Exosome coupling medicine for treating non-alcoholic steatohepatitis and preparation method thereof

Abstract

The invention relates to the technical field of biological medicines, and discloses an exosome coupling medicine for treating non-alcoholic steatohepatitis. According to the exosome coupled drug delivery system, the liver targeting of the exosome is utilized, the drug is accurately delivered to the liver lesion position, the side effect of non-target tissue is reduced, meanwhile, the exosome plays a role in protecting the drug, the stability and bioavailability of the drug are improved, the treatment effect is remarkably enhanced, the advantages of targeting and long-term effect are shown, and the exosome coupled drug delivery system has a good application prospect. An innovative path is provided for the treatment of the non-alcoholic steatohepatitis. The preparation method of the exosome coupling drug is simple and feasible, and is suitable for further industrial mass production.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and more specifically, to an exosome-coupled drug for treating non-alcoholic steatohepatitis and a preparation method thereof. Background Art

[0002] Nonalcoholic steatohepatitis (NASH), also known as metabolic steatohepatitis, is an inflammatory subtype of nonalcoholic fatty liver disease (NAFLD). NASH has a high prevalence in adults, adolescents, and children. NAFLD is the most common liver disease worldwide, affecting 20-25% of the adult population. Approximately 30% of NAFLD patients progress to NASH, which is characterized by hepatocellular damage and liver inflammation with or without fibrosis, and is at risk for progression to end-stage liver disease. NASH is closely associated with metabolic disorders such as obesity, insulin resistance, type 2 diabetes, and hyperlipidemia. NASH has become a major public health challenge, yet suitable drugs specifically for the treatment of NASH remain limited. Existing treatments (such as lifestyle interventions and antioxidants) have limited effectiveness, and there is an urgent need to develop liver-targeted drugs that combine anti-inflammatory and metabolic regulation.

[0003] GLP-1R agonists have shown significant potential in the treatment of non-alcoholic steatohepatitis (NASH). Their mechanisms include improving insulin resistance, inhibiting hepatic fatty acid synthesis, and protecting liver cells by regulating mitochondrial function, reducing oxidative stress and inflammatory responses. In addition, it also has a certain effect on delaying the progression of liver fibrosis. Clinical trials have shown that GLP-1R agonists such as liraglutide and semaglutide can significantly reduce liver fat content, improve hepatocyte ballooning and lobular inflammation, and delay the progression of fibrosis. Although its improvement in liver fibrosis still needs long-term research verification, overall, GLP-1R agonists provide a new treatment option for NASH patients, especially with significant advantages in improving liver steatosis and inflammation.

[0004] Tirzepatide is a novel dual GLP-1 and glucose-dependent insulinotropic peptide (GIP) receptor agonist that has been approved for the treatment of type 2 diabetes and obesity. Its potential mechanisms (such as improving insulin resistance and reducing liver fat synthesis) can also improve NASH-related biomarkers in patients with T2DM and delay the progression of liver fibrosis in patients with NASH. It may be effective for NASH, but its targeting is insufficient. After systemic administration, only a small amount of drug is accumulated in the liver, and high doses are required to induce side effects (the most common adverse events are gastrointestinal events, such as nausea, vomiting, decreased appetite, abdominal distension and diarrhea, which are considered to be mild to moderate in severity). Therefore, how to construct a stable and efficient drug-loaded targeted formulation to improve the therapeutic efficiency of drugs for NASH is a key scientific issue that needs to be solved urgently.

[0005] Exosomes are tiny vesicles released by cells, typically ranging in diameter from 30 to 150 nm. Exosomes possess unique advantages, including a hollow lipid bilayer structure, negative charge, small size, natural biocompatibility, tissue penetrability, low immunogenicity, and long circulation time. They are highly anticipated as the next generation of revolutionary natural nanocarriers for drug delivery. Furthermore, compared to traditional nanocarriers, exosomes' unique membrane structure exhibits superior affinity and adaptability for peptide drugs. Furthermore, during the encapsulation process, they are more compatible with peptide drugs that resemble intracellular substances, thereby increasing drug encapsulation efficiency and achieving efficient targeted drug delivery.

[0006] Hepatocyte-derived exosomes (HEx) serve as drug delivery vehicles for NASH, leveraging their ability to home to liver tissue to reach sites of NASH disease. HEx can reduce phagocytosis by liver macrophages, reducing immune clearance. HEx can also increase fusion with hepatocytes, promoting cellular internalization of encapsulated drugs and effectively delivering NASH therapeutics to diseased liver cells. Plant-derived exosomes (PELNs) exhibit high compositional heterogeneity, making extraction susceptible to polysaccharide / polyphenol contamination. Immature separation techniques result in low recovery rates and high levels of impurities. Cross-species delivery is inefficient, with a lack of targeted proteins and unclear mechanisms of action for plant miRNAs. Safety evidence is insufficient, and PELNs may trigger immune responses. Large-scale production is significantly affected by environmental factors, leading to significant batch-to-batch variability. Compared to animal-derived exosomes, PELNs exhibit inferior uniformity, purity, and delivery efficiency.

