorescence. Quantitative analysis was then performed using a fluorescence microscope.

[0056] Cell proliferation and toxicity assay (CCK-8 assay): The experiment was conducted using a cell counting kit-8. After cell treatment, CCK-8 solution was added to cells in 96-well plates, and the cells were incubated for 1–4 h. The absorbance was measured at 450 nm, and cell viability was calculated.

[0057] Apoptosis detection: The Annexin V-FITC / PI apoptosis detection kit was used in combination with flow cytometry to distinguish live cells, early apoptotic cells, late apoptotic cells and necrotic cells.

[0058] 5. Biochemical indicator testing The activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mouse serum were detected using a kit from Beyotime Biotechnology Co., Ltd. The determination was performed by fluorescence or colorimetric methods based on the amount of pyruvate / oxaloacetate produced.

[0059] Alkaline phosphatase, gamma-glutamyl transferase (GGT / γ-GT), albumin, total bilirubin (TBIL), urea (BUN), red creatinine, creatine kinase, lactate dehydrogenase (LDH), mouse tumor necrosis factor-α (TNF-α), mouse interleukin-6 (IL-6), mouse interleukin-1β (IL-1β), mouse interleukin-10 (IL-10), mouse complement component 3a (C3a), and mouse complement component 5a (C5a) were detected using a serum assay kit.

[0060] The levels of triglycerides and total cholesterol were determined using an enzymatic reaction combined with fluorescence / colorimetric method.

[0061] The content of residual hydrogen peroxide in the system was detected by colorimetry to assess catalase activity; the total superoxide dismutase activity was determined by the water-soluble tetrazolium salt-8 (WST-8) method based on the inhibition of superoxide anion generation; and the total glutathione content was quantitatively determined by monitoring the kinetic changes at a wavelength of 412 nm using the glutathione reductase cycle reaction.

[0062] The malondialdehyde (MDA) content was determined using the thiobarbituric acid reactant (TBARS) method, which served as a marker for lipid peroxidation damage.

[0063] 6. Histopathological analysis Liver tissue was fixed in 4% paraformaldehyde solution, embedded in paraffin, sectioned, and then stained with hematoxylin-eosin to observe the gross morphology and structure of the liver tissue.

[0064] Liver tissue sections were stained using an Oil Red O staining kit to visually represent the deposition of neutral lipids within the tissue.

[0065] 7. Western blot detection Total cellular protein was extracted using pre-cooled radioimmunoprecipitation (RIPA) lysis buffer containing 1% phenylmethylsulfonyl fluoride (PMSF) protease inhibitor. Protein concentration was determined using a quinolinic acid (BCA) protein quantification kit. The protein sample concentration was leveled by adding 5-fold sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer, and then denatured at 100°C.

[0066] Equal volumes of protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the proteins were then transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was placed in a Tween 20 solution containing 5% skim milk powder and blocked at room temperature for 1 hour. Subsequently, the membrane was incubated overnight at 4°C with the following primary antibodies: breast cancer susceptibility gene 1 antibody (BRCA1), breast cancer susceptibility gene-associated protein 1 antibody (BARD1), liver X receptor α antibody (LXRα), sialic acid glycoprotein receptor 1 antibody (ASGR1), interleukin-1β antibody (IL-1β), interleukin-6 antibody (IL-6), differentiation cluster antigen 9 antibody (CD9), differentiation cluster antigen 81 antibody (CD81), tumor susceptibility gene 101 antibody (TSG101), apoptosis connexin antibody (Alix), and glyceraldehyde-3-phosphate dehydrogenase antibody (GAPDH).

[0067] After washing the membrane, incubate it with the corresponding horseradish peroxidase (HRP)-labeled secondary antibody at room temperature for 1 hour, and then use the BeyoECLPlus ultrasensitive chemiluminescence kit to develop and observe the protein bands.

[0068] 8. Imaging technology Laser confocal scanning microscopy (CLSM) was used to observe the cellular uptake and localization of nanomaterials using a triple staining protocol. The staining reagents used included: 4',6-diamidinyl-2-phenylindole (DAPI, for nuclear staining), a green fluorescent probe for cell membranes (DiOplus, for cell membrane staining), a red fluorescent probe for cell membranes (Dil, for labeling nanomaterials), and a near-infrared fluorescent probe for cell membranes (DiR, for cell membrane staining).

[0069] Small animal in vivo imaging: DiR-labeled nanomaterials were injected into mice via the tail vein. Mice were anesthetized with isoflurane (anesthetic gas flow rate of 2-3 L / min), and then whole-body fluorescence imaging of mice was performed using the IVIS Lumina XRMS Ⅲ in vivo imaging system. The imaging exposure time was uniformly set to 20 s to analyze the accumulation of nanomaterials in liver tissue.

