WO2021147924A1 - Application of vesicle in preparing drug for treating or preventing liver disease - Google Patents

Abstract

Disclosed in the present application is an application of an induced extracellular vesicle in preparing a drug for treating or preventing a liver disease. The disease does not comprise thioacetamide-induced damaged liver fibrosis. The induced extracellular vesicle can target a liver, maintain liver homeostasis, promote tissue regeneration after partial hepatectomy (PHx), prevent acetaminophen (APAP)-induced acute liver failure, prevent non-alcoholic steatohepatitis, treat non-alcoholic fatty liver disease (NAFLD), and treat autoimmune hepatitis and liver fibrosis.

Description

Application of vesicle in preparation of medicine for treating or preventing liver disease Technical field

The present disclosure belongs to the field of biomedicine, and relates to the application of vesicles in the preparation of drugs for treating or preventing liver diseases.

Background technique

China is a big country with liver disease. The number of patients with end-stage liver disease caused by acute drug-induced liver failure, chronic fatty liver and hepatitis, fibrosis, etc., is newly increased by 5 million annually. It is also due to insufficient regeneration capacity of the diseased liver and limited healthy liver supply. It becomes a disease with high incidence and incurable disease. Therefore, studying new liver disease prevention methods and strategies for the regulation of liver homeostasis is a major medical and scientific problem that needs to be resolved urgently.

Summary of the invention

In some embodiments, the present disclosure provides the use of vesicles in the preparation of drugs for the treatment or prevention of liver diseases. The vesicles are induced vesicles.

In some embodiments, the present disclosure provides a composition comprising an inducible vesicle and a drug for treating or preventing liver disease.

In some embodiments, the present disclosure provides a method for treating or preventing liver disease in a subject, comprising administering to the subject an effective amount of a vesicle-containing drug or the composition, the vesicle It is an induced vesicle.

In some embodiments, the IEVs in the embodiments of the present disclosure are short for inducible vesicles, which can be called induced vesicles, or induced extracellular vesicles (IEVs).

During the research of the inventor, it was accidentally discovered that GalNAc glycoprotein is present on the surface of IEVs.

When detecting the distribution of IEVs in the organs/tissues of mice by using what is considered to be a relatively accurate method of labeling and detecting vesicle-like substances, the inventors found that IEVs exhibited an astonishing degree of enrichment in the liver. In some embodiments, 48 hours after the injection of IEVs, the total intake of IEVs in the liver reaches 25.7% ± 2.4% of the total injection, and the average intake per gram of liver tissue reaches 16.9% ± 1.6% of the average injection. ; Comparing bone, pancreas, teeth, muscle, brain and fatty organs/tissues, it is 61.0±14.2 times the total bone intake, 14.1±3.3 times the average intake, and 133.8±24.8 times the total pancreatic intake, the average 15.8±2.9 times the total amount of tooth intake, 145.7±11.5 times the total intake of teeth, 21.1±1.7 times the average intake, 272.5±51.0 times the total muscle intake, and 39.4±7.4 times the average intake, which is the total brain intake It is 108.1±16.1 times of the fat intake, 41.2±6.2 times of the average amount, 642.3±256.7 times of the total fat intake, and 54.9±22.0 times of the average amount.

The inventor speculates that the liver-targeting properties of induced vesicles (specifically the uptake of hepatocytes) may be related to the GalNAc on the surface. Hepatocytes express specific asialoglycoprotein receptor (ASGPR), which mediates the endocytosis of glycoprotein conjugates containing N-acetylgalactosaminyl (GalNAc) residues, and is its regulation An important way for the body's metabolism.

Fortunately, after further research, in some specific examples, the inventor found that IEVs have a good therapeutic effect on a variety of liver diseases. On the one hand, IEVs have the ability to treat the liver, and on the other hand, they are just abundantly enriched in the liver, making them particularly suitable for the treatment of liver-related diseases.

In some embodiments, the more accurate method for labeling and detecting vesicles is the ⁶⁴ Cu isotope labeling and tracking method.

In some embodiments, the present disclosure provides an application of a vesicle in the preparation of a drug for the treatment or prevention of liver diseases, the vesicle being an inducible vesicle, and the disease does not include thioacetamide-induced damage Liver fibrosis.

In some embodiments, the vesicles are used to prepare hepatoprotective or hepatoprotective drugs.

In some embodiments, the drug is used to maintain liver homeostasis.

In some embodiments, the drug is used to maintain the homeostasis of glucose metabolism or lipid metabolism of the liver.

In some embodiments, the drug is used to maintain the homeostasis of one or more levels of liver glycogen, liver lactic acid, blood glucose, blood lactic acid, liver triglycerides, liver cholesterol, blood triglycerides, and blood cholesterol.

In some embodiments, the drug is used to maintain structural homeostasis of liver tissue.

In some embodiments, the drug is used to maintain the homeostasis of the organelles or cytoskeleton of liver parenchymal cells.

In some embodiments, the medicament is used to promote liver regeneration.

In some embodiments, the drug is used to promote tissue regeneration after partial hepatectomy (PHx).

Partial hepatectomy is to remove the local lesions of the liver, including liver tumors, liver trauma, liver abscesses, intrahepatic bile duct stones, liver cysts, etc., using surgical techniques to perform liver segment, liver lobe and hemihepatectomy, and retain enough Normal liver tissue that maintains function. It is also used for liver donation during partial liver transplantation. A process of liver tissue regeneration occurs after partial hepatectomy.

In some embodiments, the medicament is used to prevent or treat liver failure.

In some embodiments, the liver failure includes acute liver failure or chronic liver failure.

In some embodiments, the medicament is used to prevent acute liver failure induced by acetaminophen.

In some embodiments, the medicament is used to treat liver damage.

In some embodiments, the medicament is used to treat fatty liver disease.

Fatty liver disease generally refers to fatty liver, fatty liver (fatty liver) refers to the pathological changes of excessive accumulation of fat in liver cells due to various reasons. It is a common liver pathological change, rather than an independent disease. Fatty liver disease is seriously threatening the health of Chinese people. It has become the second largest liver disease after viral hepatitis, and it is also a precursor disease of steatohepatitis. The incidence rate is increasing, and the age of onset is getting younger. Normal human liver tissue contains a small amount of fat, such as triglycerides, phospholipids, glycolipids, and cholesterol. Its weight is about 3% to 5% of the liver weight. If the accumulation of fat in the liver is too much, it will exceed 5% of the liver weight. Or when histologically more than 50% of liver cells have fatty degeneration, it can be called fatty liver. Effective treatment at the stage of fatty liver disease will help control the liver disease from progressing to the more serious stage of fatty liver disease.

Steatohepatitis is a progressive lesion of fatty liver disease. In addition to significant hepatocyte steatosis, it also has liver cell damage (such as balloon degeneration, vitreous corpuscles in the cytoplasm, etc.), inflammatory cell infiltration, liver blood Pathological features such as fibrous tissue hyperplasia and cholestasis around the sinuses. Once fatty liver disease progresses to steatohepatitis, the severity of liver pathology increases and is difficult to reverse or cure. It can also continue to progress, leading to liver fibrosis, liver cirrhosis, portal hypertension, liver cancer, and liver failure. It has become the first adult liver transplant reason.

In some embodiments, the fatty liver disease is non-alcoholic fatty liver disease (NAFLD).

Non-alcoholic fatty liver disease (NAFLD) refers to a clinicopathological syndrome characterized by excessive fat deposition in hepatocytes caused by alcohol and other clear liver damage factors, and is closely related to insulin resistance and genetic susceptibility. Metabolic stress liver injury is a kind of fatty liver disease, mainly including simple fatty liver (SFL). With the globalization of obesity and its related metabolic syndrome, non-alcoholic fatty liver disease has become an important cause of chronic liver disease in developed countries such as Europe and the United States and wealthy areas of my country. The prevalence of NAFLD in ordinary adults is 10%-30%. Among them, 10%-20% are NASH (Non-alcoholic steatohepatitis (NASH)), and the incidence of liver cirrhosis in the latter is as high as 25% within 10 years.

