Exosome fusion liver-targeting liposome drug delivery system, and preparation method and application thereof

Abstract

The present application relates to an exosome fusion liver-targeting liposome drug delivery system and its preparation method and application, and belongs to the field of polymer materials and pharmaceutical preparations. The present application discloses an exosome fusion liver-targeting liposome drug delivery system, which uses high-concentration PEG 8000 and Nycodenz in combination to synergistically improve the exosome and liposome fusion efficiency, form biomimetic vesicles with uniform size and high drug loading. The exosome not only can play the characteristics of anti-inflammatory and antioxidant, but also plays a role in resisting harsh gastrointestinal environment and crossing biological barriers. In addition, the liposome is modified by cholic acid derivative by using EDC / NHS mediated amidation reaction, which realizes precise targeting of liver and sufficient accumulation in liver. The above-mentioned effects synergistically realize the treatment of type II diabetes and non-alcoholic fatty liver disease.

Description

Technical Field

[0001] This invention relates to an exosome-fused liver-targeting liposome drug delivery system, its preparation method, and its application, belonging to the fields of polymer materials science and pharmaceutical formulation. Background Technology

[0002] Type 2 diabetes is a chronic disease caused by insufficient or inefficient insulin secretion. Furthermore, the accumulation of inflammatory lipids and cytokines in diabetic patients can lead to liver fibrosis and apoptosis, triggering non-alcoholic steatohepatitis (NASH). The NLRP3 inflammasome, primarily found in innate immune cells, has been identified as a key factor promoting NASH. NLRP3 plays a crucial role in the synthesis of mature interleukin-1β (IL1β), initiating metabolic inflammation. Moreover, it not only promotes the transformation of hepatic macrophages to the pro-inflammatory M1 phenotype but also induces the release of additional NLRP3 and inflammatory factors, leading to a harmful cycle. Inhibition of excessive NLRP3 has been shown to alleviate liver inflammation. However, there are currently no specific drug treatments for obesity-induced type 2 diabetes and NASH.

[0003] Berberine is an isoquinoline alkaloid with antioxidant, immunomodulatory, and anti-fibrotic properties, and it can effectively control postprandial blood glucose in patients with early-stage type II diabetes. Berberine, administered via injection, can reduce lipid accumulation, improve mitochondrial function, and alleviate oxidative stress, showing promise as a potential therapeutic agent for NASH. However, its low oral bioavailability limits its application. To achieve therapeutic effects, berberine requires high-dose administration (mice: 100-250 mg / kg / day, humans: 900-1500 mg / day), which can cause potential side effects, further limiting its clinical application.

[0004] Exosomes are spherical structures composed of a phospholipid bilayer, with their main lipid components including cholesterol, saturated fatty acids, and sphingomyelin. They can transport molecules such as proteins, nucleic acids, and lipids between cells and are often considered natural carriers of signaling molecules. They can also encapsulate drugs; however, the retention characteristics and low drug loading capacity of exosomes in the intestine limit their therapeutic efficacy. Liposomes, as an effective drug delivery system, have a high drug loading capacity. Therefore, the combined use of exosomes and liposomes in drug delivery can be considered.

[0005] However, existing exosome-liposome fusion technologies generally suffer from low fusion efficiency, poor product stability, and insufficient drug loading, making it difficult to meet the needs of precision therapy. For example, chemical methods often use polyethylene glycol (PEG) as a medium, typically employing 45%–60% PEG. 4000 / PEG 6000Incubation is possible, but the drug loading capacity is limited, and the higher the relative molecular mass and the higher the volume fraction, the greater the toxicity to cells. Cells may deform or even rupture, and the final fusion may not be uniform and complete (Journal of Hubei Normal University (Natural Science), 2014, 34(1)). Currently, there are many methods for purifying and extracting exosomes, including ultracentrifugation and density gradient centrifugation. Density gradient centrifugation is cumbersome and time-consuming, while ultracentrifugation is time-consuming and has a low yield. Currently, low concentrations of Nycodenz can be used for gradient centrifugation in other cell purification processes. For example, Li et al. used 20%–34% Nycodenz to purify rat myocardial mitochondria (Journal of Zunyi Medical University, 2016, 39(5): 525-528), and Li et al. used 8%–8.2% Nycodenz to separate hepatic stellate cells (Progressin Modern Biomedicine, 2014 14(16) 3033-3037). However, there are few techniques for separating plant exosomes using Nycodenz.

[0006] Meanwhile, under this technological background, existing exosome-liposome fusion drug delivery systems also have shortcomings: when using exosomes alone to carry small molecule drugs, or when using liposome delivery systems alone, the drug delivery efficiency and therapeutic effect of the fusion system formed by combining the two using traditional techniques are limited. For example, in the study by Xiao et al. (International Journal of Biological Macromolecules, 2025, 306:141606) on the treatment of acute photodamaged skin with hyaluronic acid-modified liposome-exosome hybrid carriers (HL@Exo), the SOD level and MDA content in the HL@Exo treatment group were not significantly improved compared with the HL and Exo treatment groups; Ji et al. (ACS Nano) The clophosphonate-nintedanib hybrid exosome-liposome complex (CLD / NIN@LIEV) prepared in 2024, 18:21091-21111, showed limited performance in macrophage phenotype regulation. The CD163 / CD86 ratio in the CLD / NIN@LIEV group was not significantly different from that in the CLD-encapsulated group, indicating a small improvement. Although both have pro-inflammatory-anti-inflammatory phenotypic switching capabilities, the pro-inflammatory-anti-inflammatory capacity of the experimental group was slightly inferior. In addition, resveratrol liposomes fused with exosomes to form hybrid nanoparticles, Exo@Lip-Res, while retaining the antioxidant capacity of liposome Lip-Res, showed lower nanoparticle fusion capacity than the Lip-Res group in hydrogen peroxide, FRAP, and ROS scavenging assays. Summary of the Invention

[0007] The purpose of this invention is to provide an exosome-fused liver-targeting liposome drug delivery system, its preparation method, and its application in the treatment of non-alcoholic steatohepatitis and type II diabetes.

[0008] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows: an exosome-fused liver-targeting liposome drug delivery system, the preparation process of which is as follows: exosomes are fused with liver-targeting liposomes modified with bile acid derivatives to prepare empty nanoparticles, and drugs are encapsulated in the empty nanoparticles to prepare liver-targeting exosome drug-loaded nanoparticles. The liver-targeting exosome drug-loaded nanoparticles are the exosome-fused liver-targeting liposome drug delivery system.

[0009] In a preferred embodiment, the exosomes are derived from one of the following plants, including ginger, galangal, yam, pineapple, etc.

[0010] Furthermore, the cholic acid derivatives include ursodeoxycholic acid, chenodeoxycholic acid, deoxycholic acid, etc.

[0011] Furthermore, the drug is one of the drugs that can treat liver diseases, including berberine hydrochloride, morin, smegglutinin, etc.

[0012] This invention also provides a method for preparing an exosome-fused liver-targeting liposome drug delivery system, comprising the following steps:

[0013] (1) Preparation of liver-targeting ligands:

[0014] The bile acid derivative and reactants were placed in water, and DSPE-PEG was added. 2000 -NH2, react at 35-39℃ for 20-24h, purify, and obtain liver-targeting ligand;

[0015] (2) Extraction of exosomes:

[0016] Ginger juice was extracted, and ginger exosomes were obtained by low-temperature high-speed centrifugation and sucrose density gradient centrifugation.

[0017] (3) Preparation of liver-targeting liposomes modified with bile acid derivatives:

[0018] The lipid mixture consisting of emulsifier, liver-targeting ligand obtained in step (1) and cholesterol was dissolved in an organic reagent, and liver-targeting liposomes modified with bile acid derivatives were prepared by extrusion.