[0007] Exosome-conjugated drugs have natural targeting and low immunogenicity, enabling them to effectively penetrate biological barriers (such as the blood-brain barrier), improving drug delivery efficiency. Their phospholipid bilayer structure protects drugs from degradation and reduces systemic toxicity. While there are reports of exosome-conjugated chemotherapeutic drugs, there are no reports of using hepatocyte exosomes conjugated to GLP-1RA for the targeted treatment of NASH. Summary of the Invention

[0008] The purpose of the present invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide an exosome-coupled drug for treating non-alcoholic steatohepatitis and a preparation method thereof.

[0009] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0010] An exosome-coupled drug for treating non-alcoholic steatohepatitis, wherein the exosome-coupled drug is a drug formed by coupling a glucagon-like peptide 1 receptor agonist to exosomes via a chemical cross-linking agent.

[0011] The exosome-coupled drug, the ratio of the total exosome protein amount, the chemical cross-linking agent and the glucagon-like peptide 1 receptor agonist is 1:2:0.5-3. Preferably, the ratio of the total exosome protein amount, the chemical cross-linking agent and the glucagon-like peptide 1 receptor agonist is 1:2:1.

[0012] The exosome-coupled drug, the cross-linking agent is selected from one or more of disuccinimidyl suberate, bissulfosuccinimidyl suberate, ethylene glycol disuccinic acid N-hydroxysuccinimide ester, and 3,3'-dithiodisuccinimidyl propionate.

[0013] Furthermore, in a preferred embodiment of the present invention, the exosome-conjugated drug and the glucagon-like peptide 1 receptor agonist are selected from one or more of exenatide, liraglutide, semaglutide, benaglutide, polyethylene glycol loxenatide, tilportide, danglipil and olglipril.

[0014] Furthermore, in a preferred embodiment of the present invention, the glucagon-like peptide 1 receptor agonist is selected from telportide

[0015] Furthermore, in a preferred embodiment of the present invention, the exosome-drug conjugate is prepared by: inoculating liver cells in a culture flask, collecting the cell supernatant, centrifuging at low speed, collecting the cell supernatant and centrifuging at high speed, and then collecting the cell supernatant and centrifuging at ultrahigh speed to separate the exosomes. Finally, the exosomes are washed with PBS and centrifuged again at ultrahigh speed to remove residual impurities. Finally, the exosome precipitate is resuspended to obtain an exosome A solution for use.

[0016] Furthermore, in a preferred embodiment of the present invention, the exosome-drug conjugated cell is centrifuged at low speed at 300 g, 4 ° C for 10 minutes; at high speed at 1000 g, 4 ° C for 10 minutes; and at ultrahigh speed at 100000 g, 4 ° C for 1-2 hours.

[0017] Furthermore, the exosome-drug conjugate is prepared as follows:

[0018] 1) preparing a PBS aqueous solution of exosomes A according to claim 5 or 6, slowly adding a crosslinker solution, slowly stirring at room temperature for 20-40 minutes, and then purifying by gel column chromatography to obtain a solution of exosomes B linked to the crosslinker;

[0019] 2) mixing the exosome solution B from step 1) with a PBS solution of a glucagon-like peptide 1 receptor agonist, and continuing stirring at room temperature for 20-40 minutes to obtain an exosome solution C modified with a glucagon-like peptide 1 receptor agonist and a cross-linker;

[0020] 3) The exosome C solution was separated and purified by gel column chromatography to obtain high-purity exosome-conjugated drugs.

[0021] Furthermore, in a preferred embodiment of the present invention, the total protein concentration of the PBS aqueous solution of exosomes A is 0.5 mg / mL to 2000 mg / mL.

[0022] Furthermore, in a preferred embodiment of the present invention, the cross-linking agent solution has a mass fraction of 0.005% to 15%.

[0023] Furthermore, in a preferred embodiment of the present invention, the mass fraction of the glucagon-like peptide 1 receptor agonist is 0.001% to 20%.