In some embodiments, the medicament is used to treat autoimmune hepatitis or autoimmune liver fibrosis.

In some embodiments, the drug is used to prevent non-alcoholic steatohepatitis.

In some embodiments, the source of the vesicles includes stem cells.

In some embodiments, the stem cells are mesenchymal stem cells.

In some embodiments, the cell may be a primary cultured cell, or an existing or established cell line.

In some embodiments, the cell line refers to an immortalized cell culture that can proliferate indefinitely in a suitable fresh medium and space.

In some embodiments, the cell may be an established cell strain.

In some embodiments, the inducible vesicle is a vesicle that is produced by external factors inducing apoptosis when the stem cell is in normal survival.

In some embodiments, the inducible vesicles are produced by inducing stem cell apoptosis, and the induction methods include, but are not limited to, staurosporine, ultraviolet irradiation, starvation, or thermal stress.

In some embodiments, the stem cells are mesenchymal stem cells.

In some embodiments, the source of the mesenchymal stem cells includes bone marrow, urine, oral cavity, fat, placenta, umbilical cord, periosteum, tendon, but is not limited thereto.

In some embodiments, the inducible vesicles are produced by adding staurosporine to induce apoptosis of mesenchymal stem cells.

In some embodiments, the concentration of the staurosporine is about 1 nM to 10000 nM. In some embodiments, the staurosporium

The concentration of element is about 100nM-10000nM. In some embodiments, the concentration of the staurosporine is about 500 nM-10000 nM.

In some embodiments, the concentration of the staurosporine may be about 500-1000 nM, or about 500-900 nM, or

To about 500-800nM.

In some embodiments, the surface of the inducible vesicle carries N-acetylgalactosaminyl (GalNAc).

In some embodiments, the surface of the inducible vesicle is enriched in N-acetylgalactosamine.

In some embodiments, the inducible vesicle has the marker Syntaxin 4. In some embodiments, the inducible vesicles highly express the marker Syntaxin 4. In some embodiments, the expression of the marker Syntaxin 4 by the inducible vesicles is higher than that of MSCs or exosomes. In some embodiments, the expression level of the marker Syntaxin 4 is 3-6 times the expression level of Syntaxin 4 in exosomes derived from mesenchymal stem cells. In some embodiments, the expression level of the marker Syntaxin 4 is 3.5-5 times the expression level of Syntaxin 4 in exosomes derived from mesenchymal stem cells. In some embodiments, the expression level of the marker Syntaxin 4 is 4.45 times the expression level of Syntaxin 4 in exosomes derived from mesenchymal stem cells. In some embodiments, the marker further includes one or more of Annexin V, Flotillin-1, Cadherin 11, and Integrin alpha 5. In some embodiments, the marker is a combination of Syntaxin4, Annexin V, Flotillin-1, Cadherin 11, and Integrin alpha 5. In some embodiments, the inducible vesicles highly express the markers Annexin V, Flotillin-1, Cadherin 11, and Integrin alpha 5. In some embodiments, the expression level of the markers Annexin V, Flotillin-1, Cadherin 11, and Integrin alpha 5 of the inducible vesicle is higher than that of MSCs or exosomes. In some embodiments, the expression level of the markers Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5 in the inducible vesicle is relative to the expression level of the marker in the exosomes derived from mesenchymal stem cells Respectively 1-2 times, 2-3 times, 1-3 times and 3-4 times. In some embodiments, the expression levels of the markers Annexin V, Flotillin-1, Cadherin 11, and Integrin alpha 5 in the inducible vesicles are 1.5-2 times, 2.5-3 times, 1.5-2.5 times and 3.5-fold, respectively. 4 times. In some embodiments, the expression levels of the markers Annexin V, Flotillin-1, Cadherin 11, and Integrin alpha 5 in the vesicle are 1.76 times, 2.81 times, 2.41 times, and 3.68 times, respectively.

The inducible vesicles described in the present disclosure are essentially different from exosomes. For example, compared with exosomes, the inducible vesicles IEVs described in the present disclosure highly express Syntaxin 4, as well as their Annexin V, Flotillin-1, The expression levels of Cadherin 11 and Integrin alpha 5 were significantly higher than those of exosomes (see Example 3). In addition to the differences in the markers, the inducible vesicle IEVs also exhibit characteristics that are different from stem cells and other extracellular vesicles (such as exosomes) in terms of function or therapeutic effect. For example, IEVs can significantly shorten the in vitro clotting time of most plasma, and the procoagulant effect is better than exosomes (see Test Example 4). For example, mesenchymal stem cells can treat damaged liver fibrosis, however, induced vesicles cannot achieve therapeutic effects on these diseases (see Test Example 1). For example, MSCs can treat Sjogren’s syndrome, but the inducible vesicles described in the present disclosure have no therapeutic effect on Sjogren’s syndrome (see Test Example 2). For example, the mechanism of IEVs in treating hemophilia mice has nothing to do with PS and TF. In previous literature reports, the procoagulant effects of extracellular vesicles are highly dependent on the PS and TF on their surface (see Test Example 3).

In some embodiments, the inducible vesicle also expresses CD29, CD44, CD73, CD166; and does not express CD34, CD45. In some embodiments, the inducible vesicle also expresses one or more of CD9, CD63, CD81, and C1q.

In some embodiments, the diameter of the inducible vesicle is about 0.03-10 μm. In some embodiments, the diameter of the inducible vesicle is about 0.03-6 μm. In some embodiments, the diameter of the inducible vesicle is about 0.03-4.5 μm.

In some embodiments, the method for preparing the inducible vesicle includes the steps: (1) culturing mesenchymal stem cells; (2) collecting the medium supernatant of the mesenchymal stem cells; (3) from step (2) The vesicles were isolated from the supernatant of the culture medium.

In some embodiments, the step of culturing mesenchymal stem cells in step (1) includes: (4) separating mesenchymal stem cells from tissues; (5) adding culture medium to culturing mesenchymal stem cells; The medium of mesenchymal stem cells is exposed to an apoptosis inducer.

In some embodiments, in the step (3), the method of separating the vesicles includes using an ultracentrifugation method to separate the vesicles.

In some embodiments, the step of separating the vesicles by the ultracentrifugation method includes: (a) subjecting the collected culture supernatant to a first centrifugation, and taking the supernatant; (b) performing step (a) The collected supernatant was centrifuged for the second time, and the supernatant was taken; (c) the supernatant received in step (b) was centrifuged for the third time, and the precipitate was taken; (d) the supernatant received in step (c) The pellet was centrifuged for the fourth time, and the pellet was taken.

In some embodiments, the first centrifugation is 500-1500g for 5-30 minutes; or the first centrifugation is 500-1000g for 5-20 minutes; or the first centrifugation is 500-900g Centrifuge for 5-15 minutes. In some embodiments, the second centrifugation is 1000-3000g centrifugation for 5-30 minutes; or the second centrifugation is 1500-2500g centrifugation for 5-20 minutes; or the second centrifugation is 1500-2200g Centrifuge for 5-15 minutes. In some embodiments, the third centrifugation is 10000-30000g centrifugation for 15-60 minutes; or the third centrifugation is 12000-25000g centrifugation for 20-60 minutes; or the third centrifugation is 12000-20000g centrifugation Centrifuge for 20-40 minutes. In some embodiments, the fourth centrifugation is 10000-30000g centrifugation for 15-60 minutes, or the fourth centrifugation is 12000-25000g centrifugation for 20-60 minutes; or the fourth centrifugation is 12000-20000g centrifugation. Centrifuge for 20-40 minutes.

In some embodiments, the generation number of the mesenchymal stem cells may be 2 to 5 generations, but it is not limited thereto.