[0019] (4) Preparation of empty nanoparticle solution:

[0020] The exosomes obtained in step (2) were combined with the liver-targeting liposomes modified with bile acid derivatives obtained in step (3) and PEG. 8000 After mixing and co-incubation, the mixture was purified by density gradient centrifugation, containing PEG. 8000The concentration is 60±3% w / v; this mixture is mixed with 75-85% w / v Nycodenz to form a Nycodenz solution of the first concentration containing the mixture. The Nycodenz solution of the first concentration, the Nycodenz solution of the second concentration, and PBS form a gradient solution. The top separation layer is separated by ultracentrifugation to obtain purified empty nanoparticles; the Nycodenz concentration gradient decreases by 80% to 15% (preferably 40% to 15%, referring to the difference between the first and second concentrations); the first concentration is lower than 75-85% w / v; the second concentration is lower than the first concentration;

[0021] (5) Preparation of ligand-modified drug-loaded nanoparticles:

[0022] Equal volumes of drug solution and empty nanoparticle solution were mixed to obtain liver-targeting exosome drug-loaded nanoparticles; the concentration ratio of empty nanoparticle solution to drug solution was 1:1-3.

[0023] Ginger exosomes are rich in polysaccharides and low-immunogenic proteins, making it easier to form a hydrophilic and charge-neutral hydration layer. This reduces their binding to mucin glycoproteins and lowers the probability of being captured by mucus. Ginger exosome membranes are also more flexible than animal exosomes, while other plant exosomes (such as grapefruit exosomes) tend to have harder membranes, resulting in lower permeation efficiency.

[0024] This invention employs a direct incubation method for the fusion of ginger exosomes and bile acid liposomes. However, such methods may suffer from problems such as increased size, uneven size distribution, and low fusion efficiency. This invention incorporates a high concentration of PEG. 8000 This method achieves highly efficient purification of exosomes, enabling the fusion of intact exosomes with liposomes. Through multiple material-based experimental explorations, 80% Nycodenz (w / v) was ultimately adopted. Furthermore, density gradient centrifugation (GR-Exos) was used to purify the fusion products, ensuring the homogeneity of the nanoparticles. This targeted improvement resulted in uniform fusion of exosomes and liposomes. In this paper, Nycodenz was provided by Axis-Shield of Norway as a density gradient separation solution.

[0025] Bile acid derivatives (such as ursodeoxycholic acid) and DSPE-PEG 2000 The acylation reaction requires precise control of the molar ratio (1:1-1.5), reaction time (24 hours), and temperature (37°C). Otherwise, incomplete modification or byproduct formation will reduce liver targeting efficiency, leading to drug accumulation in non-target tissues. If the order of dosing is incorrect, the main reaction will be inhibited: DSPE-PEG 2000 After the -NH2 amino group is modified by EDC, it cannot couple with the active ester of UDCA, and the main product (DSPE-PEG) is lost. 2000 -UDCA production decreases, while byproducts accumulate.

[0026] Key parameters in the preparation method include:

[0027] Exosome extraction: Three centrifugation programs (1000-10000g, 10-40 minutes) combined with ultra-high speed centrifugation (200,000g, 2 hours), with precise sucrose gradient separation (8%-60%).

[0028] Fusion purification: Empty nanoparticles (GR-Exos) were separated using 80% Nycodenz (w / v) and gradient centrifugation (200,000 g, 2 h) with 40% Nycodenz.

[0029] Drug loading optimization: GR-Exos solution and berberine solution should be mixed at a ratio of 1:1-3. Ultrasonic parameters should be 55-70 kHz for 15-30 minutes to ensure efficient drug encapsulation without damaging the nanostructure.

[0030] Preferably, the reactants are 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC˙HCl) and N-hydroxysuccinimide (NHS); in step (1), the reactants are EDC˙HCl or NHS, bile acid derivatives, and DSPE-PEG. 2000 The molar ratio of -NH2 is 1-1.5:1:1-1.5.

[0031] Further, step (2) specifically involves: extracting ginger juice, centrifuging at 0-4℃ at 800-1200g for 5-10 min, at 2500-3500g for 15-20 min, and at 9000-11000g for 20-40 min; ultracentrifuging at 180,000-200,000g for 1-2 h, resuspending the juice, and transferring it to a sucrose solution with different gradient concentrations for centrifugation, the sucrose gradient concentrations being 8%, 15%, 30%, 45%, and 60%, respectively; then centrifuging, collecting the bands located between the 8% / 30% layer and the 30% / 45% layer, washing, centrifuging, and resuspending to obtain ginger exosomes.

[0032] In step (3), the emulsifier, liver-targeting ligand and cholesterol are mixed in a mass ratio of 1-5:1-2:2.

[0033] In step (4), exosomes, liver-targeting liposomes modified with bile acid derivatives, and PEG... 8000 The volume ratio is 1-2:1:1-2.

[0034] In step (5), the drug is encapsulated in the empty nanoparticles by ultrasound, with ultrasound parameters of 55-70 kHz and 15-30 min.

[0035] Another object of the present invention is to disclose the application of the exosome fusion liver-targeting liposome drug delivery system or the preparation method thereof in the preparation of medicaments for treating non-alcoholic steatohepatitis and type II diabetes.

[0036] This invention fuses exosomes with liposomes to form a biomimetic vesicle. This vesicle not only retains the membrane proteins of the exosomes but also increases the drug loading capacity of this carrier. Liposomes modified with bile acid derivatives have liver-targeting properties, enabling the exosomes to be directed to the liver. Drugs, such as berberine hydrochloride, can treat non-alcoholic steatohepatitis (NAH), but their oral bioavailability is low and they lack liver-targeting properties. The biomimetic vesicles prepared in this invention can encapsulate such drugs, overcoming gastrointestinal barriers to achieve liver-targeting action. Simultaneously, the drug and the vesicle work synergistically to achieve long-term drug circulation, showing potential application prospects in the treatment of NHA and type II diabetes.

[0037] Compared with the prior art, the present invention has the following advantages:

[0038] (1) The exosome drug delivery system designed in this invention uses prepared exosomes as oral drug carriers, which has advantages in terms of endogenous nature, biocompatibility and multifunctionality. Exosomes have advantages such as good drug loading capacity, unique biomolecular protection, intestinal mucus penetration and tissue bioavailability, and have great advantages in maintaining the stability of oral drugs and the permeability of the intestinal mucus layer.

[0039] (2) Liposomes modified with bile acid derivatives via EDC / NHS-mediated amidation are taken up by endocytosis mediated by apical sodium-dependent bile acid transporter (ASBT) and transported intracellularly via ileal bile acid-binding protein (IBABP). They then enter the enterohepatic circulation, ultimately specifically guiding the drug to the liver for precise hepatic targeting. The application of hepatic targeting technology can increase the accumulation of drugs in the liver, thereby achieving precise therapeutic effects, improving the bioavailability of oral administration, and achieving longer-lasting drug circulation.

[0040] (3) The biomimetic vesicles formed by fusing liposomes and exosomes in this invention not only retain the membrane proteins of exosomes but also enhance their drug-carrying capacity. GR-Exos has the ability to specifically target NLRP3 inflammatory vesicles in hepatic macrophages, and uses its anti-inflammatory and antioxidant properties to inhibit NLRP3 expression. At the same time, berberine can work synergistically to enhance the inhibition of inflammatory factors and improve NASH and type II diabetes at lower doses.