[0024] Furthermore, in a preferred embodiment of the present invention, the exosome-drug conjugate is prepared as follows:

[0025] S1: Cell culture

[0026] 1) Resuscitate AML12 cells and culture them in DMEM / F12 medium containing fetal bovine serum. Subculture the cells when they grow to 80-90% of the culture dish;

[0027] 2) Taking the cells from step 1), culture them in an extra-large dish. When the cell density reaches 70%-80%, replace the culture medium with fetal bovine serum without Exos and continue culturing for 12-24 hours. When the cell density reaches 90%-100%, collect the cell supernatant and set aside;

[0028] S2: Hepatocyte exosome extraction

[0029] 1) The supernatant was centrifuged at 300 g for 10 minutes at 0-8°C to remove cell debris and large particles, then at 1000 g for 10 minutes to remove smaller impurities, followed by ultracentrifugation at 100,000 g at 4°C for 1.5 hours to isolate exosomes A for later use;

[0030] 2) The exosomes from step 1) were washed with PBS and ultracentrifuged again at 100,000 g, 4°C, for 1 hour to remove residual impurities;

[0031] 3) Quantify the protein according to the instructions of the BCA protein quantification kit and set aside;

[0032] S3: Preparation of exosome-drug conjugates

[0033] 1) Prepare an exosome PBS aqueous solution with a total protein concentration of 0.5 mg / mL to 2000 mg / mL, add a crosslinker solution with a mass fraction of 0.005% to 15%, and slowly stir at room temperature for 20-40 minutes to obtain a mixed solution containing exosomes; set aside;

[0034] 2) Separating the exosome mixture connected to the crosslinker from the reaction system using gel column chromatography purification technology to obtain a purified exosome solution connected to the crosslinker for later use;

[0035] 3) mixing the exosome solution from step 2) with a PBS solution containing 0.001% to 20% GLP-1RA by mass, and slowly stirring at room temperature for 20-40 minutes to obtain exosome-drug conjugates containing GLP-1RA and a crosslinker, for later use;

[0036] 4) Separating and purifying the exosomes from step 3) by gel column chromatography to obtain high-purity exosome-drug conjugates.

[0037] Furthermore, the exosome-coupled drug is used in the preparation of medical drugs for treating non-alcoholic steatohepatitis.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] The hepatocyte exosome-coupled drug prepared in the present invention can not only target NASH but also reduce phagocytosis by liver macrophages, prolonging the drug's duration of action, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Schematic diagram for the preparation of Tirzepatide-loaded hepatocyte exosome-drug conjugates;

[0041] Figure 2Characterization and functional confirmation of Tirzepatide-HEx, including a. infrared spectrum; b. electron microscopy, scale 100 μm; c. western blot;

[0042] Figure 3 Biochemical diagram of mice after Tirzepatide-HEx treatment: a. Body weight / week; b. Liver weight; c. Liver weight / body weight; d. Serum TC content; e. Serum TC content; f. Serum ALT level; g. Serum AST level;

[0043] Figure 4 The following are pathological histological images of the mouse liver stained with HE, Oil Red O, and Masson staining after Tirzepatide-HEx treatment; red arrows indicate inflammatory foci, yellow arrows indicate fatty degeneration, and purple arrows indicate vesicular degeneration.

[0044] Figure 5 The levels of inflammatory factors in the liver of mice after Tirzepatide-HEx treatment. DETAILED DESCRIPTION

[0045] The following is a detailed description of the innovative content of the present invention with reference to the accompanying drawings. It should be noted that the embodiments provided herein are only used to illustrate the core content of the present invention and do not constitute any limitation. Those skilled in the relevant art should understand that without changing the design concept and protection scope of the present invention, the specific implementation scheme can be adjusted or replaced similarly, but these modifications are all within the legal scope defined by the protection requirements of this patent.

[0046] Example 1: Preparation of hepatocyte exosomes coupled to tilpoxetine

[0047] (1) Cell culture

[0048] Resuscitate AML12 cells and culture in DMEM / F12 (1:1) medium containing 10% fetal bovine serum at 37°C, 5% CO2, and 95% humidity. When cells grow to approximately 85% of the dish, passage the cells. Culture in extra-large dishes and, when the cell density reaches 70%-80%, switch to medium containing fetal bovine serum without Exos. Continue culturing for 12-24 hours. Collect the cell supernatant when the cell density reaches 90%-100%.

[0049] (2) Extraction of exosomes from hepatocytes

[0050] The supernatant was then centrifuged at 300g for 10 minutes at 4°C to remove cell debris and large particles, then at 1000g for 10 minutes to further remove smaller impurities. Exosomes were then isolated by ultracentrifugation at 100,000g for 1.5 hours at 4°C. The exosomes were washed with PBS and ultracentrifuged again at 100,000g for 1 hour at 4°C to remove residual impurities. Protein was quantified according to the BCA protein quantification kit instructions.

[0051] (3) Synthesis of hepatocyte exosome-coupled tilportide

[0052] To a 10 ml PBS solution (pH 7.4) containing hepatocyte exosomes (2 mg / mL), 1 ml of a 10 mM aqueous solution of bis(sulfosuccinimidyl) suberate crosslinker was added and the mixture was vortexed at room temperature for 30 minutes. The crosslinked exosomes were isolated and purified using a Sephadex column. The purified crosslinked exosomes (1 mg / mL) in PBS were then mixed with a 1:1 mass ratio of tilpoitide (1 mg / mL) in PBS and vortexed at room temperature for 30 minutes. Separation and purification using the Sephadex column yielded hepatocyte exosomes conjugated to the tilpoitide drug. The tilpoitide concentration in the exosomes was quantitatively determined using HPLC.