[0070] 9. Transcriptome sequencing and bioinformatics analysis Total RNA was extracted from liver tissue and, after passing quality testing, transcriptome sequencing was performed using a biological sequencing platform. Following sequencing, the data underwent quality control, sequence alignment, and quantitative gene expression analysis. Differentially expressed gene analysis was then performed using software.

[0071] II. Results and Discussion 1. Characterization of human umbilical cord mesenchymal stem cells (UC-MSCs) and their exosomes (UC-MSC-Exos) This invention successfully isolated and cultured human umbilical cord mesenchymal stem cells (UC-MSCs). Under an optical microscope, these cells exhibited a typical spindle-shaped fibroblast-like morphology, a hallmark feature of mesenchymal stem cells. To verify cell identity, flow cytometry was used to detect the expression of surface markers. The cultured cells highly expressed the classic mesenchymal stem cell markers CD73 (positive rate 99.7%), CD90 (positive rate 97.4%), and CD105 (positive rate 99.8%), while the expression levels of hematopoietic stem cell markers CD34 (positive rate 1.83%), CD45 (positive rate 3.71%), and CD19 (positive rate 0.97%) were extremely low. These results confirm that the cells isolated by this invention are high-purity human umbilical cord mesenchymal stem cells.

[0072] Subsequently, exosomes (UC-MSC-Exos) were isolated from the conditioned medium of human umbilical cord mesenchymal stem cells using ultracentrifugation. Transmission electron microscopy revealed that the isolated vesicles exhibited a clear disc- or cup-shaped morphology, a characteristic ultrastructural feature of exosomes. Furthermore, Western blotting analysis confirmed the successful detection of typical marker proteins of exosomes, further demonstrating the successful isolation of high-purity exosomes.

[0073] 2. Synthesis and Characterization of CMEPA Example 1 describes the synthesis of copper-based metal-organic framework nanoparticles (copper hexacyanoferrate, molecular formula (Cu2[Fe2(CN)2])) via a one-step self-assembly method. The preparation conditions are mild, allowing for exosome encapsulation during synthesis, trapping the proteins and nucleic acids within the metal-organic framework. The product was then modified with polydopamine to introduce protein-affinity amino groups; subsequently, an ASGR1 antibody was linked via amide bonds, ultimately yielding the self-assembled nanocomposite material CMEPA (…). Figure 2 (Figure A in the middle)

[0074] Scanning electron microscope images ( Figure 2 Figure B shows that the synthesized CMEPA nanoparticles exhibit good uniformity, with a particle size of 112 ± 15 nm (n=20), which is consistent with the hydrated particle size measured by the Malvern particle size analyzer. Elemental analysis results from high-angle annular dark-field scanning transmission electron microscopy (HETEM) are also presented.Figure 2 Figure B in the diagram confirms that elements such as carbon (C), nitrogen (N), oxygen (O), iron (Fe), copper (Cu), phosphorus (P), and sulfur (S) are uniformly distributed within the nanoparticles. Among these, carbon, nitrogen, iron, and copper primarily originate from the copper-based metal-organic framework core and the polydopamine coating, while phosphorus and sulfur originate from the phospholipids and proteins of the exosomes. This result confirms that the exosome contents have been successfully embedded within the CMEPA framework structure.

[0075] X-ray diffraction pattern ( Figure 2 Figure C shows that the characteristic diffraction peaks of CMEPA are in high agreement with those of CuMOF, indicating that the modification with the polydopamine coating did not disrupt the original phase structure of CuMOF. Zeta potential detection results ( Figure 2 Figure D shows that the CuMOF surface is strongly negatively charged, while the exosome surface is positively charged. This charge difference facilitates electrostatic interactions between the two. Compared with CuMOF, the zeta potential of CMEPA is slightly increased. UV-Vis absorption spectroscopy further confirmed the presence of characteristic absorption peaks of exosomes and proteins in the system, indicating that the relevant components have been successfully integrated into CMEPA.

[0076] Furthermore, to verify the encapsulation efficiency of CMEPA on exosomes, Western blotting was used to detect and quantify exosome marker proteins such as CD9, CD81, TSG101, and Alix. The results showed that these marker proteins were effectively detected in CMEPA, confirming that exosome proteins were successfully encapsulated in this nanocomposite material. Fourier transform infrared spectroscopy (FTIR) Figure 2 The figure (E) shows that the depth is 3500-3200 cm. -1 A distinct characteristic absorption band appears in the wavenumber range, which is correlated with the polydopamine loading; while at 1000 cm⁻¹... -1 The characteristic peaks in the vicinity indicate the presence of nucleic acid components in the system.