[0041] (4) This invention uses a combination of high concentrations of PEG 8000In conjunction with Nycodenz, the fusion efficiency of liposomes and exosomes is improved, and biomimetic vesicles with uniform size and high drug loading are obtained by centrifugation purification. The vesicles are simple to obtain, have low oral toxicity, and good biocompatibility. Attached Figure Description

[0042] Figure 1 DSPE-PEG in Example 1 2000 -UDCA 1 1H NMR spectrum (400MHz, DMSO-d6);

[0043] Figure 2 The image shows the size distribution and morphology of the nanoparticles RAL, G-Exos, GR-Exos, and GR-Exos@B in Example 3.

[0044] Figure 3 The following is a characterization diagram of the fusion of G-Exos and GR-Exos in Example 4(2): (A) Nanoparticle tracking analysis (NTA) was used to detect the size of G-Exos; (B) The integrity of GR-Exos was evaluated by FRET spectroscopy.

[0045] Figure 4 The in vitro release diagrams of RAL@B, G-Exo@B and GR-Exo@B in Example 4(3) are shown in (A) solution at pH 6.8; and (B) solution at pH 1.2.

[0046] Figure 5 This is a schematic diagram of cell viability in the Caco-2 and HT-29 cytotoxicity assays in Example 4(4), (A) HT-29; (B) Caco-2;

[0047] Figure 6 This is a schematic diagram of the drug-loaded nanoparticles penetrating the mucus in Example 4(5);

[0048] Figure 7 This is a schematic diagram showing the weight change trend of diabetic mice that were given GRExos@B, G-Exos@B and RAL@B samples for a long period of time in Example 5(1);

[0049] Figure 8 This is a schematic diagram of serum inflammatory factor levels 60 days after administration in Example 5(1), (A) LDL, (B) TG, (C) HDL, (D) AST, (E) ALP, (F) ALT;

[0050] Figure 9 Images of liver H&E staining, Masson staining, and Oil Red O staining in Example 5(1);

[0051] Figure 10For the Western blot analysis of genes such as COA and CY2EBP-1 in the liver in Example 5(1), g. The expression of COA and CY2EBP-1 in the liver was evaluated by Western blot analysis; 1: HFD; 2: NM; 3: RAL@B; 4: G-Exos@B; and 5: GRExos@B; h. The expression of SCD, SREBP-1 and CD95; numbered as above; i. Liver AMPK, p-AMPK, Nrf2, p-NRf2, HO-1 and NOQ1, numbered as above;

[0052] Figure 11 This is a schematic diagram of the levels of TNF-α, IL-6, IL-4, IL-1β and IL-10 in each drug administration group in Example 5(1);

[0053] Figure 12 This is a schematic diagram of the glucose tolerance test and insulin resistance index after 60 days in Example 5(2). (A) Insulin tolerance was tested in mice that had been fasted after 60 days of treatment with intraperitoneal injection (ip) of insulin; (B) Insulin resistance index of HFD mice treated with G-Exos@B, RAL@B and GR-Exos@B; (C) Blood glucose levels of mice 2 hours before and after meals after 60 days of treatment.

[0054] Figure 13 The levels of C-peptide in mice in each treatment group after 60 days of treatment in Example 5(2); Purple: NM, Blue: HFD, Yellow: G-Exos@B, Green: RAL@B, Red: GR-Exos@B;

[0055] Figure 14 This is a visualization of insulin immunostaining images of the pancreatic islets of mice in each treatment group after 60 days of administration in Example 5(2). Scale bar: 30.0 μm.

[0056] Figure 15 For Western blot analysis of PEPCK, PGC-1α and GLUT4 expression in the liver in Example 5(2), 1: NM; 2: HFD; 3: RAL@B; 4: G-Exos@B; and 5: GR-Exos@B;

[0057] Figure 16 In Example 5(3), the macrophage marker M1 / M2 ratio was determined in BMDM cells by flow cytometry.

[0058] Figure 17 For the Western blot assessment of NLRP3 expression in BMDM cells in Example 5(3); 1: BBR; 2: M1; 3: M2; 4: RAL; 5: G-Exos; 6: GR-Exos, and 7: GR-Exos@B. And the statistical analysis of NLRP3 protein expression levels;

[0059] Figure 18 For the staining of major organ sections of mice in each drug administration group in Example 5(4) with hematoxylin and eosin (H&E), scale bar: 40.0 μm. Detailed Implementation

[0060] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0061] The present invention discloses an exosome-fused liver-targeting liposome drug delivery system, the preparation process of which is as follows: exosomes are fused with liver-targeting liposomes modified with bile acid derivatives to prepare empty nanoparticles, and drugs are encapsulated in the empty nanoparticles to prepare liver-targeting exosome drug-loaded nanoparticles. The liver-targeting exosome drug-loaded nanoparticles are the exosome-fused liver-targeting liposome drug delivery system.

[0062] DSPE-PEG (polyethylene glycol-distearate phosphatidylethanolamine) and bile acid derivatives were amidated to obtain the ligand-modified polymer DSPE-PEG-UDCA (which is a liver-targeting ligand). Simultaneously, the material containing exosomes was repeatedly centrifuged using an ultra-high-speed refrigerated centrifuge to obtain exosomes. Subsequently, the polymer, emulsifier, and cholesterol were co-dissolved in an organic solvent, and liposomes were obtained using an extrusion apparatus. The liposomes and exosomes were mixed to prepare ligand-modified empty nanoparticles. The empty nanoparticles were then ultrasonically mixed with a drug to obtain the aforementioned exosome-fused liver-targeting liposome drug delivery system.

[0063] Furthermore, the cholic acid derivatives include ursodeoxycholic acid, chenodeoxycholic acid, deoxycholic acid, etc.

[0064] Furthermore, the exosomes are derived from one of the following plants, including ginger, galangal, yam, pineapple, etc.

[0065] Furthermore, the liver-targeting ligand is composed of polyethylene glycol-distearate phosphatidylethanolamine (DSPE-PEG). 2000 DSPE-PEG is linked to bile acid derivatives via an amidation reaction. 2000 The molar ratio with bile acid derivatives is 1-1.5:1.

[0066] Furthermore, the extraction of the exosomes involves first removing macromolecular substances from the exosome material using a low-temperature high-speed centrifuge, then centrifuging the supernatant using an ultra-high-speed refrigerated centrifuge to remove small molecules, obtaining exosome precipitate, resuspending in PBS, and transferring to a sucrose gradient centrifugation at different concentrations to collect bands between different concentrations, thus obtaining the exosome solution.

[0067] Furthermore, the preparation of the bile acid derivative-modified liver-targeting liposomes involves dissolving a lipid mixture composed of an emulsifier, a liver-targeting ligand, and cholesterol in a ratio of 1-5:1-2:2 in an organic solvent, incubating and evaporating the mixture. After separating the dried lipid membrane, the lipid membrane is hydrated, rotary evaporated at room temperature, and finally extruded using a liposome extruder to obtain the final product.

[0068] Furthermore, the exosomes were fused with liver-targeting liposomes modified with bile acid derivatives to prepare empty-loaded nanoparticles. The exosomes, liver-targeting liposomes modified with bile acid derivatives and the carrier were dissolved in solution at a ratio of 1-2:1:1-2, incubated at a certain temperature for a fixed time, and purified GR-Exos were obtained by density gradient centrifugation.

[0069] Furthermore, the exosome-fused liver-targeting liposome drug delivery system is a nanoparticle solution obtained by mixing empty nanoparticles with drugs and then sonicating them using a cell sonicator.

[0070] The preparation method of the exosome fusion liver-targeting liposome drug delivery system of the present invention includes the following steps:

[0071] (1) Preparation of liver-targeting ligands:

[0072] The bile acid derivative and reactants were placed in water, and DSPE-PEG was added. 2000 After the reaction with -NH2 was carried out, the mixture was stirred at 37°C. After the reaction was completed, the mixture was precipitated with an organic solvent and the lower precipitate was collected by centrifugation and vacuum dried to obtain the liver-targeting ligand.