[0053] Example 2: Preparation of hepatocyte exosomes coupled with semaglutide

[0054] (1) Cell culture

[0055] Resuscitate AML12 cells and culture in DMEM / F12 (1:1) medium containing 10% fetal bovine serum at 37°C, 5% CO2, and 95% humidity. When cells grow to approximately 85% of the dish, passage the cells. Culture in extra-large dishes and, when the cell density reaches 70%-80%, switch to medium containing fetal bovine serum without Exos. Continue culturing for 12-24 hours. Collect the cell supernatant when the cell density reaches 90%-100%.

[0056] (2) Extraction of exosomes from hepatocytes

[0057] The supernatant was then centrifuged at 300g for 10 minutes at 4°C to remove cell debris and large particles, then at 1000g for 10 minutes to further remove smaller impurities. Exosomes were then isolated by ultracentrifugation at 100,000g for 1.5 hours at 4°C. The exosomes were washed with PBS and ultracentrifuged again at 100,000g for 1 hour at 4°C to remove residual impurities. Protein was quantified according to the BCA protein quantification kit instructions.

[0058] (3) Synthesis of hepatocyte exosome-coupled semaglutide

[0059] To a 10 ml PBS solution (pH 7.4) containing hepatocyte exosomes (2 mg / mL), 1 ml of a 10 mM aqueous solution of ethylene glycol bis(succinic acid N-hydroxysuccinimide ester) crosslinker was added and the mixture was vortexed at room temperature for 30 minutes. The crosslinked exosomes were isolated and purified using a Sephadex column. The purified crosslinked exosomes (1 mg / mL) in PBS were then mixed with a 1:1 mass ratio of tesiparatide (1 mg / mL) in PBS and vortexed at room temperature for 30 minutes. Separation and purification using a Sephadex column yielded hepatocyte exosomes conjugated to semaglutide. The concentration of semaglutide in the exosomes was quantitatively determined using HPLC.

[0060] Example 3: Preparation of hepatocyte exosomes coupled with liraglutide

[0061] (1) Cell culture

[0062] Resuscitate AML12 cells and culture in DMEM / F12 (1:1) medium containing 10% fetal bovine serum at 37°C, 5% CO2, and 95% humidity. When cells grow to approximately 85% of the dish, passage the cells. Culture in extra-large dishes and, when the cell density reaches 70%-80%, switch to medium containing fetal bovine serum without Exos. Continue culturing for 12-24 hours. Collect the cell supernatant when the cell density reaches 90%-100%.

[0063] (2) Extraction of exosomes from hepatocytes

[0064] The supernatant was then centrifuged at 300g for 10 minutes at 4°C to remove cell debris and large particles, then at 1000g for 10 minutes to further remove smaller impurities. Exosomes were then isolated by ultracentrifugation at 100,000g for 1.5 hours at 4°C. The exosomes were washed with PBS and ultracentrifuged again at 100,000g for 1 hour at 4°C to remove residual impurities. Protein was quantified according to the BCA protein quantification kit instructions.

[0065] (3) Synthesis of hepatocyte exosome-coupled liraglutide

[0066] To a 10 ml PBS solution (pH 7.4) containing hepatocyte exosomes (2 mg / mL), 1 ml of a 10 mM aqueous solution of bis(sulfosuccinimidyl) suberate crosslinker was added and the mixture was vortexed at room temperature for 30 minutes. The crosslinked exosomes were isolated and purified using a Sephadex column. The purified crosslinked exosomes (1 mg / mL) in PBS were then mixed with a 1:1 mass ratio of tesiparatide (1 mg / mL) in PBS and vortexed at room temperature for 30 minutes. The hepatocyte exosomes conjugated to liraglutide were isolated and purified using a Sephadex column. The liraglutide concentration in the exosomes was quantitatively determined using HPLC.

[0067] Example 4: Preparation of hepatocyte exosomes coupled with exenatide

[0068] (1) Cell culture

[0069] Resuscitate AML12 cells and culture in DMEM / F12 (1:1) medium containing 10% fetal bovine serum at 37°C, 5% CO2, and 95% humidity. When cells grow to approximately 90% of the dish, passage the cells. Culture in extra-large dishes and, when the cell density reaches 70%-80%, switch to medium containing fetal bovine serum without Exos. Continue culturing for 12-24 hours. Collect the cell supernatant when the cell density reaches 90%-100%.