In some embodiments, the mesenchymal stem cells are derived from mammals, but are not limited thereto.

In some embodiments, the mammal is selected from humans or mice, but is not limited thereto.

In some embodiments, the mesenchymal stem cells are derived from bone marrow.

In some embodiments, in the process of using the inducible vesicles for disease treatment, the inducible vesicles can be optionally selected from intravenous injection, intramuscular injection, subcutaneous injection, intrathecal injection or infusion. Injection and intra-organ infusion of the group consisting of route administration. For example, as an example, for intravenous injection, it can be injected through the tail vein. Intra-organ infusion includes infusion into an anatomical space, such as, for example, the gallbladder, gastrointestinal cavity, esophagus, pulmonary system (by inhalation), and/or bladder.

As an example, for intraperitoneal injection in gastrointestinal cavity infusion, compared with tail vein injection, intraperitoneal injection can also obtain the same good therapeutic effect. The safety and operability of intraperitoneal injection are better than those of tail vein injection.

Description of the drawings

Figures 1A-1E show the flow cytometric detection results of the surface markers of isolated BMMSCs.

Fig. 2 is a flowchart of the operation of the second embodiment.

3 is the number of MSCs IEVs statistical results analyzed by flow cytometry (10 ⁶ MSCs) produced.

Figure 4A-4F shows the diameter detection of IEVs particles: Figure 4A shows the particle diameter distribution of IEVs by flow detection; Figure 4B shows the scattered light intensity of IEVs analyzed by side scattered light (SSC), showing the particle diameter distribution of IEVs; Figure 4C To analyze the scattered light intensity of IEVs with standardized small particle microspheres produced by Bangs Laboratories, showing the particle diameter distribution of IEVs; Figure 4D shows the IEVs observed by transmission electron microscopy (TEM), showing the particle diameter distribution of IEVs; Figure 4E shows the particle diameter distribution of IEVs. Tracking analysis (NTA), showing the particle diameter distribution of IEVs; Figure 4F shows the particle diameter detection of IEVs at the single vesicle level using nanoflow detection technology, showing the particle diameter distribution of IEVs.

Figures 5A-5K show the analysis results of surface membrane proteins of IEVs by flow cytometry.

Figure 6A-Figure 6G are the content analysis of IEVs: Figure 6A is the proteomic quantitative analysis results of MSCs, MSCs-Exosomes, and MSCs-IEVs by DIA quantitative technology; Figure 6B is a heat map drawn by screening IEVs-specific and highly expressed proteins Figure 6C is the GO enrichment analysis of differential proteins and the results of IEVs expressing Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5 and Syntaxin 4 molecules; Figure 6D is the western blot verifying that MSCs, MSCs-Exosomes, and MSCs-IEVs express Annexin The results of V, Flotillin-1, Cadherin 11, Integrin alpha 5 and Syntaxin 4; Figure 6E shows the specific lectin of Gal and GalNAc coupled with FITC fluorescent substance to stain IEVs, and perform fluorescence picture observation and flow analysis after staining The results showed that 61.8% of IEVs had Gal on the surface; Fig. 6F is the specific lectin of Gal and GalNAc coupled with FITC fluorescent substance to stain IEVs. After staining, fluorescence picture observation and flow cytometry analysis showed that up to 93.3% The surface of IEVs with GalNAc; Figure 6G shows the membrane components of IEVs, exosomes (Exos) and MSCs are labeled with CellMask membrane dyes, and GalNAc coupled with FITC fluorescent substance is stained with specific lectin, and the fluorescence detector is used to read the results. , The GalNAc fluorescence intensity of the unit membrane surface of IEVs was significantly higher than that of Exos and MSCs.

Figure 7A-D shows the enrichment in the liver of mice after injection of MSCs-derived IEVs: A. Copper 64 isotope-labeled IEVs, traced in vivo in mice after intravenous infusion; B. Total radiation intensity of each tissue and organ 48 hours after injection ; C. Detection of the average radiation intensity of each tissue and organ 48 hours after injection; D. Detection of the difference in uptake of IEVs and exosomes by cells, in which IEVs are mainly taken up by hepatocytes rather than hepatic macrophages.

Figure 8 shows that IEVs maintain the steady state of liver glucose and lipid metabolism: A. Liver glycogen content detection; B. Liver lactate content detection; C. Blood glucose level detection; D. Blood lactic acid level detection; E. Liver triglyceride content detection F. Liver cholesterol content test; G. Blood triglyceride level test; H. Blood cholesterol level test; WT, wild-type C57 mice; Fas ^mut , apoptosis-deficient mice with Fas gene mutations, producing EVs (normal cells) Outer vesicles) lack; mice are 8 weeks old, and the IEVs are taken 1 week after injection.

Figure 9 shows that IEVs maintain the homeostasis of liver tissue structure: A. HE staining and F-actin immunofluorescence staining; B. Hepatic parenchymal cell nucleus ratio measurement; C. Hepatic parenchymal cell chromatin content measured by flow cytometry; Mouse age At 8 weeks, IEVs were collected 1 week after injection.

Figure 10 shows that IEVs maintain the organelle and cytoskeleton homeostasis of hepatic parenchymal cells: A. IEVs co-stained with Golgi; B. IEVs co-stained with microtubule cytoskeleton; hepatic parenchymal cells were cultured in vitro and treated with IEVs for 72 h.

Figure 11 shows that IEVs promote tissue regeneration after partial hepatectomy (PHx): A. Gross map of the intact liver; B. After resection of the left and middle lobes of the liver (70% PHx); C. Normal liver regeneration for 72 hours; D. Liver regeneration 72h after IEVs injection; E. Statistics of the proportion of liver weight to body weight; mice aged 8 weeks, IEVs were injected 24h before PHx surgery.

Figure 12 shows the prevention of acute liver failure induced by Acetaminophen (APAP) by IEVs: A.1g/kg APAP survival rate test after intraperitoneal injection; NAC, antioxidant, injection 24h before APAP injection; IEVs preventive injection in APAP injection The first 24h; IEVs were injected 6h after APAP injection; B. HE staining and serum liver injury index detection (alanine aminotransferase, aspartate aminotransferase); IEVs were preventive injections.

Figure 13 shows the treatment of non-alcoholic fatty liver disease (NAFLD) with IEVs; A. Periodic acid-Schiff (PAS) reaction shows liver glycogen staining; B. Oil red staining shows fatty liver; HFD , High-fat diet; mice are fed HFD from 6 weeks of age, IEVs are injected once at 10 weeks of age, and materials are taken at 14 weeks of age.

Figure 14 shows the treatment of autoimmune hepatitis and liver fibrosis with IEVs: A. HE staining shows lymphocyte infiltration; B. Masson's staining shows collagen fiber deposition; Casp3 ^+/- , Caspase 3 gene heterozygous deficient mice, showing autoimmunity Symptoms: The mice were 8 weeks old, and the IEVs were taken 1 month after injection.

Figures 15A-E are data of MSCs-derived IEVs to prevent AMLN feed (high trans fat, high fructose, high cholesterol feed) fed steatohepatitis (NASH) data. Mouse body type and weight statistics; B. Liver histopathological section; C. Serum liver injury index detection (alanine aminotransferase, aspartate aminotransferase); D. Serum and liver triglyceride level detection ; E. Serum and liver cholesterol levels detection. Mice were fed AMLN from 6 weeks of age, IEVs were injected once at 10 weeks of age, and materials were taken at 32 weeks of age.

Figure 16 shows the data of MSCs-derived IEVs for treatment of ammonium thioacetate-induced traumatic liver fibrosis. A. Histopathological section of liver; B. Fatty liver disease score.

Figure 17 shows the treatment of Sjogren’s syndrome with IEVs: A. The effect of IEVs on the saliva flow rate of Sjogren’s syndrome (Sjogren’s syndrome); B. The results of HE staining of submandibular glands in the treatment of Sjogren’s syndrome by IEVs; C. Treatment of Sjogren’s syndrome vs. B The influence of cells.