[0073] (2) Extraction of exosomes:

[0074] Macromolecules in the material were removed by centrifugation using a low-temperature high-speed centrifuge. The supernatant was then centrifuged using an ultra-high-speed refrigerated centrifuge to remove small molecules, resulting in an exosome precipitate. This precipitate was resuspended in phosphate-buffered saline (PBS) solution and transferred to a sucrose gradient centrifugation at different concentrations. The bands between different concentrations were collected, washed with PBS, balanced, and centrifuged again. Finally, the precipitate was resuspended and stored at -80°C for later use. The resulting exosome solution was designated G-Exos.

[0075] (3) Preparation of liposomes:

[0076] The lipid mixture, consisting of an emulsifier, the ligand solution obtained in step (1), and cholesterol, was dissolved in an organic reagent. The mixture was then evaporated in a water bath. The organic reagent was added to dissolve the dried lipid membrane. The lipid membrane was rehydrated and then extruded sequentially through a polycarbonate porous membrane using a liposome extruder to obtain a liposome solution.

[0077] (4) Preparation of empty nanoparticle solution:

[0078] The exosomes obtained in step (2) are combined with the liposomes and PEG obtained in step (3). 8000 After mixing and co-incubation, the mixture was purified by density gradient centrifugation. This mixture was then mixed with a certain concentration of Nycodenz (w / v) in PBS to produce a slightly lower concentration mixture. This solution, the appropriate concentration of Nycodenz in PBS, and the PBS solution were introduced into a cryogenic ultracentrifuge for centrifugation. The top separation layer yielded a purified solution of empty nanoparticles.

[0079] (5) Preparation of liver-targeting exosome-loaded nanoparticles:

[0080] Prepare a drug solution with a concentration of 20 mg / mL in advance. When preparing the drug-loaded nanoparticle solution GR-Exos@B, mix the drug solution with the empty nanoparticle solution finally obtained in steps (1)-(4), sonicate, and remove the unencapsulated drug by ultrafiltration to obtain liver-targeted exosome drug-loaded nanoparticles.

[0081] Further, in step (1), the reactants are 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC˙HCl) and N-hydroxysuccinimide (NHS);

[0082] Furthermore, in step (1), the reactants are EDC˙HCl or NHS, bile acid derivatives, and DSPE-PEG. 2000 The molar ratio of -NH2 is 1-1.5:1:1;

[0083] Furthermore, in step (2), the high-speed centrifuge is set to a program of 1000-10000g and 10-40min three times;

[0084] Furthermore, in step (2), the program for separating and purifying exosomes using an ultra-high speed refrigerated centrifuge is set to 180,000-220,000g for 60-120min;

[0085] Furthermore, in step (2), the program for precipitating and purifying exosomes using an ultra-high speed refrigerated centrifuge is set to 180,000-220,000g for 60-120min;

[0086] Furthermore, in step (2), the sucrose gradients of different concentrations are 8%, 15%, 30%, 45%, and 60% (20mM Tris-Cl, pH 7.2);

[0087] Furthermore, in step (3), the organic solvent is a mixture of chloroform and methanol, a mixture of trichloroethane and methanol, or anhydrous diethyl ether;

[0088] Furthermore, in step (3), the emulsifier is soybean lecithin, egg yolk lecithin, or rice bran lecithin;

[0089] Furthermore, in step (3), the pore size of the polycarbonate porous membrane is 150-250 nm;

[0090] Furthermore, in step (4), the program setting for the low-temperature ultra-high-speed centrifuge is 180,000-220,000g, 60-120min;

[0091] Furthermore, in step (5), when GR-Exos is mixed with the drug solution, the concentration ratio is 1:1-3;

[0092] Furthermore, in step (5), the ultrasonic program of the cell ultrasonic disruptor is 55-70 kHz for 15-30 min.

[0093] Furthermore, in application, exosome solutions and liposome solutions are mixed and incubated to prepare empty-loaded nanoparticles. A drug solution is then prepared, mixed with the empty-loaded nanoparticles, and incubated, followed by sonication to prepare liver-targeting exosome-liposome synergistic drug-loaded nanoparticles. The nanoparticles are administered orally.

[0094] Previous studies have shown that bile acid derivative-modified nanoparticles (NPs) can reach the liver to exert therapeutic effects through endocytosis, intracellular transport, and enter the enterohepatic circulation (Chemical Engineering Journal. 2024, 485:150129, CN115531309A). This invention utilizes an EDC / NHS-mediated amidation reaction to modify liposomes with bile acid derivatives. This overcomes the limitations of traditional physical mixing methods while achieving precise targeting to the liver and sufficient accumulation within the liver.

[0095] This invention designs and prepares GR-Exos@B nanoparticles based on the fusion of exosomes and liver-targeting liposomes and encapsulation of drugs, and innovatively uses high concentrations of PEG. 8000 When used in combination with Nycodenz, it works synergistically to improve the fusion efficiency of exosomes and liposomes, resulting in uniform and complete fusion with low cytotoxicity, thus solving the problem of balancing fusion efficiency and product purity in traditional methods. GR-Exos@B has highly efficient transmembrane intestinal transport capabilities, effectively overcoming the gastrointestinal barrier to achieve precise targeting of the liver and sufficient accumulation to exert anti-inflammatory and antioxidant effects, thereby synergistically improving drug delivery efficiency and enhancing the bioavailability of the encapsulated drug.

[0096] Example 1

[0097] DSPE-PEG 2000 The synthesis of UDCA is as follows:

[0098] To synthesize DSPE-PEG incorporating ursodeoxycholic acid (UDCA) 2000 -UDCA, EDC˙HCl (0.432 mmol), NHS (0.432 mmol), and UDCA (0.36 mmol) were added to 6 mL of water with stirring for 30 minutes, followed by the addition of DSPE-PEG. 2000 -NH2 (0.36 mmol) was added, and the reaction was carried out at 37 °C for 24 hours. The resulting product was then transferred to a dialysis bag for one day (molecular weight cutoff (MWCO): 2.0 kDa) to remove UDCA, precipitated in cold diethyl ether, and dried under vacuum at room temperature. 325.3 mg of the precipitate was weighed. DSPE-PEG 2000 -UDCA yield was approximately 86.8%. The DSPE-PEG prepared in this example... 2000 -UDCA was used for proton nuclear magnetic resonance analysis, and the results are as follows Figure 1 As shown.

[0099] Example 2

[0100] Extraction of ginger exosomes: The extraction process is as follows:

[0101] The ginger purchased for the experiment was fresh ginger. The extraction method was gradient centrifugation, using a low-temperature high-speed centrifuge. First, the fresh ginger was thoroughly washed with deionized water, chopped, and then mixed with pH 7.4 PBS (NaCl, KCl, Na₂HPO₄, KH₂PO₄; 8.0g NaCl, 0.2g KCl, 1.44g Na₂HPO₄, and 0.24g KH₂PO₄ were dissolved in 800mL distilled water, the solution was adjusted to pH 7.4 with HCl, and finally distilled water was added to bring the volume to 1L to obtain the PBS buffer; all PBS used in this study was prepared in this manner) for 30 minutes to extract ginger juice (the ratio of chopped ginger to PBS was 1g:4ml). The obtained ginger juice was centrifuged at 1000g for 10 minutes, 3000g for 20 minutes, and 10000g for 40 minutes at 4℃, and the supernatant was collected. The precipitate was then centrifuged at 200,000g for 2 hours using an ultracentrifuge, resuspended in PBS, and transferred to sucrose gradients of different concentrations (8%, 15%, 30%, 45%, and 60% sucrose in 20mM Tris-Cl solutions at pH 7.2). The mixture was then centrifuged at 200,000g for 2 hours, and bands located between the 8% / 30% and 30% / 45% layers were collected. These bands were washed with PBS and centrifuged at 200,000g for 1 hour at 4°C, then resuspended in PBS again. The final liquid obtained was the exosome solution G-Exos (the concentration of G-Exos in the exosome solution was controlled to 10 mg / mL by adjusting the amount of PBS used for resuscitation).