[0070] (2) Extraction of exosomes from hepatocytes

[0071] The supernatant was then centrifuged at 300g for 10 minutes at 4°C to remove cell debris and large particles, then at 1000g for 10 minutes to further remove smaller impurities. Exosomes were then isolated by ultracentrifugation at 100,000g for 1.5 hours at 4°C. The exosomes were washed with PBS and ultracentrifuged again at 100,000g for 1 hour at 4°C to remove residual impurities. Protein was quantified according to the BCA protein quantification kit instructions.

[0072] (3) Synthesis of hepatocyte exosome-coupled exenatide

[0073] To a 10 ml PBS solution (pH 7.4) containing hepatocyte exosomes (2 mg / mL), 1 ml of a 10 mM aqueous solution of ethylene glycol bis(succinic acid N-hydroxysuccinimide ester) crosslinker was added and the mixture was vortexed at room temperature for 30 minutes. The crosslinked exosomes were isolated and purified using a Sephadex column. The purified crosslinked exosomes (1 mg / mL) in PBS were then mixed with a 1:1 mass ratio of tesiparatide (1 mg / mL) in PBS and vortexed at room temperature for 30 minutes. Separation and purification using a Sephadex column yielded hepatocyte exosomes conjugated to exenatide. The exosome concentration of exenatide was quantitatively determined using HPLC.

[0074] Example 5: Preparation of hepatocyte exosomes coupled with benaglutide

[0075] (1) Cell culture

[0076] Resuscitate AML12 cells and culture in DMEM / F12 (1:1) medium containing 10% fetal bovine serum at 37°C, 5% CO2, and 95% humidity. When cells grow to approximately 80% of the dish, passage the cells. Culture in extra-large dishes and, when the cell density reaches 70%-80%, switch to medium containing fetal bovine serum without Exos. Continue culturing for 12-24 hours. Collect the cell supernatant when the cell density reaches 90%-100%.

[0077] (2) Extraction of exosomes from hepatocytes

[0078] The supernatant was then centrifuged at 300g for 10 minutes at 4°C to remove cell debris and large particles, then at 1000g for 10 minutes to further remove smaller impurities. Exosomes were then isolated by ultracentrifugation at 100,000g for 1.5 hours at 4°C. The exosomes were washed with PBS and ultracentrifuged again at 100,000g for 1 hour at 4°C to remove residual impurities. Protein was quantified according to the BCA protein quantification kit instructions.

[0079] (3) Synthesis of hepatocyte exosome-conjugated benaglutide

[0080] To a 10 ml PBS solution (pH 7.4) containing hepatocyte exosomes (2 mg / mL), 1 ml of a 10 mM 3,3'-dithiobis(succinimidyl propionate) crosslinker solution was added and the mixture was vortexed at room temperature for 30 minutes. The crosslinked exosomes were isolated and purified using a Sephadex column. The purified crosslinked exosomes (1 mg / mL) in PBS were then mixed with a 1:1 mass ratio of tesiparatide (1 mg / mL) in PBS and vortexed at room temperature for 30 minutes. Separation and purification using a Sephadex column yielded hepatocyte exosomes conjugated with benaglutide. The concentration of benaglutide in the exosomes was quantitatively determined using HPLC.

[0081] Example 6: Preparation and investigation of different coupling agents coupled to telomeric peptide in hepatocyte exosomes

[0082] The term "particle size D90" refers to the particle size corresponding to the 90% cumulative particle size distribution percentage of a sample. Its physical meaning is that 90% of the particles have a particle size smaller than this.

[0083] Table 1

[0084]

[0085] Table 2 Investigation of exosome-drug conjugation with different coupling agents and dosages

[0086]

[0087]

[0088] Table 2 shows particle size analysis. Particle sizes ①-④ showed a D90 diameter of ≤120 nm, while those ⑤-⑥ showed a D90 diameter of ≤200 nm. Product yield was >85%. The ratio of total exosome protein, chemical crosslinker, and glucagon-like peptide 1 receptor agonist was 1:2:0.5-3. Among all crosslinkers, the ratio of total exosome protein, chemical crosslinker, and glucagon-like peptide 1 receptor agonist was 1:2:1, which yielded the best overall particle size and yield.

[0089] Validation Example 1: Characterization of Hepatocyte Exosomes Conjugated to Telportide

[0090] (1) Experimental methods

[0091] The structure of hepatocyte exosomes coupled with tilpotide was investigated by Fourier transform infrared technology; the morphology of hepatocyte exosomes coupled with tilpotide was observed by transmission electron microscopy; and the expression levels of exosome surface markers (CD9, CD63, CD81 and TSG101) were detected by western blot technology.