[0077] Exosomes are prone to membrane structure damage, content leakage, and functional loss during repeated freeze-thaw cycles, a deficiency that severely limits their storage and clinical application. To evaluate the stability of CMEPA under low-temperature conditions, CMEPA and free exosomes were subjected to multiple freeze-thaw cycles, followed by quantitative analysis of the retention rates of marker proteins using Western blotting. The results showed that compared with the free exosome group, the CMEPA group had significantly higher retention rates of proteins such as CD9 and TSG101 (p<0.01), indicating that the nanocomposite structure of CMEPA can effectively protect the encapsulated exosome contents and alleviate functional decline caused by freeze-thaw processes. This advantage provides important support for the long-term storage and practical application of CMEPA.

[0078] Nitrogen adsorption-desorption isotherm ( Figure 2The figure (Figure F) shows that, compared with HCF, CMEPA has a significantly reduced specific surface area, which is due to the infiltration of exosomal proteins and nucleic acids into its framework structure. Thermogravimetric analysis results (Figure F) Figure 2 Figure G shows that after heating the sample to 1200℃, the residual mass percentage of CuMOF was 53.9% and the residual mass percentage of CMEPA was 36.7%, which means that exosomes and polydopamine contributed about 17.2% to the mass of CMEPA.

[0079] X-ray photoelectron spectroscopy analysis ( Figure 2 The H-plot confirms that CMEPA contains carbon (C), nitrogen (N), oxygen (O), sulfur (S), phosphorus (P), iron (Fe), and copper (Cu). The copper 2p orbital spectrum (…) Figure 2 (Figure I) at 935.1 eV (Cu 2p) 3 / 2 ) and 955.3 eV (Cu2p 1 / 2 A characteristic peak appears at 932.9 eV, accompanied by a satellite peak at 932.9 eV. This is consistent with the presence of divalent copper ions (Cu) in the system. 2+ ) and monovalent copper ions (Cu + The characteristics are consistent with those of the LMM Auger spectrum of copper. Figure 2 Figure J confirms that in this metal-organic framework material, copper mainly exists as divalent copper ions (Cu). 2+ It exists in the form of ). Finally, the sulfur 1s orbital spectrum ( Figure 2 The characteristic peak at approximately 164 eV (as shown in the middle K figure) is a characteristic peak of thiol groups, confirming that the exosome contents have been successfully encapsulated in the CuMOF framework structure.

[0080] 3. Multienzyme-like activity of CMEPA CMEPA, due to its abundant copper-nitrogen (Cu-N) catalytic sites and regular crystal structure, exhibits excellent superoxide dismutase (SOD)-like activity and catalase (CAT)-like activity. Figure 3 (See Figure A). To evaluate the antioxidant properties of CMEPA, this example uses a standard NBT reduction experiment. Figure 3 As shown in Figure B, both CuMOF and CMEPA significantly inhibited the reduction rate of NBT. UV-Vis spectroscopy confirmed that the colorimetric intensity of the CuMOF-treated group was significantly reduced, while the CMEPA-treated group showed a comparable inhibitory effect. Figure 3 (Figure C in the middle)

[0081] Electron paramagnetic resonance spectroscopy analysis results using DMPO as a spin trapping agent show that ( Figure 3(Figure D) Both materials effectively scavenge superoxide anion radicals, specifically as evidenced by a significant decrease in the signal intensity of the DMPO-OOH adduct. Although CMEPA exhibits slightly lower activity than CuMOF, this slight decrease in activity can be attributed to the steric hindrance effect of the polydopamine coating, which may, to some extent, hinder the contact between superoxide anion radicals and the catalytic site. With increasing CMEPA additive content, the absorbance signal intensity at 560 nm decreases in a dose-dependent manner, providing direct evidence that CMEPA possesses superoxide dismutase-like activity. Figure 3 (China E map).