Figure 18 shows the procoagulant effects of IEVs in hemophilia A mice.

Figures 19A-19D show the changes in the levels of various coagulation factors after injection of IEVs into hemophilia A mice: Figure 19A shows the changes in coagulation factor VIII; Figure 19B shows the changes in vWF factor; Figure 19C shows the changes in tissue factor ( TF) changes; Figure 19D shows the changes of prothrombin.

Figures 20A-20B show the effects of PS and TF on the in vivo therapeutic effects of IEVs in the hemophilia A mouse model.

Figure 21 is a comparison of the therapeutic effects of IEVs and Exosomes derived from the same MSCs on hemophilia A mice.

Note: In the drawings, WT is a wild-type mouse; HA group is a hemophilia A mouse model; HA+IEVs is a hemophilia A mouse model given IEVs treatment; HA+PS-IEVs is hemophilia A mouse model is given PS negative IEVs; HA+TF-IEVs is a hemophilia A mouse model given TF-negative IEVs; HA+Exosomes is a hemophilia A mouse model given Exosomes treatment.

Figures 22A-22C show that IEVs can be excreted through the skin and hair: Figure 22A is a schematic diagram of the dynamic metabolism of IEVs on the skin surface. Figure 22B shows that over time, IEVs gradually move from the subcutaneous tissue to the dermis and epidermis. Figure 22C shows that PKH26-IEVs were found in the hair follicles in the plucked hair from the mouse body on day 7.

Detailed ways

The technical solutions of the present disclosure are further described below through specific examples, and the specific examples do not represent a limitation on the protection scope of the present disclosure. Some non-essential modifications and adjustments made by others based on the concept of this disclosure still fall within the protection scope of this disclosure.

IEVs in the embodiments of the present disclosure are short for inducible vesicles, which can be called induced vesicles, or induced extracellular vesicles (IEVs), and IEVs are the same as IEVs. Inducible extracellular vesicles refer to a type of subcellular product produced when precursor cells (such as stem cells) survive normally and are interfered or induced to cause apoptosis. Usually this type of subcellular product has a membrane structure, expresses apoptotic markers, and partly contains genetic material DNA. The inventor found that inducible extracellular vesicles are a class of substances distinguished from cells and conventional extracellular vesicles (such as exosomes, etc.). In some embodiments, the cells that survive normally are, for example, non-apoptotic cells, non-senescent cells, non-aging cells that have stagnated proliferation, cells that are not recovered after cryopreservation, or abnormally proliferating without malignant transformation. Cells or non-damaged cells, etc. In some embodiments, the normally viable cells are taken from cells that are in contact with 80-100% of the fusion during the cell culture process. In some embodiments, the cells that survive normally are taken from cells in the log phase. In some embodiments, the cells that survive normally are obtained from primary culture and subculture cells derived from human or murine tissue. In some embodiments, the cells that survive normally are taken from established cell lines or cell lines. In some embodiments, the precursor cells are taken from early cells.

Normal extracellular vesicles (EVs) refer to nano-sized small bodies with membrane structures that are spontaneously secreted by cells during normal culture or under physiological conditions in vivo, with diameters ranging from 40-1000nm, mainly composed of microvesicles ( Microvesicles (MV) and exosomes (Exosomes), containing a variety of RNA, protein and other signal molecules.

The STS in the present disclosure is staurosporine. In this disclosure, Exosomes refers to exosomes.

BMMSCs (Bone marrow mesenchymal stem cells) refer to bone marrow mesenchymal stem cells.

"Comprising" or "including" is intended to mean that the composition (e.g., medium) and method include the listed elements, but do not exclude other elements. When used to define compositions and methods, "consisting essentially of" means excluding other elements that have any significance for the combination for the stated purpose. Therefore, a composition consisting essentially of the elements defined herein does not exclude other materials or steps that do not materially affect the basic and novel features of the claimed disclosure. "Consisting of" refers to the elimination of trace elements and substantial method steps of other components. The embodiments defined by each of these transition terms are within the scope of this disclosure.

As used herein, the terms "high expression" and the like are intended to include increasing the expression of nucleic acid or protein to a level higher than the level contained in existing vesicles (for example, exosomes).

In the present disclosure, the components in the "composition" may exist in a mixed form, or they may be packaged separately. Separately packaged components may also contain their respective adjuvants. The adjuvant refers to a means that can assist the curative effect of drugs in pharmacy. When the components in the composition are packaged separately, the separately packaged components can be administered simultaneously or in any order, in which the patient is treated with one drug first, and then another drug is administered . The said patient refers to a mammalian subject, especially a human being.

In the present disclosure, the "composition" may also exist in a form in which one component is wrapped by another component. In some embodiments, in the composition, the inducible vesicle is used as a drug carrier, and drugs for treating or preventing liver diseases are encapsulated in the inducible vesicle, and the inducible vesicle has liver targeting. Therefore, the drug contained in it can be presented to the liver, so as to achieve the effect of curing liver diseases.

In the present disclosure, an "effective amount" is an amount sufficient to achieve beneficial or desired results, such as enhanced immune response, treatment, prevention or improvement of medical conditions (disease, infection, etc.). The effective amount can be administered in one or more administrations, applications, or doses. The appropriate dosage will vary depending on body weight, age, health, disease or condition to be treated, and route of administration.

In the present disclosure, the corresponding reagent sources are as follows: penicillin/streptomycin solution (BIOSOURCE; P303-100); glutamine (BIOSOURCE; P300-100); dexamethasone sodium phosphate (Sigma; D-8893); α- MEM (Gibco; 12571-063); 2-ME (GIBCO; 21985-023).

In the examples herein, ⁶⁴ Cu isotope labels are used to track the biological distribution of IEVs, specifically: the collected IEVs are combined with NOTA chelating agent under the action of HEPES buffer, and ⁶⁴ CuCl _{2 is} added to react at room temperature for 1 hour, and the free isotope and isotopes are removed by centrifugal chromatography. Chelating agent. The labeling efficiency was detected by transient thin-layer chromatography in a citric acid solution environment, and then injected into mice via the tail vein. After injection, PET was used to track the biological distribution of IEVs in real time. After the mice were sacrificed to separate multiple tissues, a portable gamma counter was used to detect the radiation intensity. The traditional labeling methods for MSCs and EVs mainly use membrane fluorescent dyes such as PKH, but when this fluorescent dye is inserted into the membrane, it will damage the membrane lipids, and the staining is easy to contaminate and quench. The sensitivity and specificity for tracking in vivo Sex needs to be strengthened. The principle of the isotope labeling method is based on the chemical reaction of the amino groups of the protein on the surface of the membrane, and the tracking method based on the detection of radiation intensity, combined with non-toxicity, non-contaminated leakage, and higher detection accuracy and sensitivity. However, the previous isotope labeling was mainly done successfully in Liposome because it is convenient to add chelating agents such as NOTA and other metal isotopes during the synthesis of Liposome, but no effective method has been established for biologically active vesicles such as EVs.

In this article, F4/80 is a mature mouse macrophage marker mouse EGF-like module-containing mucin-like hormone receptor-ike 1 (mouse containing growth factor-like motif mucin-like hormone receptor-ike 1), which is A cell surface glycoprotein, a member of the EGFTM7 protein family, has 68% amino acid identity with human EMR1 protein. Studies have found that during the maturation and activation of macrophages, the expression of F4/80 protein changes significantly. F4/80 protein is expressed in a variety of mature macrophages, such as Kupffer cells, Langerhans cells, microglia cells and located in the peritoneum, intestinal lamina propria, spleen red pulp, lung, chest trace, bone marrow stroma In the macrophages.