[0092] (2) Experimental results

[0093] like Figure 2 The infrared spectrum shown ( Figure 2 A) shows: Tirzepatide at 2500 cm -1 ~40000cm -1 There are obvious characteristic peaks, while hepatocyte exosomes (HEx) and bis(sulfosuccinimidyl) suberate (BS) have no characteristic peaks here, but the prepared sample (Tirzepatide-HEx) has obvious characteristic peaks here. Electron microscope images show ( Figure 2 B) presents a saucer shape, which is a characteristic of exosomes. WB( Figure 2 C) The results showed that both groups of exosomes highly expressed the characteristic exosome marker proteins CD9, CD63, CD81, and TSG101. In summary, we successfully prepared hepatocyte exosomes coupled with telportin.

[0094] Verification Example 2: Therapeutic Effect of Hepatocyte Exosomes-Conjugated Telportide on NASH in Mice

[0095] 1. Experimental methods:

[0096] Six-week-old male C57BL / 6J mice were randomly divided into a control group and a modeling group after one week of adaptive feeding. The control group was fed with ordinary feed, and the modeling group was fed with 60% high-fat + 2% high-cholesterol feed for 5 months. Healthy mice and NASH mice were randomly divided into four groups: HC group (healthy control group), NASH group, Tirzepatide group (10nM), and Tirzepatide-HEx group (10nM), with 6 mice in each group. They were administered subcutaneously once a week for 3 consecutive months. The weight changes of the mice were observed every week. The mice in the healthy group continued to be fed with ordinary feed, and the other groups continued to be fed with 60% high-fat + 2% high-cholesterol feed and drank normal water.

[0097] After the experiment, each mouse was anesthetized with 1% sodium amobarbital intraperitoneally, and the thoracotomy was opened to collect about 0.5 ml of blood from the heart. The blood was placed in an EP tube without anticoagulant and allowed to stand at room temperature for 2 h at 3000 r / min. -1 After centrifugation for 15 minutes, the supernatant serum was collected and stored in a -80°C refrigerator. After blood was collected from the mice, the tissues were perfused with normal saline, and the liver tissue was removed and weighed. A portion was stored in a -80°C refrigerator, and the other portion was fixed and stored in 4% paraformaldehyde.

[0098] 2. Index detection

[0099] 2.1 Determination of serum biochemical enzymes AST and ALT

[0100] The levels of TC, TG, ALT and AST in mouse serum were detected according to the instructions of the TC, TG, ALT and AST kits provided by Nanjing Jiancheng Bioengineering Institute.

[0101] 2.2 HE staining of liver tissue

[0102] Liver tissue fixed with 4% paraformaldehyde (0.5 cm × 0.5 cm × 0.2 cm) was obtained, dehydrated with gradient alcohol, transparentized with xylene, and embedded in paraffin to prepare 5 μm thick sections. The sections were then dewaxed, rehydrated, and stained with hematoxylin-eosin (HE). The fatty degeneration and inflammatory level of the liver tissue were observed under a microscope.

[0103] 2.3 Oil Red O staining of liver tissue

[0104] Frozen sections of 5-8 μm were dried at room temperature for 10 min, fixed with 4% paraformaldehyde for 10 min, rinsed twice with distilled water, and stained with Oil Red O solution for 10-15 min in the dark. The sections were adjusted with 60% isopropanol, washed with water, counterstained with hematoxylin, washed with running water to return to blue, and mounted with glycerol gelatin. The changes in lipid droplets in the liver tissue of each group were observed under a microscope.

[0105] 2.4 Detection of liver fibrosis

[0106] 4 μm paraffin sections were dewaxed to water, stained with Weigert iron hematoxylin for 5 min, quickly washed with water, differentiated with 1% hydrochloric acid alcohol for 5 s, rinsed with distilled water, back to blue, stained with Ponceau acid fuchsin solution for 5 min, differentiated with phosphomolybdic acid for 2 min, counterstained with aniline blue solution for 2 min, washed with 1% glacial acetic acid aqueous solution for 1 min, dehydrated with gradient ethanol, transparentized with xylene, and mounted with neutral resin glue. The fibrosis of the liver tissue in each group was observed under a microscope.

[0107] 2.5 PCR detection of liver tissue

[0108] ① RNA extraction: Remove liver tissue, add 1ml of Trizol reagent, homogenize the tissue, add 200µl of chloroform, swirl and mix thoroughly, let stand for 10 minutes, and centrifuge at 12,000 rpm at 4°C for 15 minutes. Transfer the supernatant to a new centrifuge tube, add an equal volume of isopropanol solution, gently invert and mix thoroughly, let stand for 10 minutes, and centrifuge at 12,000 rpm at 4°C for 15 minutes. Discard the supernatant, wash the pellet once with 75% ethanol, and centrifuge at 12,000 rpm at 4°C for 15 minutes. Discard the supernatant, and evaporate the pellet at room temperature with the lid uncovered for 5-10 minutes. Add 30-50µl of DEPC-treated water to dissolve the RNA. Determine sample concentration and purity by spectrophotometry.