[0082] Furthermore, this experimental example evaluates the CAT-like activity of CuMOF and CMEPA by detecting their ability to scavenge hydrogen peroxide. Figure 3 As shown in Figure F, the addition of both CuMOF and CMEPA significantly increased the dissolved oxygen content in the solution, and the oxygen release increased in a concentration-dependent manner with increasing CMEPA concentration. Figure 3 (See Figure G). Furthermore, as the hydrogen peroxide concentration increases, the oxygen release from the system also exhibits a concentration-dependent increase (see Figure G). Figure 3 (See Figure H), which confirms that CMEPA possesses excellent CAT-like activity. Notably, under physiological pH conditions (pH=7.4), CMEPA retains 84.4% of its activity, demonstrating good biocompatibility. Figure 3 (See Figure I in the original text). This experiment also investigated the long-term storage stability of the CMEPA nanozyme solution. After 20 days of storage, the SOD-like and CAT-like activities of CMEPA remained above 90%, demonstrating good stability. These results indicate that CMEPA is a highly efficient antioxidant system with great application potential in the biomedical field.

[0083] 4. Biocompatibility of CMEPA To assess the biocompatibility of CMEPA, this study used the CCK-8 assay to detect the cell viability of the normal hepatocyte cell line MIHA after treatment with CMEPA at concentrations ranging from 0 to 50 μg / mL for 24 h. The results showed that while cell viability decreased slightly with increasing CMEPA concentration, it remained above 80% even at the highest concentration of 50 μg / mL. Within the tested concentration range in this study, CMEPA showed no significant cytotoxicity to normal hepatocytes, demonstrating good biocompatibility.

[0084] To comprehensively evaluate the long-term biosafety of CMEPA, a systematic in vivo toxicological assessment was conducted in this study. Healthy mice were injected intravenously with CMEPA (10 μg / mL, 200 μL), while control mice were injected with an equal volume of saline. Twelve weeks after injection, histopathological examinations were performed on the major organs (heart, brain, liver, kidney, lung, small intestine, and spleen) of the mice. The results showed no significant pathological changes in any organ compared to the control group. Specifically: the myocardial fiber structure is intact with clear striations, showing no signs of inflammatory infiltration or necrosis; the neuronal structures of the cerebral cortex and hippocampus are well preserved, with no signs of edema or degeneration; the liver tissue structure is normal, with clear demarcation between the hepatic cords and central vein, and no fatty degeneration or intralobular inflammation; the glomeruli and tubules of the kidneys are intact, with no signs of tubular damage or interstitial nephritis; the alveolar structure of the lung tissue is clear, with no congestion, hemorrhage, or inflammatory cell infiltration; the villi and crypt structures of the small intestinal mucosa are normal, with no signs of erosion or ulceration; and the red and white pulp structures of the spleen remain intact.

[0085] In addition, a comprehensive serological analysis was performed on mice in this experiment. The results showed that there were no significant differences in the levels of a range of biomarkers compared with the control group, covering liver function indicators (alanine aminotransferase, aspartate aminotransferase, gamma-glutamyl transferase, total bilirubin, albumin, and alkaline phosphatase), kidney function indicators (blood urea nitrogen and creatinine), muscle and tissue damage indicators (creatine kinase and lactate dehydrogenase), key pro-inflammatory cytokines (interleukin-1β, interleukin-6, interleukin-10, and tumor necrosis factor-α), and complement components (complement 3a and complement 5a).

[0086] In summary, histopathological and serological examination results indicate that CMEPA does not cause detectable acute or long-term toxicity, confirming its excellent biosafety and suitability for in vivo application.

[0087] 5. Sialic acid glycoprotein receptor 1-mediated CMEPA hepatocyte targeting First, the hepatocyte targeting ability of the constructed CMEPA was evaluated. Laser confocal scanning microscopy analysis on the normal human hepatocyte cell line MIHA showed that the material can achieve efficient and precise binding to cells. Figure 4 As shown in Figure A, after 2 hours of incubation, CMEPA labeled with the red fluorescent probe (DiL) (orange fluorescence) and the cell membrane stained with the green fluorescent probe (DiO-PLUS) (green fluorescence) exhibited extensive co-localization, forming a distinct yellow fluorescent signal at the cell edges. This clear spatial overlap directly confirms that CMEPA has been successfully anchored to the surface of the hepatocyte membrane.

[0088] Flow cytometry was used to quantitatively analyze the targeting efficiency. Detection results of MIHA cells and the hepatocellular carcinoma line Huh7 treated with CMEPA labeled with a near-infrared cell membrane fluorescent probe (DiR) indicated that the introduction of the ASGR1 antibody was a key factor in achieving targeting. The final CMEPA formulation showed significantly enhanced binding ability to cells, with its average fluorescence intensity in MIHA and Huh7 cells increasing by approximately 4.5 times compared to the control group without targeting function (p<0.001). Figure 4 (Figure B in the middle). This confirms that ASGR1-mediated action plays a central role in achieving efficient hepatocyte-specific delivery.