Example 1 Isolation and culture of MSCs

_{The mice were killed with excessive CO 2} according to the guidance of the animal ethics committee. Under aseptic conditions, the tibia and femur were removed, and the muscle and connective tissue attached to it were peeled off. The metaphysis was further separated and the bone marrow cavity was exposed. 10 mL was used. A sterile syringe draws PBS containing 10% fetal calf serum to repeatedly wash the bone marrow cavity. After filtering with a 70μm pore size cell strainer, centrifuge at 500g for 5min. After removing the supernatant, collect the cell pellet at the bottom, resuspend in PBS, and centrifuge again at 500g. 5min, collect the final cell pellet. Then the cells were sorted by flow cytometry, and BMMSCs were sorted using CD34- and CD90+ as sorting criteria. Finally, the cells were resuspended in Dex(-) medium and seeded in a 10 cm diameter cell culture dish, incubated at 37°C and 5% CO ₂ . After 24 hours, the supernatant non-adherent cells were aspirated, washed with PBS, and Dex(-) medium was added to continue the culture. One week later, the same amount of Dex(+) culture medium was added, and one week later, dense primary BMMSCs colonies were seen. The BMMSCs were digested with trypsin at 37°C and subcultured and expanded. Then, the Dex(+) medium was changed every 3 days, and then subcultured after full growth. Use P2 generation BMMSCs for follow-up experiments.

Among them, the composition of Dex(-) culture solution is shown in Table 1, and the composition of Dex(+) culture solution is shown in Table 2:

Table 1 Dex(-) medium composition table

Table 2 Formulation of Dex(+) culture solution

Flow cytometry was used to analyze the surface markers to evaluate the purity of the isolated BMMSCs. For surface marker identification, after trypsin digestion to collect P2 generation BMMSCs, wash once with PBS, ^{resuspend the cells in 3% FBS-containing PBS at a density of 5×10 5} /mL, and add 1 μL of PE fluorescence-conjugated CD29, CD44, CD90, CD45 and CD34 antibodies were not added to the blank group. Incubate at 4°C in the dark for 30 minutes, wash with PBS twice, and then test on the machine. The results of flow cytometry are shown in Figures 1A-1E. It can be seen that the isolated cells are BMMSCs (Bone Marrow Mesenchymal Stem Cells).

Example 2 Obtaining Inducible Vesicles (IEVs)

The MSCs (MSCs derived from bone marrow) cultured in Example 1 to the second passage were cultured with the medium in Example 1 (Dex(+) culture medium) until the cells were 80%-90% confluent, then rinsed with PBS Two times, 500nM STS-containing serum-free medium (α-MEM medium) was added to induce apoptosis, incubated at 37°C for 24 hours, and the cell supernatant was collected for separation and extraction of IEVs.

IEVs are separated and extracted from the collected culture supernatant. The operation process is shown in Figure 2. The specific steps include: 800g centrifugation for 10 minutes—collecting the supernatant—2000g centrifugation for 10 minutes—collecting the supernatant—16000g centrifugation for 30 Minutes—Remove the supernatant and resuspend the IEVs in sterile PBS—centrifuge at 16000g for 30 minutes—Remove the supernatant and resuspend the IEVs in 300-500ul sterile PBS.

Comparative Example 1 Isolation and extraction of exosomes derived from the same MSCs

MSCs (MSCs derived from bone marrow) cultured in Example 1 to the second generation were cultured with the medium in Example 1 until the cells were 80%-90% confluent, washed twice with PBS, and added serum-free medium Incubate at 37°C for 48h, and collect the cell supernatant for separation and extraction of Exosomes.

The extraction steps include: 800g centrifugation for 10 minutes-collection of supernatant liquid-2000g centrifugation for 10 minutes-collection of supernatant liquid-16000g centrifugation for 30 minutes-collection of supernatant liquid-120,000g centrifugation for 90 minutes-remove the supernatant, and resuspend in sterile PBS Precipitation—Centrifuge again at 120,000g for 90 minutes, remove the supernatant, collect the Exosomes at the bottom, and resuspend in sterile PBS.

Example 3 Analysis of IEVs

(1) Quantification of IEVs and analysis of membrane proteins

Flow cytometry of IEVs obtained in Example 2 was quantitatively analyzed, IH measurement time point for the first, second 4h, the first 8h, and 16h of the 24h, 10 ⁶ The results show the induction of MSCs through IH, first 4h, After 8h, 16h and 24h, 0.76×10 ⁸ , 1.29×10 ⁸ , 1.95×10 ⁸ , 2.48×10 ⁸ , 3.14×10 ⁸ IEVs can be produced respectively. It can be seen from this that induced After 24h, a single MSCs can produce 300 IEVs (Figure 3).

In addition, flow cytometry found that the particle diameter distribution of IEVs were all concentrated below 1 μm, accounting for 94.97% (Figure 4A).

The side scattered light (SSC) analysis results also show that the scattered light intensity of IEVs is concentrated in the range below 1 μm (Figure 4B).

Furthermore, the scattered light intensity of IEVs was analyzed by standardized small particle microspheres (0.2 μm, 0.5 μm, 1 μm) produced by Bangs Laboratories, and the results showed that the particle diameters of IEVs were all below 0.2 μm (Figure 4C).

The results of transmission electron microscopy (TEM) observation are similar to the results of flow cytometry, and most of the vesicles are 200nm and below 200nm in diameter (Figure 4D).

The results of Nanoparticle Tracking Analysis (NTA) are consistent with the results of transmission electron microscopy, and the average particle diameter of IEVs is 169nm (Figure 4E).

The state-of-the-art nanoflow detection technology was used for particle size detection at the single vesicle level, and the results also showed that the average particle diameter of IEVs was 100.63 nm (Figure 4F).

The surface membrane proteins of the IEVs extracted in Example 3 were analyzed by flow cytometry. The results showed that IEVs derived from MSCs can express surface proteins similar to MSCs, namely CD29, CD44, CD73, CD166 positive, CD34, CD45 negative. At the same time, IEVs can express the universal surface proteins CD9, CD63, CD81 and C1q of extracellular vesicles (Figure 5A-5K).

(2) Content analysis of IEVs

Quantitative proteomic analysis of MSCs, MSCs-Exosomes (extracted in Comparative Example 1), and MSCs-IEVs (obtained in Example 2) was completed using protein DIA quantitative technology. The results showed that the protein content expression of MSCs-Exosomes and MSCs-IEVs had a high overlap with that of parent cells, and 170 proteins were specifically and highly expressed in IEVs (Figure 6A). Through bioinformatics analysis, IEVs were screened for specific and highly expressed proteins, and a heat map was drawn (Figure 6B), and further combined with the GO enrichment analysis results of differential proteins, it is clear that IEVs can specifically and highly express Annexin V, Flotillin-1, and Cadherin 11 , Integrin alpha 5 and Syntaxin 4 molecules (Figure 6C). Compared with Exosomes derived from the same MSCs, the expression levels of the five characteristic molecules of IEVs are significantly up-regulated, specifically: the markers Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5 and Syntaxin 4 in IEVs The expression levels of the corresponding markers in Exosomes were 1.76 times, 2.81 times, 2.41 times, 3.68 times and 4.45 times, respectively. Finally, the western blot technology was used for verification again, and the results were consistent with the quantitative analysis results of DIA (Figure 6D).

MSCs-Exosomes: Refers to exosomes derived from MSCs.

MSCs-IEVs: Refers to IEVs derived from MSCs.

The MSCs used in the content analysis and the MSCs extracted from Exosomes and IEVs are the same cell line.

(3) Analysis of sugar-based substances on the surface of IEVs

The IEVs obtained in Example 2 were stained with specific lectins of galactose (Gal) and N-acetylgalactosamine (GalNAc) coupled with FITC fluorescent substances. After staining, fluorescence picture observation and flow analysis were performed, and the results showed 61.8% of IEVs have Gal on the surface (Figure 6E), and up to 93.3% of IEVs have GalNAc on the surface (Figure 6F).