[0102] Example 3

[0103] (1) Preparation of liposomes (RAL) and drug-loaded liposomes (RAL@B)

[0104] Liver-targeting liposomes loaded with berberine hydrochloride (RAL@B) were prepared using a previously reported thin-film hydration method. A lipid mixture consisting of soybean lecithin (10 mg / mL), DSPE-PEG-UDCA (10 mg / mL), and cholesterol (10 mg / mL) in a 5:1:2 volume ratio was dissolved in a chloroform:methanol mixture (3:1, v / v) to form an organic solution with a liposome concentration of 10 mg / mL. 50 μL of this organic solution was incubated in water at 40 °C for 1 h, evaporated at 37 °C and 50 rpm for 30 min, and the synthesized lipid membrane was placed under nitrogen for 30 min. The dried lipid membrane was dissolved by adding 3 mL of anhydrous diethyl ether. The berberine hydrochloride solution (20 mg / mL, 50 μL; the 20 mg / mL berberine hydrochloride solution in this article was obtained by dissolving berberine hydrochloride in DMF) was then slowly injected below the liquid surface using a syringe. The mixture was sonicated for 10 min to form a W / O primary solution, and then evaporated at room temperature (RT) for 30 min. The berberine hydrochloride-containing lipid membrane was hydrated by adding PBS, followed by rotary evaporation at room temperature for 1 h. The liposomes were then sequentially extruded through a 200 nm polycarbonate porous membrane using a liposome extruder. This extrusion process was repeated a total of 20 times to form 10 mg / mL RAL@B with a uniform particle size.

[0105] The preparation steps for RAL are basically the same as those for RAL@B, except that water is added instead of berberine solution, thus preparing 10 mg / mL RAL.

[0106] The specific process is as follows: A lipid mixture consisting of soybean lecithin (10 mg / mL), DSPE-PEG-UDCA (10 mg / mL), and cholesterol (10 mg / mL) in a volume ratio of 5:1:2 was dissolved in a chloroform:methanol mixture (3:1, v / v) to form an organic solution with a liposome concentration of 10 mg / mL. 50 μL of this organic solution was incubated in water at 40 °C for 1 h, evaporated at 37 °C and 50 rpm for 30 min, and the synthesized lipid membrane was placed under nitrogen for 30 min. The dried lipid membrane was dissolved by adding 3 mL of anhydrous diethyl ether. Then, 50 μL of water was added dropwise, and the mixture was sonicated for 10 min to form a W / O primary solution. The mixture was then evaporated at room temperature (RT) for 30 min. The lipid membrane was hydrated by adding PBS, followed by rotary evaporation at room temperature for 1 h. The liposomes were then extruded sequentially through a 200 nm polycarbonate porous membrane using a liposome extruder. This extrusion process was repeated a total of 20 times to form 10 mg / mL RAL with a uniform particle size.

[0107] (2) Preparation of G-Exos@B, GR-Exos, and GR-Exos@B nanoparticles

[0108] The 10 mg / mL G-Exos solution prepared in Example 2 and the 20 mg / mL berberine hydrochloride solution were mixed in equal volumes and ultrasonicated at 100 W power and 60 kHz frequency with a 2-second on / off cycle for 20 min. Then, the unencapsulated berberine hydrochloride was removed by ultrafiltration with a molecular weight cutoff value (MWCO) of 100 kDa to obtain G-Exos@B.

[0109] The previously prepared 10 mg / mL G-Exos, 10 mg / mL RAL and 60% (w / v) PEG were used. 8000 The mixture was prepared at a volume ratio of 1:1:2 and incubated at 40°C for 2 hours. PEG was included in the mixture. 8000 Dissolved in PBS to obtain 60% (w / v) PEG 8000 The mixture was purified by density gradient centrifugation according to the aforementioned method: centrifuged at 1000g for 10 min, 3000g for 20 min, and 10000g for 40 min at 4°C, and the supernatant was collected; then, it was ultracentrifuged at 200,000g for 2 h using an ultracentrifuge, the precipitate was resuspended in PBS, and transferred to different concentrations of sucrose gradient (sucrose concentrations of 8%, 15%, 30%, 45%, and 60% in pH 7.2 and 20mM Tris-Cl solutions, respectively). The mixture was then centrifuged at 200,000 g for 2 h, and bands located between the 8% / 30% and 30% / 45% layers were collected. The bands were washed with PBS and centrifuged at 200,000 g for 1 h at 4 °C. The precipitate was resuspended in PBS to obtain the mixture. This PBS-resuspended mixture was mixed with 80% Nycodenz (w / v) at a 1:1 (v / v) ratio to produce a 40% solution (i.e., a solution containing the mixture and 40% Nycodenz (w / v)). A mixture consisting of 4 mL of the 40% solution, 2 mL of 15% Nycodenz (w / v) in PBS (prepared by mixing PBS and 80% Nycodenz (w / v) in a specific ratio), and 1 mL of PBS solution was introduced into a centrifuge tube. The mixture was then centrifuged at 200,000 g for 2 h using a low-temperature ultracentrifuge. The top layer yielded purified GR-Exos, which was diluted or concentrated with PBS to a concentration of 10 mg / mL.

[0110] In step (2) of this embodiment, the GR-Exos@B preparation method involves mixing equal volumes of GR-Exos at a concentration of 10 mg / mL and berberine hydrochloride at a concentration of 20 mg / mL, and then performing ultrasonic treatment for 20 min at a frequency of 60 kHz with a 2-second on / off cycle using 100 W power. Subsequently, unencapsulated berberine hydrochloride is removed by ultrafiltration using a molecular weight cutoff value (MWCO) of 100 kDa to obtain GR-Exos@B.

[0111] The nanoparticles prepared in this embodiment were subjected to particle size analysis, and the results are as follows: Figure 2 As shown.

[0112] Figure 2 The particle size distribution of RAL@B, RAL, G-Exos, G-Exos@B, and GR-Exos@B nanoparticles in Example 3 is shown below. Figure 2 It can be seen that the average particle size is around 200-300 nm.

[0113] The nanoparticles prepared in this embodiment were subjected to scanning electron microscopy and transmission electron microscopy (TEM), and the results are as follows: Figure 2 As shown in AD, RAL, G-Exos, GR-Exos, and GR-Exos@B are spherical with similar sizes, with average values ​​of 235.3±17.1 nm, 118.4±10 nm, 297.5±21.4 nm, and 324.5±10 nm, respectively, indicating that the drug-loaded nanoparticles are uniform in size.

[0114] Example 4

[0115] (1) Drug loading and encapsulation efficiency of nanoparticles

[0116] Encapsulation efficiency (EE) and drug loading (LC) are commonly used to express the drug-carrying capacity of nanoparticles. Drug loading is the percentage of drug loaded in the nanoparticles to the total mass (carrier and drug load), while encapsulation efficiency refers to the percentage of drug loaded in the nanoparticles to the total mass of the drug.

[0117] The encapsulation efficiency and drug loading of the berberine hydrochloride nanoparticles prepared in this embodiment were calculated, and the results are shown in Table 1. Compared with ginger exosomes and liposomes loaded with berberine, the fusion carrier GR-Exos@B has higher berberine loading capacity (i.e., drug loading LC) and encapsulation efficiency (i.e., mass encapsulation efficiency EE).