[0109] ② Reverse transcription: Based on TAKARA's PrimeScript TMThe extracted RNA was reverse transcribed according to the RT Master Mix (Perfect Real Time) kit instructions. The conditions were: 37°C for 15 min, 85°C for 5 sec, and stored at 4°C.

[0110] ③RT-PCR: The reverse transcribed cDNA was subjected to real-time fluorescence quantitative PCR detection. The Fast qPCR Mix kit was used for the operation. The reaction system was as follows: 2×SYBR Green Fast qPCR Mix 12.5 μl, 10 μM forward and reverse primer mixture 2 μl, cDNA template 2 μl, and sterile water 8.5 μl. The reaction conditions were: 1 cycle of 95°C for 30 seconds, and 40 cycles of 95°C for 5 seconds and 60°C for 10 seconds.

[0111] The obtained data were normalized with internal reference genes, and the expression levels of IL-1β, IL-6, and TNF-α in the liver tissues of each group were calculated.

[0112] 3. Experimental results:

[0113] 3.1 Serum biochemistry results showed that Figure 3 :

[0114] After 12 weeks of treatment, both the Tirzepatide and Tirzepatide-HEx groups significantly reduced mouse body weight compared to the NASH group, and by the end of the 12th week of treatment, their body weights were close to those of the HC group. Similarly, compared to the NASH group, the Tirzepatide and Tirzepatide-HEx groups showed significantly reduced liver weight / body weight percentages and serum TC, TG, ALT, and AST levels. Compared to the Tirzepatide group, the Tirzepatide-HEx group showed significantly reduced liver weight / body weight percentages and serum TC, TG, ALT, and AST levels, approaching those of the HC group. Therefore, we believe that Tirzepatide can alleviate dyslipidemia and liver damage to a certain extent, with Tirzepatide-HEx being more effective.

[0115] 3.2 Liver histology results showed that Figure 4 :

[0116] HE staining revealed normal liver structure in the HC group, with no hepatocyte steatosis, clear hepatic lobule structure, distinct centrally located nuclei, and no inflammatory foci. In the NASH group, liver tissue showed a significant decrease in the number of nuclei within the visual field, with widespread diffuse hepatocyte steatosis, ballooning, and numerous inflammatory foci. Tirzepatide and Tirzepatide-HEx administration significantly improved these lesions, including steatosis, ballooning, and inflammatory foci. In particular, Tirzepatide-HEx treatment nearly eliminated steatosis, clarified nuclei, eliminated ballooning, and significantly reduced inflammatory foci. Oil Red O staining of liver tissue revealed increased lipid deposition in the NASH group. Tirzepatide treatment significantly reduced lipid droplet size, while Tirzepatide-HEx treatment essentially eliminated these droplets. Masson staining, assessing the degree of liver fibrosis, showed significant improvement in liver fibrosis compared to NASH in the Tirzepatide-HEx group, while improvement in liver fibrosis in the Tirzepatide group was modest. Taken together, these results support the protective effect of Tirzepatide-HEx against NASH by reducing hepatocyte steatosis, lipid deposition, lobular inflammation, and fibrosis, and its effect is superior to Tirzepatide.

[0117] 3.3PCR results showed that Figure 5 :

[0118] Compared with the HC group, the levels of liver inflammatory factors IL-1β, IL-6 and TNF-α in the NASH group were significantly increased, while compared with the NASH group, the levels of liver inflammatory factors IL-1β, IL-6 and TNF-α in the Tirzepatide and Tirzepatide-HEx groups were significantly decreased. Compared with the Tirzepatide group, the levels of liver inflammatory factors IL-6 and TNF-α in the Tirzepatide-HEx group were significantly different and closer to those in the HC group, indicating that Tirzepatide can attenuate the inflammatory level of mice with non-alcoholic fatty liver disease, and the effect of Tirzepatide-HEx is more obvious.

[0119] In summary, this study reports for the first time that hepatocyte exosomes coupled to tilportide improve the pathological conditions of lipid accumulation, inflammation, and liver fibrosis in mice induced by high-fat and 2% high cholesterol, significantly reducing serum liver weight / body weight, TC, TG, AST, and ALT levels, and significantly reducing liver tissue levels of the inflammatory factors IL-1β, IL-6, and TNF-α. Therefore, this study has the potential to be developed as a drug for NASH.

Claims

1. An exosome-drug conjugate for treating non-alcoholic steatohepatitis, characterized in that: The exosome-coupled drug is a drug formed by coupling a glucagon-like peptide 1 receptor agonist to exosomes through a chemical cross-linking agent.

2. The exosome-drug conjugate according to claim 1, characterized in that Calculated by weight, the ratio of the total exosome protein amount, the chemical crosslinker and the glucagon-like peptide 1 receptor agonist is 1:2:0.5-3. Preferably, the ratio of the total exosome protein amount, the chemical crosslinker and the glucagon-like peptide 1 receptor agonist is 1:2:

1.