Furthermore, the membrane components of IEVs, exosomes (Exos) and MSCs are labeled with CellMask membrane dyes, and specific lectin staining of Gal and GalNAc coupled with FITC fluorescent substances is performed. Using a fluorescence detector to read, it was found that the GalNAc fluorescence intensity on the surface of the unit membrane of IEVs was significantly higher than that of Exos and MSCs (Figure 6G).

Example 4 Enrichment of IEVs in vivo

For every 20 g mouse body weight, ^{IEVs derived from 1×10 7} MSCs (prepared in Example 2) were resuspended in 200 μL PBS+20 μL heparin sodium solution (0.2% w/v). Mix well, place on ice, and complete the injection via tail vein within 30 minutes. Then the application analysis was performed on the mice injected with IEVs.

(1) Detection steps or methods: IEVs are prepared and combined with NOTA chelating agent, ^{labeled with the radioisotope 64} CuCl ₂ and injected into mice via the tail vein. Positron emission tomography (PET) monitors the presence of isotope-labeled IEVs in real time. Distribution in the body, after 48 hours of injection, the tissues were separated for radiation dose reading and testing. IEVs were prepared with PKH26-labeled membranes, and the specimens were taken 72 hours after tail vein injection. Liver tissues were subjected to frozen section and immunofluorescence staining to detect the co-localization of hepatocytes, hepatic macrophages and IEVs.

(2) Results: As shown in Figures 7A-7D and Table 3, the results show that the IEVs derived from the MSCs are significantly enriched in liver tissue. Among them, IEVs are mainly taken up by hepatocytes, while exosomes can also migrate to the liver but are mainly taken up by hepatic macrophages. Among them, the enrichment of stem cells after injection: After intravenous injection, it first enters the pulmonary circulation through the heart, a large amount of blockage in the capillaries of the lungs, and very little entry into the liver tissue (Lee et al., Cell Stem Cell, 2009). The condition of exosomes being enriched in the liver is similar to that of IEVs, but it is speculated that because of its low expression of GalNAc on the surface, it is engulfed and lysed by hepatic macrophages instead of being mainly taken up by hepatocytes to play a biological role.

Table 3 Enrichment of IEVs in vivo

Example 5 Research on IEVs to maintain liver homeostasis

For every 20 g mouse body weight, ^{IEVs derived from 1×10 7} MSCs (prepared in Example 2) were resuspended in 200 μL PBS+20 μL heparin sodium solution (0.2% w/v). Mix well, place on ice, and complete the injection via tail vein within 30 minutes. Then the application analysis was performed on the mice injected with IEVs.

1. IEVs maintain the homeostasis of liver glucose and lipid metabolism

(1) Detection steps or methods: Take 8-week-old wild-type (WT) and Fas gene mutations (Fas ^mut ) induced endogenous IEVs secretion defects mice injected with IEVs via the tail vein, once a week, after the third injection 7 Day (total time is 1 month), collect liver tissue and grind with 100mg tissue: 900mL PBS, centrifuge to extract tissue supernatant; separate and collect serum. A kit was used to detect liver glycogen, lactic acid, triglyceride and cholesterol content, as well as blood sugar, lactic acid, triglyceride and cholesterol levels.

(2) Results: As shown in Figure 8, the results show that compared with WT mice, Fas ^mut mice's glucose metabolism indicators (sugar and lactic acid content) are significantly reduced, while lipid metabolism indicators (triglycerides, cholesterol) are significantly increased. The trends in liver and blood are the same. IEVs injection can significantly restore the ^{balance of glucose and lipid metabolism in Fas mut} mice, but it has no effect on normal WT mice, showing a maintenance effect on homeostasis.

2. IEVs maintain the homeostasis of liver tissue structure

(1) Detection steps or methods: 8 weeks old WT and Fas ^mut mice were injected with IEVs via the tail vein, once a week, 7 days after the third injection (total time: 1 month). Collect liver tissues, dehydrated by gradient ethanol, embed in paraffin, and observe the tissue morphology by HE staining; frozen section after sucrose treatment, F-actin staining to mark the outline of liver tissue, combined with DAPI to label nuclear DNA, and count the ratio of monocytes to binuclear cells ; Separate liver cells for propidium iodide (PI) staining, flow cytometric analysis of the ratio of chromatin diploid (2N), tetraploid (4N), and octoploid (8N).

(2) Results: As shown in Figure 9, the results show that Fas ^mut mice with defective endogenous IEVs secretion have disordered liver tissue structure, and the ratio of binuclear and 4N hepatocytes is significantly increased. IEVs injection can significantly restore liver tissue structure and ploidy Steady state.

3. IEVs maintain the organelles and cytoskeleton homeostasis of liver parenchymal cells

(1) Detection steps or methods: Isolate and culture mouse hepatocytes via portal vein reverse perfusion. Hepatocytes were derived from 8-week-old WT and Fas ^mut mice. ^{The hepatocytes from Fas mut} mice were treated with IEVs in vitro, and the dosage was ^{2×10 5} hepatocytes were treated for 1×10 ⁶ MSCs-derived IEVs, and the treatment time was 3 days. At the end of the treatment, hepatocytes were fixed with paraformaldehyde, Golgi and microtubule markers were stained by immunofluorescence, and binuclear hepatocytes were selected for laser confocal microscope observation.

(2) Results: As shown in Figure 10, the results show that the dinuclear hepatocytes of Fas ^mut mice with defects in endogenous IEVs secretion have discrete Golgi and perinuclear microtubule cytoskeleton cavities. IEVs treatment can help the assembly and reconstruction of Golgi organelles. Induces perinuclear microtubule remodeling and cell division mediated by it.

Example 6 IEVs promote tissue regeneration after partial hepatectomy (PHx)

For every 20 g mouse body weight, ^{IEVs derived from 1×10 7} MSCs (prepared in Example 2) were resuspended in 200 μL PBS+20 μL heparin sodium solution (0.2% w/v). Mix well, place on ice, and complete the injection via tail vein within 30 minutes. Then the application analysis was performed on the mice injected with IEVs.

1. Detection steps or methods: select 8 weeks old WT mice, 24 hours after IEVs tail vein infusion, open the abdominal cavity under isoflurane anesthesia, and perform ligation and resection of the middle and left lobe of the liver in sequence (the weight of the removed tissue is the whole liver 70%), suture the wound, and observe the liver regeneration after 72 hours.

2. Results: As shown in Figure 11, the results show that compared with normal livers, only the right lobe and tail lobe are left after 70% PHx operation. IEVs injection significantly promotes liver tissue regeneration 72h after hepatectomy in mice.

Example 7 IEVs prevent acute liver failure induced by acetaminophen (APAP)

For every 20 g mouse body weight, ^{IEVs derived from 1×10 7} MSCs (prepared in Example 2) were resuspended in 200 μL PBS+20 μL heparin sodium solution (0.2% w/v). Mix well, place on ice, and complete the injection via tail vein within 30 minutes. Then the application analysis was performed on the mice injected with IEVs.

1. Detection steps or methods: select 8 week-old WT mice, and intraperitoneally inject APAP at a dose of 1 g/kg. Antioxidant NAC injection dose is 1200mg/kg, preventive infusion of IEVs is 24h before APAP injection, and therapeutic infusion of IEVs is 12h after APAP injection. The lethality rate was observed and statistically observed in the experiment. The samples were taken 24 hours after APAP injection, the liver tissue was dehydrated with gradient ethanol, embedded in paraffin, and stained with HE; the serum was separated, and the kit was used to detect alanine aminotransferase (ALT) and aspartate aminotransferase (AST) Concentration.

2. Results: As shown in Figure 12, the results show that APAP injection induces fatal acute liver failure, the antioxidant NAC cannot save it, while prophylactic injection of IEVs can maintain the life of mice, and therapeutic injection of IEVs can prolong the survival of mice. Staining of liver tissue sections and analysis of serum transaminase concentration confirmed that IEVs can significantly maintain liver tissue integrity and reduce liver tissue damage.