[0118] Table 1. Drug loading, encapsulation efficiency, size, and polymer dispersibility index of different sample amounts

[0119]

[0120] (2) Characterization of G-Exos and GR-Exos fusion

[0121] To evaluate the hydrodynamic diameters and zeta potentials of G-Exos, RAL, G-Exos@B, RAL@B, and GR-Exos@B, dynamic light scattering analysis (DLS) was employed, followed by imaging using transmission electron microscopy (TEM). The protein composition of G-Exos, RAL, and GR-Exos was analyzed using Coomassie staining. G-Exos, RAL, and GR-Exos were prepared in loading buffer after assays using a BCA kit. Samples were then heated to 95°C for 5 min, and 40 μg of sample was loaded onto an 8% SDS-polyacrylamide gel. Samples were electrophoresed at 85 V for 30 min and 120 V for 60 min. After staining with Coomassie blue reagent for 1 h, washing overnight, and then observation were performed. G-Exos were labeled using DiR (red). The labeled G-Exos and RAL were then mixed with 60% PEG. 8000 G-Exos were fused (10 mg / m³ LG-Exos labeled with DiR, stirred for 12 h, dialyzed) and filtered through a 0.2 μm polycarbonate membrane using an extruder. The integration of G-Exos with RAL was visualized using confocal laser scanning microscopy (CLSM) and captured using a flow detector. To further explore GR-Exos fusion, G-Exos were labeled with DiR and fused GR-Exos were prepared as described above. After filtration through nanoparticles, FRET was detected by microplate reader under 420 nm excitation.

[0122] The results are as follows Figure 3 As shown in Figure A, the average size of G-Exos measured by nanoparticle tracking analysis (NTA) is 110 nm. The morphology is spherical and the size is uniform, indicating the successful separation and purification of G-Exos. Figure 3 In B of the study, the FRET study confirmed the complete fusion of GR-Exos.

[0123] (3) In vitro acid-responsive release assay of drug-loaded nanoparticles

[0124] RAL@B, G-Exo@B, and GR-Exos@B were prepared according to Example 3 and incubated for 8 hours at pH levels of 1.2 and 6.8. The size of the nanoparticles was measured using DLS. To simulate the gastrointestinal environment, RAL@B, G-Exo@B, and GR-Exo@B were introduced into dialysis bags with a molecular weight cutoff (MWCO) of 12000 Da. These bags were then immersed in 5 ml of different release media: simulated gastric juice (SGF) containing pepsin at pH 1.2 and simulated intestinal juice (SIF) containing trypsin at pH 6.8. The bags were continuously shaken at 80 rpm and maintained at 37°C. At specific time intervals, 200 μL of release medium was sampled and replaced with an equal volume of fresh medium. The concentration of berberine was assessed using HPLC. Furthermore, the physical stability of the nanoparticles was assessed by monitoring changes in DLS during 7 days of refrigeration (4°C).

[0125] Results of in vitro acid-responsive cumulative release of drug-loaded nanoparticles are shown in [reference needed]. Figure 4 .like Figure 4 As shown in Figure B, after prolonged incubation at pH 1.2, G-Exos@B and GR-Exos@B exhibited a cumulative release of less than 30% of berberine, while RAL@B showed quenched release of berberine. This indicates that the ginger exosomes prepared in this invention can release berberine relatively continuously, effectively preventing premature drug release. Compared with G-Exos@B and RAL@B, GR-Exos@B has a slower release rate, but at pH 6.8 (… Figure 4 In A), the cumulative release amounts of GR-Exos@B, G-Exos@B, and RAL@B within 28 hours were similar, at 61.2%, 73.1%, and 78.5%, respectively, which further demonstrates that the ginger exosomes prepared in this invention have a relatively sustained release capacity.

[0126] (4) Cytotoxicity assay of drug-loaded nanoparticles (MTT)

[0127] G-Exos@B, RAL@B, and GR-Exos@B solutions were prepared according to steps (1) and (2) of Example 3. MTT assays were performed on human clonal colon adenocarcinoma (Caco-2) and human colon adenocarcinoma (HT-29). Based on the cell type and the type of nanoparticles added, the cells were divided into two groups: Group A was designated as G-Exos@B(HT-29), RAL@B(HT-29), and GR-Exos@B(HT-29); Group B was designated as G-Exos@B(Caco-2), RAL@B(Caco-2), and GR-Exos@B(Caco-2). Results are shown below. Figure 5 . Figure 5 In this context, A represents the HT-29 cytotoxicity assay. Figure 5The diagram in Figure B shows the cell viability in the Caco-2 cytotoxicity assay. It indicates that cells treated with different concentrations (50-500 μg / mL) of RAL@B, G-Exos@B, and GR-Exos@B maintained viability above 80% after 24 hours of incubation. Therefore, this demonstrates that the nanoparticles exhibit low cytotoxicity and are suitable for oral administration in mice without undesirable side effects.

[0128] (5) Mucus penetration experiment of drug-loaded nanoparticles

[0129] 1 mL of LG-Exos, RAL, and GR-Exos (1 mg / mL) were mixed with 100 μL of the fluorescent dye DiO (1 mg / mL, dissolved in DMSO) and stirred overnight at 250 rpm. During this process, DiO entered the exosome cell membrane or liposomes and was encapsulated therein. After dialysis, G-Exos@DiO, RAL@DiO, and GR-Exos@DiO were obtained, thus producing specific fluorescence. The mixture was then divided into 5 × 10⁻⁶ cells per well in a 12-well plate. 4 HT-29 cells were cultured at a density of [number] cells and incubated until complete confluence was achieved. Subsequently, cells were exposed to 100 μg / m³ LG-Exos@DiO, RAL@DiO, and GR-Exos@DiO for 2 hours. After this incubation period, the cell culture medium was removed, and the cells were washed three times with PBS. The mucinous layer cells (HT-29) were then stained with 200 μL Alexa 594-WGA diluted 1:1000 for 1 hour, followed by three washes with PBS. During observation, three-dimensional images were captured using a confocal scanning microscope, and the data were reconstructed in 3D using ZEN software.

[0130] Nanoparticle mucus penetration as follows Figure 6 As shown (a three-dimensional image of a cell monolayer after incubation of HT-29 cells with RAL, G-Exos, and GR-Exos), green fluorescence represents nanoparticles, and red fluorescence represents the mucus layer. It can be seen that the green fluorescence of G-Exos and GR-Exos exhibits a wide distribution, while the co-localization of red fluorescence in the cell layer with the green fluorescence of the nanoparticles is poor, indicating that both G-Exos and GR-Exos penetrated the HT-29 cell layer, exhibiting enhanced mucus permeability, with GR-Exos showing the best penetration efficiency. The co-localization of red fluorescence in the mucus layer with green fluorescence from RAL indicates that its mucus penetration efficiency is second best.

[0131] Example 5

[0132] Long-term drug efficacy

[0133] (1) Long-term in vivo administration of GR-Exos@B for the treatment of NASH

[0134] Mice with high-fat diet-induced non-alcoholic hepatitis (NAAH) were randomly divided into five groups of three mice each. In vivo experiments were conducted by orally administering 30 mg / kg of G-Exos@B, RAL@B, and GR-Exos@B prepared in Example 3 three times a week to mice with NAAH induced by a high-fat diet. G-Exos@B, RAL@B, and GR-Exos@B were dissolved in water for administration, and 30 mg / kg refers to the dosage of G-Exos@B, RAL@B, or GR-Exos@B per kg of mouse (calculated as dry weight). Normal-diet mice (NM) served as the control group. Mouse body weight was recorded at 8:00 AM daily. Weight changes were recorded after 60 days of administration.