3. The exosome-drug conjugate according to claim 1, characterized in that The cross-linking agent is selected from one or more of disuccinimidyl suberate, bissulfosuccinimidyl suberate, ethylene glycol disuccinic acid N-hydroxysuccinimide ester, and 3,3'-dithiodisuccinimidyl propionate.

4. The exosome-drug conjugate according to claim 1, characterized in that The glucagon-like peptide 1 receptor agonist is selected from one or more of exenatide, liraglutide, semaglutide, benaglutide, polyethylene glycol loxenatide, tilportide, danglipril and olglipril. Preferably, the glucagon-like peptide 1 receptor agonist is selected from tilportide.

5. The exosome-drug conjugate according to claim 1, characterized in that The exosomes were prepared by inoculating liver cells into a culture flask, collecting the cell supernatant, centrifuging at low speed, collecting the cell supernatant and centrifuging at high speed, and then collecting the cell supernatant and centrifuging at ultrahigh speed to separate the exosomes. The exosomes were washed with PBS and centrifuged again at ultrahigh speed to remove residual impurities. The exosome precipitate was finally resuspended to obtain an exosome A solution for use.

6. The exosome-drug conjugate according to claim 5, characterized in that The low-speed centrifugation is: 300g, 4°C, 10 minutes; the high-speed centrifugation is: 1000g, 4°C, 10 minutes; the ultra-high-speed centrifugation is: 100000g, 4°C, 1-2 hours.

7. The exosome-drug conjugate according to claim 5, characterized in that The preparation of the exosome-drug conjugate is specifically as follows: 1) preparing a PBS aqueous solution of exosomes A according to claim 5 or 6, slowly adding a crosslinker solution, slowly stirring at room temperature for 20-40 minutes, and then purifying by gel column chromatography to obtain a solution of exosomes B linked to the crosslinker; 2) mixing the exosome solution B from step 1) with a PBS solution of a glucagon-like peptide 1 receptor agonist, and continuing stirring at room temperature for 20-40 minutes to obtain an exosome solution C modified with a glucagon-like peptide 1 receptor agonist and a cross-linker; 3) The exosome C solution was separated and purified by gel column chromatography to obtain high-purity exosome-conjugated drugs.

8. The exosome-drug conjugate according to claim 7, characterized in that The total protein concentration of the PBS aqueous solution of the exosome A is 0.5 mg / mL to 2000 mg / mL; the mass fraction of the cross-linking agent solution is 0.005% to 15%; and the mass fraction of the glucagon-like peptide 1 receptor agonist is 0.001% to 20%.

9. The exosome-drug conjugate according to claim 7, characterized in that The preparation of exosome-drug conjugates is as follows: S1: Cell culture 1) Resuscitate AML12 cells and culture them in DMEM / F12 medium containing fetal bovine serum. Subculture the cells when they grow to 80-90% of the culture dish; 2) Taking the cells from step 1), culture them in an extra-large dish. When the cell density reaches 70%-80%, replace the culture medium with fetal bovine serum without Exos and continue culturing for 12-24 hours. When the cell density reaches 90%-100%, collect the cell supernatant and set aside; S2: Hepatocyte exosome extraction 1) The supernatant was centrifuged at 300 g for 10 minutes at 0-8°C to remove cell debris and large particles, then at 1000 g for 10 minutes to remove smaller impurities, followed by ultracentrifugation at 100,000 g at 4°C for 1.5 hours to isolate exosomes A for later use; 2) The exosomes from step 1) were washed with PBS and ultracentrifuged again at 100,000 g, 4°C, for 1 hour to remove residual impurities; 3) Quantify the protein according to the instructions of the BCA protein quantification kit and set aside; S3: Preparation of exosome-drug conjugates 1) Prepare an exosome PBS aqueous solution with a total protein concentration of 0.5 mg / mL to 2000 mg / mL, add a crosslinker solution with a mass fraction of 0.005% to 15%, and slowly stir at room temperature for 20-40 minutes to obtain a mixed solution containing exosomes; set aside; 2) Separating the exosome mixture connected to the crosslinker from the reaction system using gel column chromatography purification technology to obtain a purified exosome solution connected to the crosslinker for later use; 3) mixing the exosome solution from step 2) with a PBS solution containing 0.001% to 20% GLP-1RA by mass, and slowly stirring at room temperature for 20-40 minutes to obtain exosome-drug conjugates containing GLP-1RA and a crosslinker, for later use; 4) Separating and purifying the exosomes from step 3) by gel column chromatography to obtain high-purity exosome-drug conjugates.

10. Use of the exosome-drug conjugate according to any one of claims 1 to 9 in the preparation of a medical drug for treating non-alcoholic steatohepatitis.

Images