[0089] 6. Verification of the intracellular protective effects of copper-based metal-organic frameworks on various cell types To comprehensively evaluate the biocompatibility and antioxidant potential of the nanozyme component in CMEPA, the protective effect of CuMOF against hydrogen peroxide-induced damage to the human cervical cancer cell line HeLa was further investigated. Figure 4 As shown in Figure C, under oxidative stress, the cell viability of cells pretreated with CuMOF was significantly enhanced, and this material can effectively alleviate hydrogen peroxide-induced cell membrane damage and cell death. This result not only confirms that CuMOF, as the catalytic core, possesses good biosafety and antioxidant function even in non-hepatocyte systems, but also provides experimental evidence for the synergistic protective effect of CMEPA in multicellular environments.

[0090] 7. CMEPA improves palmitic acid-induced hepatic steatosis, oxidative stress, and apoptosis in vitro. After clarifying the targeting efficiency of CMEPA, its therapeutic potential in intervening in hepatic steatosis was verified. To simulate the lipotoxic damage in the core pathogenesis of metabolic-associated fatty liver disease, palmitic acid was used to treat the Huh7 liver cancer cell line. As a saturated fatty acid, palmitic acid can strongly induce multiple core features of the disease, including severe oxidative stress, endoplasmic reticulum stress, and significant apoptosis, thus providing a more rigorous and pathologically accurate experimental environment for efficacy evaluation. Oil Red O staining results confirmed this, showing that palmitic acid successfully induced a large accumulation of lipid droplets in the cells. Figure 4 (D diagram).

[0091] CMEPA intervention significantly improved the above-mentioned hepatic steatosis phenotype. Intracellular red lipid droplet staining signal was significantly reduced. Quantitative analysis showed that, compared with the model group, the area of ​​Oil Red O staining positivity in the CMEPA-treated group was reduced by more than 70% (p<0.0001), and its improvement effect was significantly better than that of other incomplete formulations. Figure 4 (See Figure E). This potent lipid-lowering effect fully demonstrates the synergistic therapeutic advantages of this multi-component system.

[0092] However, the therapeutic effects of CMEPA are not limited to lipid-lowering. Given that palmitic acid-induced lipotoxic damage is primarily driven by oxidative stress, it is hypothesized that CMEPA's integrated antioxidant defense system is the key mechanism by which it exerts its therapeutic effects. To verify this hypothesis, intracellular reactive oxygen species (ROS) levels were measured. As expected, CMEPA treatment most effectively inhibited palmitic acid-induced ROS bursts (…). Figure 4 (Figure F) reduced fluorescence intensity to near baseline levels, and its effect was significantly better than all other groups ( Figure 4 (Figure G in the middle). This result directly confirms that nanozyme components play an indispensable role in alleviating lipotoxicity-related oxidative damage.

[0093] The reduction of lipid overload and oxidative stress ultimately significantly improved cell viability. Apoptosis was detected by flow cytometry. Figure 4 The results of the H-plot and live / dead cell staining experiments confirmed the comprehensive protective effect of CMEPA. Palmitic acid-induced apoptosis exceeded 25%, while CMEPA treatment significantly reduced the apoptosis rate to below 5%. This indicates that CMEPA can achieve comprehensive cytoprotective effects by simultaneously targeting multiple interrelated pathological drivers of MASLD.

[0094] 8. In vivo liver targeting and pharmacokinetic characteristics of CMEPA To verify the targeting efficacy of CMEPA in living organisms, its real-time in vivo distribution was first tracked using a small animal in vivo imaging system. CMEPA labeled with a near-infrared cell membrane fluorescent probe, administered via tail vein injection, exhibited significant liver targeting. Figure 5 As shown in the sequential imaging results in Figure A, a significant fluorescence signal appeared in the liver region 6 hours after injection; the fluorescence intensity reached its peak at 24 hours; subsequently, the signal gradually faded; at 72 hours, the fluorescence intensity decreased by about half, indicating that the in vivo half-life of the material is approximately 3 days. At 168 hours after injection, the fluorescence signal was almost completely dissipated, demonstrating that the material possesses good biodegradability and in vivo clearance characteristics. This efficient clearance ability is consistent with previous histopathological observations—experimental animals exposed to this material for extended periods did not show abnormal lesions in their major organs, thus effectively reducing concerns about long-term tissue accumulation and systemic toxicity of the material.