[0135] The process of constructing high-fat diet-induced non-alcoholic hepatitis (NASH) mice was as follows: All mice were housed in a specific pathogen-free controlled environment and fed a standard diet for the first two weeks. Starting from week three, their diets were supplemented with a high-fat diet, comprising 60% of the total calories, obtained from Synergy Biotechnology Inc. The proportion of the high-fat diet in the overall rodent diet was gradually increased until week six, after which the mice were fed a high-fat diet exclusively for another eight weeks. Simultaneously, starting from week three, the mice were administered a solution containing 10% D-fructose in autoclaved tap water, with the D-fructose concentration gradually increasing to 25% by week six. As a result, the mice developed obesity and insulin resistance by week 15, and non-alcoholic steatohepatitis (NASH) was induced after week 16, with mice weighing approximately 55-60 g and exhibiting fasting glucose levels exceeding 250 mg / dL. Mouse body weight and liver wet weight were measured, and NASH-related factors (alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α)) in mouse serum were detected using an ELISA kit to validate the mouse model. This model also served as a diabetic mouse model.

[0136] Figure 7 This is a schematic diagram of the body weight levels of diabetic mice. Figure 7 It was found that the administration of RAL@B, G-Exos@B, and GR-Exos@B led to significant weight loss. The HFD group experienced continuous weight gain over two months, while the experimental groups all achieved a final weight loss of more than 40% compared to the HFD group.

[0137] After 60 days of treatment, changes in the secretion of inflammatory factors were studied, and the results were as follows: Figure 8As shown, compared with the HFD, RAL@B, and G-Exos@B groups, the GR-Exos@B group exhibited significantly lower low-density lipoprotein (LDL) levels and liver function indices (ALT, ALP, AST, and TG). For example, the TG level in the RAL@B group was 1.17 mmol / L, and in the G-Exos@B group it was 1.2 mmol / L, while the GR-Exos@B group was already lower than the normal group level (1.0 mmol / L), indicating that GR-Exos@B treatment reduced the secretion of inflammatory factors and promoted the recovery of liver function.

[0138] Sixty days after treatment, the mice were dissected, and liver sections were taken for analysis. The results showed that although RAL@B and G-Exos@B treatments reduced inflammatory factor secretion and promoted liver function recovery, Masson staining, Oil Red O staining, and H&E staining of the liver sections indicated only partial recovery of hepatic steatosis. Conversely, GR-Exos@B treatment showed almost complete normalization of hepatic steatosis. Figure 9 ).

[0139] To explore the mechanism of GR-Exos@B's anti-NASH effect in vivo, the levels of typical proteins involved in oxidative stress and lipid metabolism pathways were investigated. Western blot analysis of the livers of NASH mice treated with GR-Exos@B showed significant downregulation of lipid metabolism markers (SREBP-1, SCD, COA, and FAS) and oxidative stress marker (CY2EP1), ultimately improving NASH symptoms. Furthermore, Western blot analysis revealed significantly reduced protein expression of p-Nrf2 / Nrf2, NOQ-1, and HO-1 due to an HFD diet. GR-Exos@B intervention demonstrated a beneficial effect by increasing the protein levels of pNrf2 / Nrf2, NOQ-1, and HO-1. Figure 10 Furthermore, GR-Exos@B in the liver of NASH mice led to an increased p-AMPK / AMPK protein expression ratio. Figure 10 These results are consistent with protein expression findings observed at the cellular level. Furthermore, in the GR-Exos@B treatment group, mRNA levels of SREBP-1 and FAS / CD95 were decreased, while HO-1 mRNA levels showed a significant increase. Figure 10 ).

[0140] After 60 days of long-term administration, serum pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) levels showed varying degrees of decline compared to the control group in the RAL@B, G-Exos@B, and GR-Exos@B treatment groups. The GR-Exos@B group exhibited the most significant inhibitory effect, with a statistically significant difference in the degree of reduction. Anti-inflammatory factors such as IL-4 and IL-10 showed a similar trend; compared to the control group, the RAL@B, G-Exos@B, and GR-Exos@B treatment groups all showed varying degrees of increase, with the GR-Exos@B group exhibiting the most significant promoting effect. This indicates that GR-Exos@B has the ability to reduce intestinal inflammation and improve NASH symptoms. Figure 11 ).

[0141] (2) GR-Exos@B reverses the diabetic symptoms of NASH.

[0142] The successfully modeled diabetic mice were randomly divided into five groups of three mice each. Three mice were orally administered 30 mg / kg of G-Exos@B, RAL@B, and GR-Exos@B prepared in Example 3 three times a week for in vivo experiments. Normally fed mice (NM) served as the control group. After 60 days of administration, insulin was injected intraperitoneally (ip) into the mice that had been fasting for 60 days. Blood glucose levels were continuously monitored for 120 minutes using a glucometer. The results are as follows... Figure 12 As shown, GR-Exos@B treatment resulted in a steady decrease in fasting blood glucose levels, while the fasting blood glucose levels of G-Exos@B and RAL@B were similar to those of the HFD group. GR-Exos@B treatment effectively normalized fasting blood glucose levels, indicating recovery of insulin resistance. Figure 12 (A) It also showed a low insulin resistance index in the GR-Exos@B group, such as Figure 12 In the B group, the fasting GLs of diabetic mice in the HFD group after 60 days were 1.56 times that of diabetic mice in the GR-Exos@B treatment group after 60 days. Furthermore, as... Figure 12 As shown in C, the blood glucose fluctuations in the treatment group gradually increased 2 hours after feeding, indicating that GR Exos@B treatment improved fasting GLs during the extended period of NASH drug treatment and showed significant long-term hypoglycemic effects.

[0143] Bloodstream C-peptide concentration was used as a marker to assess the secretory activity of insulin-producing β-cells. On the morning of the first day after the end of long-term treatment (60 days), blood was collected from the tail vein of mice, and serum C-peptide levels were measured using a kit. Compared with the high-fat diet (HFD) group, serum C-peptide concentrations were significantly increased in both the G-Exos@B and RAL@B intervention groups. The increase in C-peptide levels was greater in the G-Exos@B group than in the RAL@B group, suggesting that the exosome delivery system has superior efficacy in promoting C-peptide secretion. Notably, the C-peptide concentration (4.1 ng / mL) of GR-Exos@B, formed by the fusion of the two delivery systems, was significantly higher than that of the single delivery system groups, confirming that the biological effects of the fused drug delivery system were not simply additive, but rather produced a synergistic effect. Figure 13 ).

[0144] Mice were sacrificed after long-term treatment, and pancreas was collected. Immunohistochemical analysis (IHC) using insulin antibodies was performed to detect insulin expression. Results are as follows: Figure 14 As shown, the liver-targeting characteristics of RAL enhance the effects of berberine, thereby further stimulating insulin secretion. The most significant increase was observed in the β-cell region near this NM in GR-Exos@B. Figure 14 ).

[0145] To explore the in vivo mechanism of GR-Exos@B in treating diabetes, we investigated the protein levels involved in the gluconeogenesis pathway. Mouse liver extracts (i.e., livers from mice euthanized after treatment, after grinding and post-processing to obtain protein samples) prepared using Western bubbling and IP buffer were separated on a 10% gel by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified proteins were then transferred to a polyvinylidene fluoride (PVDF) membrane (Roche), blocked in 5% BSA solution, and incubated overnight with primary antibody for detection of the desired proteins. The proteins were then incubated with HRP-conjugated secondary antibody and analyzed using an ECL assay kit, normalized for GAPDH or β-tubulin.

[0146] The results are as follows Figure 15 As shown, Western blot analysis of GR-Exos@B revealed decreased expression of proteins associated with gluconeogenesis pathways (PGC-1α and PEPCK) and enhanced expression of insulin receptor (GLUT4) protein, indicating that ginger exosomes and UDCA-modified liposomes can synergistically enhance the promoting effect and enable insulin-sensitive tissues to respond rapidly to changes in blood glucose. Figure 15 ).