Example 8 IEVs treatment of non-alcoholic fatty liver disease (Non-alcoholic fatty liver disease, NAFLD)

For every 20 g mouse body weight, ^{IEVs derived from 1×10 7} MSCs (prepared in Example 2) were resuspended in 200 μL PBS+20 μL heparin sodium solution (0.2% w/v). Mix well, place on ice, and complete the injection via tail vein within 30 minutes. Then the application analysis was performed on the mice injected with IEVs.

1. Detection steps or methods: select 6-week-old WT male mice to feed high-fat diet (HFD) for 8 weeks. IEVs were injected via the tail vein at the 4th week of feeding. After the experiment, samples were taken. Paraffin sections of liver tissue were stained with periodic acid-Schiff (PAS) to show liver glycogen, and frozen sections were stained with oil red to show liver fat.

2. Results: As shown in Figure 13, the results show that HFD significantly induces liver glycogen and fat accumulation, showing typical symptoms of NAFLD. IEVs injection can significantly relieve NAFLD.

Example 9 Treatment of autoimmune hepatitis and liver fibrosis with IEVs

For every 20 g mouse body weight, ^{IEVs derived from 1×10 7} MSCs (prepared in Example 2) were resuspended in 200 μL PBS+20 μL heparin sodium solution (0.2% w/v). Mix well, place on ice, and complete the injection via tail vein within 30 minutes. Then the application analysis was performed on the mice injected with IEVs.

1. Detection steps or methods: Take 8-week-old WT and Caspase 3 heterozygous (Casp3 ^+/- ) mice, in which Casp3 ^+/- mice receive IEVs injection, once a week, 3 times in total, after the last injection Samples were taken on the 7th, and paraffin sections of liver tissue were stained with HE and Masson's.

2. Results: As shown in Figure 14, the results show that Casp3 ^+/- mice have massive infiltration of inflammatory lymphocytes in the liver tissue and fibrous collagen deposition around the liver sinusoids. IEVs injection can significantly alleviate the symptoms of hepatitis and liver fibrosis.

Example 10 IEVs prevent non-alcoholic steatohepatitis (Non-alcoholic steatohepatitis, NASH)

For every 20 g mouse body weight, ^{IEVs derived from 1×10 7} MSCs (prepared in Example 2) were resuspended in 200 μL PBS+20 μL heparin sodium solution (0.2% w/v). Mix well, place on ice, and complete the injection via tail vein within 30 minutes. Then the application analysis was performed on the mice injected with IEVs.

1. Detection steps or methods: select 6-week-old WT male mice to feed AMLN feed (high-trans fat, high-fructose, high-cholesterol feed) for a period of 26 weeks. IEVs were injected via the tail vein at the 4th week of feeding. Before the end of the experiment, the mice were photographed and weighed under anesthesia; the liver tissue was dehydrated with gradient ethanol, embedded in paraffin, and stained with HE; the serum was separated, and the kit was used to detect alanine aminotransferase (ALT) and aspartate amino transfer Enzyme (AST) concentration: Separate the serum, grind the liver and centrifuge to take the supernatant, and test the triglyceride and cholesterol concentration with the kit.

2. Results: As shown in Figure 15A-E, the results show that AMLN-fed mice have increased body weight, massive infiltration of inflammatory lymphocytes in liver tissue, fatty liver parenchyma, fibrous collagen deposition around hepatic sinusoids, and elevated serum liver injury indicators. Blood and liver lipid metabolism indicators are disordered, and IEVs injection can significantly alleviate symptoms of fatty liver, liver injury, hepatitis and liver fibrosis.

Test example 1

MSCs and exosomes can treat the damaging liver fibrosis induced by carbon tetrachloride or Thioacetamide (TAA) (Mehrabani et al., Arch Razi Inst, 2019; Sabry et al., Int J Stem Cells, 2019; Rong et al., Stem Cell Res Ther, 2019).

(1) Detection steps or methods: Take 8-week-old C57 mice, and intraperitoneally inject 100mg/kg TAA 3 times a week for 8 weeks to create a model of traumatic liver fibrosis. During TAA modeling, IEVs were infused via the tail vein every 2 weeks. After 8 weeks, samples were taken, liver tissues were paraffin sectioned and HE stained.

(2) Results: As shown in Figure 16, TAA injection induced liver damage, tissue destruction around hepatic sinusoids, and formation of vitreous fibrosis. IEVs injection had no obvious therapeutic effect.

Test example 2

(1) Detection steps or methods: Take 8-week-old Sjogren’s syndrome (SS) model mice, inject MSCs and IEVs through the tail vein system, take samples 4 weeks after injection, measure saliva flow rate, collect salivary gland samples for paraffin section HE Staining and B-cell marker B220 staining.

(2) Results: As shown in Figure 17A-17C, the results show that the effect of the treatment of Sjogren’s syndrome (Sjogren’s syndrome) on salivary flow rate by mouse bone marrow mesenchymal stem cells and IEVs derived therefrom, mesenchymal stem cell treatment Saliva flow rate recovered slightly after IEVs treatment (*p<0.05 compared with WT group, ###p<0.001 compared with MSCs group). IEVs injection did not change the inflammatory infiltration of the salivary glands and the accumulation of B cells.

Test example 3

The in vitro coagulation experiment was used to detect the in vitro coagulation-promoting effect of the IEVs obtained in Example 2 and the Exosomes extracted in Comparative Example 1. The results are shown in Table 4, IEVs can significantly shorten the in vitro clotting time of most plasma, and the procoagulant effect is better than Exosomes.

However, for plasma lacking factor II, V, and X, IEVs cannot play an in vitro coagulation effect, indicating that the in vitro coagulation effect of IEVs is more concentrated in the upstream of the common blood coagulation pathway.

Table 4

Using hemophilia A mice (deficiency of coagulation factor VIII) as a model, 9×10 ⁸ IEVs were injected through the tail vein to observe the procoagulant effect of IEVs in vivo. The results are shown in Figure 18. After treatment with IEVs, the bleeding tendency of hemophilia mice can be significantly improved, and the therapeutic effect can be sustained and stably maintained for 14 days.

Experimental results show that IEVs can play a significant role in promoting coagulation in vitro. And after in vivo injection, it can significantly improve the bleeding tendency, and can be used to improve the bleeding tendency caused by hemophilia A. At the same time, the levels of various coagulation factors in mouse plasma were detected, and it was found that coagulation factor VIII, vWF factor, tissue factor (tissue factor, TF) and prothrombin (prothrombin) did not change significantly (Figure 19A, Figure 19B, Figure 19C) , Figure 19D).

In a mouse model of hemophilia A, normal IEVs, PS-negative IEVs and TF-negative IEVs were injected respectively, and the tail trimming experiment was performed 7 days later. The results are shown in Figure 20A and Figure 20B. The blocking of PS and TF did not affect the body of IEVs. The therapeutic effect preliminarily shows that the mechanism of IEVs in treating hemophilia mice has nothing to do with PS and TF. In previous literature reports, the procoagulant effects of extracellular vesicles are highly dependent on the PS and TF on their surface, and the in vivo experimental results of IEVs are inconsistent with previous studies. This suggests that IEVs may have a new mechanism of action in the in vivo environment. Procoagulant effect.

In the hemophilia A mouse model, IEVs derived from the same MSCs (obtained in Example 2) and Exosomes (extracted from Comparative Example 1) were injected respectively (9×10 ⁸ ). The results showed that IEVs can be significantly Corrected the bleeding tendency of mice, while Exosomes had no obvious therapeutic effect (Figure 21).