[0095] To quantitatively analyze the contribution of ASGR1-mediated active targeting to in vivo distribution, rigorous flow cytometry analysis was performed. Mice were injected via tail vein with different DiR-labeled agents, sacrificed 24 h later, and liver tissue was harvested to prepare single-cell suspensions for immediate analysis. The results provided clear comparative evidence. Figure 5Figure B in the middle section confirms that, compared with non-targeted agents, formulations conjugated with ASGR1 antibodies (especially the final CMEPA) significantly increase the proportion of DiR-positive cells in liver tissue. This experiment clearly demonstrates that, in the complex physiological environment of living animals, the ASGR1 domain is a key factor endowing this nanomaterial with hepatocyte-specific delivery capabilities.

[0096] 9. Systemic transcriptome remodeling is the core mechanism by which CMEPA exerts its therapeutic efficacy. After confirming that CMEPA can achieve efficient liver delivery, whole transcriptome sequencing was used to elucidate the molecular mechanisms regulating its therapeutic effect. Principal component analysis showed that the liver transcriptomes of the control group and the CMEPA-treated group mice exhibited a significant separation trend. Figure 5 Figure C shows that this intervention can fundamentally reconstruct the gene expression pattern in the liver.

[0097] Differential gene expression analysis results showed that CMEPA treatment could synergistically transform the liver gene expression profile from pathological features to a normal state. Figure 5 (See Figure D). Its therapeutic effects are manifested in its ability to effectively inhibit a series of key genes driving steatosis, inflammatory responses, and disease progression. Notably, the expression of the Cyp4a14 gene was significantly downregulated—previous studies have confirmed that inhibiting the expression of this gene can reverse high-fat diet-induced hepatic steatosis and fibrosis. Simultaneously, CMEPA treatment significantly inhibited the expression of acute-phase reactive protein genes Saa1 and Saa2, and inhibiting the synthesis of these two proteins can alleviate liver inflammation by reducing intrahepatic platelet aggregation. Furthermore, the expression level of the Lcn2 gene was significantly reduced; this gene encodes a pleiotropic factor, and inhibiting its expression can alleviate inflammatory responses and lipid metabolism disorders. In addition, the expression of Cidec, a key gene promoting lipid droplet amplification, was also inhibited; the loss of this gene expression can effectively alleviate hepatic steatosis. The Ciart gene, which is highly expressed in hepatocellular carcinoma tissue, was also significantly downregulated after CMEPA treatment, suggesting that CMEPA may have the potential to delay the progression of metabolic-related fatty liver disease to hepatocellular carcinoma. At the level of positive regulation of gene expression, CMEPA can significantly upregulate the expression of the Egr1 gene—a recognized core factor in transcriptional regulation that can antagonize the accumulation of fatty acids in hepatocytes. This systemic gene expression response outlines the molecular transition process of liver tissue from a diseased state to homeostasis.

[0098] This transformation is further reflected in changes in the immune microenvironment. Immune cell infiltration score heatmaps showed that liver macrophages underwent beneficial phenotypic remodeling: after CMEPA treatment, macrophages polarized from the pro-inflammatory M1 type to the pro-tissue repair M2 type. Figure 5(Figure E in Chinese). This phenotypic shift signifies the activation of the inflammatory resolution process, which does not lead to widespread dysfunction of other immune cell populations, thus highlighting the precision and safety of this therapeutic intervention.

[0099] To elucidate its therapeutic mechanism at the systemic level, a comprehensive pathway enrichment analysis was conducted based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) and the Hallmark database. KEGG pathway analysis confirmed that inflammatory response pathways were significantly inhibited, providing direct evidence for the anti-inflammatory effect of CMEPA at the pathway level. Figure 5 (Figure F in the middle). Gene set enrichment analysis based on the Hallmark database (GSEA) further comprehensively revealed the multi-target action mode of CMEPA. Figure 5 (Figure G) The results showed that CMEPA synergistically inhibited a pathway network encompassing multiple pathological processes. Specifically, it inhibited key inflammatory signaling pathways (interleukin-2 / signal transduction and activating factor 5 pathway, interleukin-6 / Janus kinase / signal transduction and activating factor 3 pathway); suppressed pathways driving adipogenesis and fatty acid metabolism; and reduced oxidative stress and cell damage by inhibiting reactive oxygen species, peroxisome, and apoptosis pathways. Furthermore, the activity of energy metabolism-related pathways such as glycolysis and oxidative phosphorylation was also inhibited, suggesting a normalization of cellular metabolic pathways. Notably, CMEPA also attenuated the activation level of the KRAS signaling pathway—a pathway proven to have pro-cancer effects—suggesting that CMEPA may have the potential to inhibit the progression of metabolic-associated fatty liver disease to cancer. Simultaneously, this treatment also enhanced the function of cholesterol homeostasis regulation pathways. This global transcriptome remodeling, validated by KEGG and HALLMARK database analysis, demonstrates that CMEPA is a comprehensive therapeutic strategy capable of addressing the complex pathogenesis of metabolic-associated fatty liver disease.