[0147] (3) GR-Exos@B reduces hepatic macrophage infiltration and regulates macrophage polarization.

[0148] Hepatic macrophages were briefly stained with an appropriate antibody for 30 minutes at room temperature and under light-limited conditions. The cells were then washed with PBS containing 2% FBS, followed by flow cytometry to determine the proportion of CD86+ cells indicating M1 macrophages and the proportion of CD206+ cells indicating M2 macrophages.

[0149] The phenotype of hepatic macrophages (CD86 and CD206) was assessed by flow cytometry to investigate the effects of berberine, RAL, G-Exos, GR-Exos, and GR-Exos@B (administered at a concentration of 100 μg / ml, incubated at 37°C for 4 h). After treatment, the percentage of M2 macrophages increased compared to M1 macrophages in each group. The expression level of the M2 macrophage marker CD206 was significantly increased in the GR-Exos@B treatment group compared to the control group. Furthermore, the downregulation of the M1 macrophage marker CD86 induced by GR-Exos@B treatment was most significant in the GR-Exos@B treatment group compared to the RAL, G-Exos, and GR-Exos groups, indicating that pro-inflammatory macrophages gradually transformed into anti-inflammatory macrophages. This transformation promoted hepatocyte recovery and reduced inflammation. Figure 16 ).

[0150] 250 μL of Westing and IP buffer was added to BMDM cells cultured in 6-well plates (culture procedure: berberine, RAL, G-Exos, GR-Exos, or GR-Exos@B at a concentration of 100 μg / ml, incubated at 37°C for 4 h). Cells were then scraped off and incubated at 4°C, 10000 rpm for 10 min. The supernatant was then added to cell lysis buffer, and the cells were incubated at 37°C on a shaker. Absorbance was measured at 562 nm, and the absorbance was calculated. Protein supernatant buffer was then added. The cell extracts were then subjected to SDS-PAGE, blocked, and incubated overnight with primary antibody, followed by incubation with secondary antibody. Results are as follows: Figure 17 As shown.

[0151] Effects of GR-Exos@B on NLRP3 expression: Compared with the M1 group, NLRP3 protein expression was significantly reduced in G-Exos, RAL, GR-Exos, and GR-Exos@B, while berberine protein expression was unaffected. Furthermore, GR-Exos@B showed the lowest NLRP3 expression level compared to other experimental groups, indicating that GR-Exos@B has the ability to alleviate inflammation by inhibiting NLRP3 signaling. Figure 17 ).

[0152] (4) Long-term toxicity in the body

[0153] Heart, liver, spleen, lung, intestine, pancreas, stomach, and kidney sections were taken from mice that had undergone the above-mentioned 60-day long-term drug administration. The results of organ hematoxylin and eosin (H&E) staining were recorded under a microscope. Normal mice (NM) served as the control group.

[0154] H&E staining analysis revealed no significant tissue damage or organ toxicity in the GR-Exos@B treatment group. Figure 18 The oral drug delivery system designed in this invention has no obvious toxic side effects, demonstrating its great potential as an oral drug delivery carrier.

[0155] The above embodiments are only used to illustrate the technical solutions of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any way. All technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

Claims

1. The application of an exosome-fused liver-targeting liposome drug delivery system in the preparation of drugs for treating non-alcoholic steatohepatitis and type II diabetes, characterized in that, The preparation process of the exosome-fused liver-targeting liposome drug delivery system is as follows: exosomes are fused with liver-targeting liposomes modified with bile acid derivatives to prepare empty nanoparticles; drugs are then encapsulated within the empty nanoparticles to prepare liver-targeting exosome drug-loaded nanoparticles. These liver-targeting exosome drug-loaded nanoparticles constitute the exosome-fused liver-targeting liposome drug delivery system; the drug is berberine hydrochloride. The preparation of the exosome-fused liver-targeting liposome drug delivery system includes the following steps: (1) Preparation of liver-targeting ligands: The bile acid derivative and reactants were placed in water, and DSPE-PEG was added. 2000 -NH2, react at 35-39℃ for 20-24h, purify, and obtain liver-targeting ligand; (2) Extraction of exosomes: Ginger juice was extracted, and ginger exosomes were obtained by low-temperature high-speed centrifugation and sucrose density gradient centrifugation. (3) Preparation of liver-targeting liposomes modified with bile acid derivatives: The lipid mixture consisting of emulsifier, liver-targeting ligand obtained in step (1) and cholesterol is dissolved in an organic reagent, and liver-targeting liposomes modified with bile acid derivatives are prepared by extrusion. (4) Preparation of empty nanoparticle solution: The exosomes obtained in step (2) were combined with the liver-targeting liposomes modified with bile acid derivatives obtained in step (3) and PEG. 8000 After mixing and co-incubation, the mixture was purified by density gradient centrifugation, containing PEG. 8000 The concentration was 60±3% w / v; this mixture was mixed with 75-85% w / v Nycodenz to form a Nycodenz solution of the first concentration containing the mixture. The Nycodenz solution of the first concentration, the Nycodenz solution of the second concentration, and PBS formed a gradient solution. The top separation layer was separated by ultracentrifugation to obtain purified empty nanoparticles; the Nycodenz concentration gradient decreased by 80%~15%; the first concentration was lower than 75-85% w / v; the second concentration was lower than the first concentration; (5) Preparation of ligand-modified drug-loaded nanoparticles: Equal volumes of drug solution and empty nanoparticle solution were mixed to obtain liver-targeting exosome drug-loaded nanoparticles; the concentration ratio of empty nanoparticle solution to drug solution was 1:1-3.

2. The application of the exosome fusion liver-targeting liposome drug delivery system according to claim 1 in the preparation of drugs for treating non-alcoholic steatohepatitis and type II diabetes, characterized in that, In step (1), the reactants are 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC˙HCl) and N-hydroxysuccinimide (NHS); reactants EDC˙HCl or NHS, bile acid derivatives, and DSPE-PEG. 2000 The molar ratio of -NH2 is 1-1.5: 1: 1-1.

5.

3. The application of the exosome fusion liver-targeting liposome drug delivery system according to claim 1 in the preparation of drugs for treating non-alcoholic steatohepatitis and type II diabetes, characterized in that, Step (2) is as follows: extract ginger juice, centrifuge at 800-1200 g for 5-10 min at 0-4℃, centrifuge at 2500-3500 g for 15-20 min, centrifuge at 9000-11000 g for 20-40 min; ultracentrifuge at 180,000-200,000 g for 1-2 h, resuspend, and transfer to sucrose solutions with different gradient concentrations for centrifugation, the sucrose gradient concentrations are 8%, 15%, 30%, 45% and 60% respectively; then centrifuge, collect the bands located between the 8% / 30% layer and the 30% / 45% layer, wash, centrifuge, resuspend, and obtain ginger exosomes.

4. The application of the exosome fusion liver-targeting liposome drug delivery system according to claim 1 in the preparation of drugs for treating non-alcoholic steatohepatitis and type II diabetes, characterized in that, In step (3), the emulsifier, liver-targeting ligand and cholesterol are mixed in a mass ratio of 1-5: 1-2:

2.

5. The application of the exosome fusion liver-targeting liposome drug delivery system according to claim 1 in the preparation of drugs for treating non-alcoholic steatohepatitis and type II diabetes, characterized in that, In step (4), exosomes, liver-targeting liposomes modified with bile acid derivatives, and PEG... 8000 The volume ratio is 1-2: 1: 1-2.

6. The application of the exosome fusion liver-targeting liposome drug delivery system according to claim 1 in the preparation of drugs for treating non-alcoholic steatohepatitis and type II diabetes, characterized in that, In step (5), the drug is encapsulated in the empty nanoparticles by ultrasound with ultrasound parameters of 55-70 kHz for 15-30 min.

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