Example 11 IEVs can be excreted through the skin and hair

Take 4×10 ⁶ IEVs prepared in Example 2 labeled with DIR, resuspend them in 200 μl PBS, and inject them into nude mice BALB/c-nu/nu systemically through the tail vein. Observe 1, 3, and 7 days later and use them in vivo The imaging instrument detects the distribution of IEVs on the skin surface, and the results are shown in Figures 22A-22C.

Figure 22A shows that IEVs can reach the surface of the skin, the number is highest on the 3rd day, and basically disappears on the 7th day, showing the dynamic metabolic process of IEVs on the skin surface (Figure 22A). Immunofluorescence results showed that PKH26-IEV gradually moved from the subcutaneous tissue to the dermis and epidermis over time after systemic injection of C57 mice. A large number of IEVs were observed in the stratum corneum of the skin surface on the 7th day, suggesting that the systemic injection of IEVs can be excreted as the stratum corneum of the skin falls off (Figure 22B). At the same time, PKH26-IEV was found in the hair follicles in the hair plucked from the surface of the mouse on day 7, indicating that the systemically injected IEVs could also be metabolized as the hair fell off (Figure 22C).

This example shows that IEVs can be excreted through the skin and hair, indicating that it is safe to inject or increase the content of IEVs in the body.

Claims (17)

1. The use of vesicles in the preparation of drugs for treating or preventing liver diseases, characterized in that the vesicles are inducible vesicles; the disease does not include thioacetamide-induced destructive liver fibrosis.
2. The application according to claim 1-2, wherein the medicine is used to protect the liver;

   Preferably, the drug is used to maintain liver homeostasis;

   More preferably, the drug is used to maintain the homeostasis of liver glucose metabolism or lipid metabolism.
3. The application according to claim 1-2, characterized in that the medicine is used to maintain liver glycogen, liver lactic acid, blood sugar, blood lactic acid, liver triglycerides, liver cholesterol, blood triglycerides and blood cholesterol. One or more levels of steady state;

   Preferably, the drug is used to maintain the homeostasis of the liver tissue structure;

   More preferably, the drug is used to maintain the homeostasis of the organelles or cytoskeleton of liver parenchymal cells;

   Preferably, the medicine is used to promote liver regeneration;

   Preferably, the drug is used to promote tissue regeneration after partial hepatectomy.
4. The application according to claims 1-3, wherein the medicine is used to prevent liver failure;

   More preferably, the liver failure includes acute liver failure or chronic liver failure;

   More preferably, the drug is used to prevent acute liver failure induced by acetaminophen.
5. The application according to claim 1-4, wherein the medicine is used to treat liver injury;

   Preferably, the medicine is used to treat non-alcoholic fatty liver disease;

   Or preferably, the medicine is used to treat autoimmune hepatitis or autoimmune liver fibrosis;

   Preferably, the medicine is used to prevent non-alcoholic steatohepatitis.
6. The application according to claims 1-5, wherein the inducible vesicles are vesicles produced by external factors inducing apoptosis when the stem cells are in normal survival;

   Preferably, the inducible vesicles are produced by inducing stem cell apoptosis, and the induction method includes addition of staurosporium, ultraviolet irradiation, starvation method, or thermal stress method;

   More preferably, the stem cells are mesenchymal stem cells;

   More preferably, the source of the mesenchymal stem cells includes bone marrow, urine, oral cavity, fat, placenta, umbilical cord, periosteum, and tendon.
7. The use according to claims 1-6, wherein the mesenchymal stem cells are derived from mammals;

   Preferably, the mammal is selected from humans or mice.
8. The use according to claims 1-7, wherein the diameter of the inducible vesicle is about 0.03-10 μm;

   Preferably, the diameter of the inducible vesicle is about 0.03-6 μm;

   More preferably, the diameter of the inducible vesicle is about 0.03-4.5 μm.
9. The application according to claims 1-8, wherein the medicine is selected from injections, oral preparations or external preparations;

   Preferably, the medicine is an injection;

   More preferably, the drug is selected from intravenous injection, intramuscular injection, subcutaneous injection or intrathecal injection;

   Or preferably, the medicament further includes a pharmaceutically acceptable pharmaceutical carrier;

   More preferably, the pharmaceutical carrier includes one or more of diluents, excipients, fillers, binders, disintegrants, surfactants and lubricants.
10. A composition, characterized in that it comprises an inducible vesicle and a medicine for treating or preventing liver disease;

    Preferably, the inducible vesicle is a vesicle produced by external force inducing apoptosis during the normal survival period of the stem cell or somatic cell;

    Preferably, the inducible vesicles are produced by inducing stem cells or stem cell apoptosis by adding staurosporium, ultraviolet irradiation, starvation method, or thermal stress method or a combination of one or more of them;

    Preferably, the diameter of the inducible vesicle is about 0.03-10 μm;

    Preferably, the diameter of the inducible vesicle is about 0.03-6 μm;

    More preferably, the diameter of the inducible vesicle is about 0.03-4.5 μm.
11. A method for treating or preventing liver disease in a subject, which is characterized in that it comprises administering to the subject an effective amount of a drug containing vesicles or the composition of claim 10, and the vesicles are inductive Vesicles

    Preferably, the disease does not include non-alcoholic steatohepatitis and thioacetamide-induced destructive liver fibrosis;

    Preferably, the treatment or prevention includes protecting the liver;

    Preferably, the treatment or prevention includes maintaining liver homeostasis;

    More preferably, the treatment or prevention includes maintaining the homeostasis of glucose metabolism or lipid metabolism of the liver;

    More preferably, the treatment or prevention includes maintaining the homeostasis of one or more levels of liver glycogen, liver lactic acid, blood sugar, blood lactic acid, liver triglycerides, liver cholesterol, blood triglycerides, and blood cholesterol;

    Preferably, the treatment or prevention includes maintaining the homeostasis of the liver tissue structure;

    More preferably, the treatment or prevention maintains the homeostasis of the organelles or cytoskeleton of liver parenchymal cells;

    Preferably, the treatment or prevention includes promoting liver regeneration;

    Preferably, the treatment or prevention includes promoting tissue regeneration after partial hepatectomy.
12. The method of claim 11, wherein the liver disease comprises liver failure;

    Preferably, the liver failure includes acute liver failure or chronic liver failure;

    Preferably, the liver disease includes acute liver failure induced by acetaminophen.
13. The method of claims 11-12, wherein the liver disease comprises liver damage;

    Preferably, the liver disease includes non-alcoholic fatty liver disease;

    Or preferably, the liver disease includes autoimmune hepatitis or autoimmune liver fibrosis.
14. The method according to claims 11-13, wherein the inducible vesicle is a vesicle produced by external factors inducing apoptosis when the stem cells are in normal survival;

    Preferably, the inducible vesicles are produced by inducing stem cell apoptosis, and the induction method includes addition of staurosporium, ultraviolet irradiation, starvation method, or thermal stress method;

    More preferably, the stem cells are mesenchymal stem cells;

    More preferably, the source of the mesenchymal stem cells includes bone marrow, urine, oral cavity, fat, placenta, umbilical cord, periosteum, and tendon.
15. 14. The method of claims 11-14, wherein the mesenchymal stem cells are derived from mammals;

    Preferably, the mammal is selected from humans or mice.
16. The method of claims 11-15, wherein the diameter of the inducible vesicle is about 0.03-10 μm;

    Preferably, the diameter of the inducible vesicle is about 0.03-6 μm;

    More preferably, the diameter of the inducible vesicle is about 0.03-4.5 μm.
17. The method according to claims 11-15, wherein the drug is selected from the group consisting of injections, oral preparations or topical preparations;

    Preferably, the medicine is an injection;

    More preferably, the drug is selected from intravenous injection, intramuscular injection, subcutaneous injection or intrathecal injection;

    Or preferably, the medicament further includes a pharmaceutically acceptable pharmaceutical carrier;

    More preferably, the pharmaceutical carrier includes one or more of diluents, excipients, fillers, binders, disintegrants, surfactants and lubricants.

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