[0100] 10. Integration analysis of exosomal microRNAs and liver transcriptome reveals potential regulatory networks. To elucidate the molecular mechanism of action of CMEPA, this experiment first characterized the microRNA profile carried by exosomes derived from human umbilical cord mesenchymal stem cells using small RNA sequencing technology. Subsequently, the exosomal miRNA profile was integrated with the liver transcriptome dataset of mice treated with CMEPA to screen for candidate miRNAs that may mediate changes in the liver transcriptome.

[0101] This multi-omics integrated analysis provides a comprehensive molecular mechanism explanation for the therapeutic efficacy of CMEPA. The study demonstrates that human umbilical cord mesenchymal stem cell-derived exosomes can serve as natural delivery vectors, carrying a sophisticated miRNA regulatory network. This network can systematically target and inhibit key nodes in metabolic, inflammatory, and oxidative stress-related pathways, thereby mediating the overall remodeling of the liver transcriptome and ultimately achieving therapeutic reversal of metabolic-related fatty liver disease.

[0102] 11. CMEPA demonstrated significant therapeutic efficacy in a diet-induced mouse model of metabolic-associated fatty liver disease. Based on sufficient in vitro experimental and transcriptomic evidence, this study further confirmed the therapeutic efficacy of CMEPA in a diet-induced mouse model of metabolic-associated fatty liver disease.

[0103] The study first examined key serum biochemical indicators reflecting liver function and metabolic health. For example... Figure 6 As shown in Figure A, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglycerides (TG), and total cholesterol (TCHO) were significantly elevated in the positive control group (model group), confirming the successful establishment of a model of liver damage and dyslipidemia. All treatment groups showed varying degrees of improvement in the above indicators, with the CMEPA nanocomposite showing the most significant effect: compared to the model group, CMEPA significantly reduced the levels of all four indicators, demonstrating superior therapeutic efficacy compared to all other tested agents.

[0104] Subsequently, this study assessed the antioxidant status of mouse livers by detecting the activities of two key antioxidant enzymes—SOD and CAT. Consistent with the overall improvement trend, the activities of SOD and CAT in the livers of mice treated with CMEPA were significantly increased. Figure 6 (Figure B in the original text) and the increase was significantly better than that of other treatment groups. This result provides direct biochemical evidence that CMEPA effectively alleviates hepatic oxidative stress (a core pathological feature of metabolic-associated fatty liver disease).

[0105] Histopathological evaluation of liver tissue directly confirmed the therapeutic effect of CMEPA at the morphological level. Hematoxylin-eosin (H&E) staining results showed that the model group mice exhibited severe microvesicular and macrovesicular steatosis in their livers, with swollen hepatocytes and numerous vacuoles in the cytoplasm; in stark contrast, the liver tissue structure of the CMEPA-treated mice was significantly improved, and the number of lipid vacuoles was greatly reduced. Oil Red O staining further verified the reversal effect of steatosis. Figure 6 (Figure C): The liver tissue in the model group was filled with bright red lipid droplets, while the lipid deposition in the CMEPA-treated group showed the largest reduction among all treatment groups. Quantitative analysis of Oil Red O staining results confirmed that the relative lipid droplet area in the liver tissue of the CMEPA group was significantly reduced (Figure C).Figure 6 The figure (D) provides objective data support for CMEPA's excellent lipid-lowering ability.

[0106] Finally, to validate the relevant molecular mechanisms at the protein level, this experimental example performed Western blotting detection on key signaling molecules. Figure 6 (Figure E). CMEPA, by targeting ASGR1, significantly downregulated the expression levels of BRCA1 and BARD1 proteins, confirming its successful activation of the target pathway ASGR1-BRCA1 / BARD1-LXRα signaling axis. Simultaneously, CMEPA treatment also significantly inhibited the expression of key pro-inflammatory factors such as IL-1β and IL-6. Figure 6 (See Figure F in the middle). This synergistic regulation of protein expression provides conclusive evidence for the multi-target mechanism of action of CMEPA. This nanocomposite can effectively improve the core pathological process of metabolic-associated fatty liver disease by simultaneously promoting cholesterol homeostasis and inhibiting inflammatory responses.

[0107] This invention successfully developed and systematically evaluated CMEPA, a multifunctional nanotherapy platform. It ingeniously integrates the catalytic therapy of nanozymes, the biological regulation of exosomes, and the molecular targeting function of antibodies, addressing the complex pathological network of MASLD through multi-mechanism synergistic action. This invention not only provides an effective candidate therapeutic material but also offers a multi-omics perspective for understanding the combined therapeutic mechanism of exosomes and nanozymes, possessing significant theoretical importance and translational application potential.

[0108] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A self-assembled nanocomposite material, characterized in that, It includes a copper-based metal-organic framework, exosomes encapsulated within the copper-based metal-organic framework, polydopamine encapsulated outside the copper-based metal-organic framework, and an ASGR1 antibody modified on the surface of the polydopamine.

2. The self-assembled nanocomposite material according to claim 1, characterized in that, The copper-based metal-organic framework is formed by the self-assembly of ferrous ions, copper ions and cyanide ions, and contains both divalent and monovalent copper ions. Preferably, the chemical formula of the copper-based metal-organic framework is Cu2[Fe2(CN)2]; Preferably, the exosomes are umbilical cord mesenchymal stem cell exosomes.

3. The self-assembled nanocomposite material according to claim 1, characterized in that, The particle size of the self-assembled nanocarrier is 95~130 nm.

4. The self-assembled nanocomposite material according to claim 1, characterized in that, The total mass of exosomes and polydopamine is 15% to 20% of the mass of the self-assembled nanocomposite material. Preferably, in the self-assembled nanocomposite material, the loading amount of exosomes is 12-17% of the mass of the copper-based metal-organic framework, the loading amount of polydopamine is 2%-5% of the mass of the copper-based metal-organic framework, and the loading amount of ASGR1 antibody is 0.1%-1% of the mass of the copper-based metal-organic framework.

5. A method for preparing a self-assembled nanocomposite material according to any one of claims 1-4, characterized in that, include: Potassium ferrocyanide and citric acid were dissolved in water, and exosome suspension and copper salt aqueous solution were added sequentially under stirring to obtain a reaction mixture. The reaction mixture was stirred at room temperature to obtain a copper-based metal-organic framework-exosome precipitate. Copper-based metal-organic framework-exosome precipitate was dispersed in water, and dopamine solution and Tris-HCl buffer were added sequentially under stirring. The reaction was stirred to obtain copper-based metal-organic framework-exosome-polydopamine complex. The copper-based metal-organic framework-exosome-polydopamine complex was resuspended in phosphate buffer, and an activator and ASGR1 antibody were added. The mixture was then shaken to react. Bovine serum albumin was added to the system to block unreacted binding sites. After centrifugation, the self-assembled nanocomposite material was obtained.

6. The preparation method according to claim 5, characterized in that, The copper salt includes any one or more of copper chloride or its hydrate, copper sulfate or its hydrate; Preferably, the copper salt is copper sulfate pentahydrate, and the mass ratio of potassium ferrocyanide, citric acid, exosomes and copper sulfate pentahydrate is (3~4):(28~32):(0.1~0.5):(3~7), more preferably (3.5~4):30:(0.3~0.5):(4~6), and even more preferably 3.7:30:0.4:

5.

7. The preparation method according to claim 5, characterized in that, The reaction mixture was stirred at room temperature for 15-20 h, washed, and copper-based metal-organic framework-exosome precipitate was obtained.

8. The preparation method according to claim 5, characterized in that, The mass ratio of copper-based metal-organic framework-exosomes to dopamine is (9~11):(0.3~0.4), preferably (9~11):(0.35~0.4), and more preferably 10:0.

375.

9. The preparation method according to claim 5, characterized in that, The copper-based metal-organic framework-exosome precipitate was dispersed in water, and dopamine solution was added while stirring. After stirring, Tris-HCl buffer was added while stirring. The pH of the Tris-HCl buffer solution is 8-9, preferably 8.5; Preferably, dopamine solution and Tris-HCl buffer are added sequentially under stirring. After stirring and reacting, the product is collected by centrifugation, washed, and resuspended in phosphate buffer to obtain a copper-based metal-organic framework-exosome-polydopamine complex. Preferably, the activator is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and the mass ratio of the copper-based metal-organic framework-exosome-polydopamine complex, the activator and the ASGR1 antibody is 100:(6000~6500):(1~5), more preferably 100:(6200~6300):(2~4), and even more preferably 100:6300:

3.

10. The use of the self-assembled nanocomposite material according to any one of claims 1-4 in the preparation of a drug for treating fatty liver; Preferably, the fatty liver includes non-alcoholic fatty liver disease.

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