A fibrin gel cardiac patch encapsulating wolfberry exosomes and its preparation method

Abstract

The present invention belongs to the field of biomedical materials and tissue repair after myocardial infarction, and discloses a fibrin gel cardiac patch encapsulating wolfberry exosomes and a preparation method thereof. Among them, the wolfberry exosome fibrin gel for myocardial repair comprises fibrinogen, wolfberry exosomes and thrombin, and is obtained by uniformly mixing a fibrinogen solution, a wolfberry exosome solution and a thrombin solution. The present invention constructs a wolfberry exosome fibrin gel by using fibrinogen, wolfberry exosomes and thrombin, and is particularly applicable to myocardial repair. The present invention can alleviate and treat cardiomyocyte apoptosis, myocardial fibrosis, heart function decline and ventricular remodeling caused by myocardial infarction, thereby solving the technical problem of low survival rate after myocardial infarction, and providing a more highly safe and efficient targeted myocardial treatment strategy.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical materials and tissue repair after myocardial infarction, and more specifically, relates to a fibrin gel cardiac patch encapsulating wolfberry exosomes and a preparation method thereof. Background Art

[0002] Currently, clinical treatment methods for myocardial infarction (such as drug treatment, coronary artery bypass grafting, stent implantation, etc.) usually focus on restoring blood supply to the infarcted area. Although timely intravenous thrombolysis or interventional treatment can significantly reduce the mortality of patients with acute myocardial infarction, it cannot restore the necrotic or fibrotic myocardium to normal and cannot completely control ventricular remodeling after myocardial infarction. In the past decade, many efforts have been made to identify drugs that can promote recovery after myocardial infarction, such as renin-angiotensin-aldosterone system inhibitors, inflammatory regulators, β-blockers, endothelin antagonists, and transforming growth factor β (TGF-β) inhibitors. However, due to the lack of cardiac-specific targeting, the application of these drugs often leads to side effects related to systemic toxicity. For example, although TGF-β inhibitors play a key role in recovery after myocardial infarction, systemic administration of the TGF-β inhibitor pirfenidone approved by the US Food and Drug Administration may cause serious adverse events, including gastrointestinal adverse reactions, cardiac valve defects, and systemic photosensitivity. Therefore, there is an urgent need to find potential treatment methods that directly target the myocardium to improve the safety and effectiveness of myocardial infarction treatment.

[0003] With high bioavailability, slow release, specificity, and fewer side effects, the nanoscale drug delivery system has become a valuable model for administering small molecules and biologics after myocardial infarction. The research group where the inventors of the present invention are located previously reported the application of wolfberry extracellular vesicles in promoting tissue repair or growth (see Chinese Patent Application CN116726090A for details), and also published a paper "Gouqi-derived nanovesicles (GqDNVs) inhibited dexamethasone-induced muscle atrophy associating with AMPK / SIRT1 / PGC1α signaling pathway", which elaborated in detail the role of wolfberry exosomes in skeletal muscle repair and regeneration. However, there is currently no study confirming the therapeutic effect of wolfberry exosomes on myocardial infarction.

[0004] Different from skeletal muscle, myocardium has a unique automatic rhythm, that is, the heart can automatically and rhythmically generate excitation without external stimulation. Moreover, myocardial cells are connected by low-impedance intercalated disc structures, and excitation can be directly transmitted from one cell to another, so the myocardium is a "functional syncytium". When any cell in the atrium or ventricle generates excitation, it can quickly transmit the excitation to the entire atrial muscle or ventricular muscle, so myocardial repair is more difficult. Summary of the Invention

[0005] In view of the above defects or improvement requirements of the prior art, the purpose of the present invention is to provide a fibrin gel cardiac patch encapsulating wolfberry exosomes and its preparation method, which constructs wolfberry exosome fibrin gel by using fibrinogen, wolfberry exosomes and thrombin, and can be particularly used for myocardial repair. The present invention can alleviate and treat cardiomyocyte apoptosis, myocardial fibrosis, heart function decline and ventricular remodeling caused by myocardial infarction, thereby solving the technical problem of low survival rate after myocardial infarction, and providing a more highly safe and efficient targeted myocardial treatment strategy.

[0006] To achieve the above object, according to the first aspect of the present invention, there is provided a wolfberry exosome fibrin gel for myocardial repair, which is characterized by comprising fibrinogen, wolfberry exosomes and thrombin, and is obtained by uniformly mixing a fibrinogen solution, a wolfberry exosome solution and a thrombin solution.

[0007] According to the second aspect of the present invention, there is provided a precursor of the wolfberry exosome fibrin gel for myocardial repair, which is characterized by comprising discrete fibrinogen solution, wolfberry exosome solution and thrombin solution; when in use, by uniformly mixing the fibrinogen solution, the wolfberry exosome solution and the thrombin solution, the wolfberry exosome fibrin gel for myocardial repair can be obtained.

[0008] According to the third aspect of the present invention, there is provided a precursor of the wolfberry exosome fibrin gel for myocardial repair, which is characterized by comprising discrete first solution and second solution, wherein the first solution is obtained by uniformly mixing a fibrinogen solution and a wolfberry exosome solution; the second solution is a thrombin solution; when in use, by uniformly mixing the first solution and the second solution, the wolfberry exosome fibrin gel for myocardial repair can be obtained.

[0009] As a further preference of the present invention, the fibrinogen solution is obtained by dispersing freeze-dried fibrinogen powder in PBS buffer, the wolfberry exosome solution is obtained by resuspending wolfberry exosomes in PBS solution, and the thrombin solution is obtained by dispersing freeze-dried thrombin powder in physiological saline for injection;

[0010] In the mixed system obtained by mixing fibrinogen solution, wolfberry exosome solution and thrombin solution, the final concentration of fibrinogen satisfies 3.75-7.5 mg / mL, and the final concentration of wolfberry exosomes satisfies 0.125-0.375×10 9 particles / 200 μL, and the final concentration of thrombin satisfies 31.25-125 IU / mL.

[0011] According to the fourth aspect of the present invention, the present invention provides a wolfberry exosome fibrin gel cardiac patch for myocardial repair, which is characterized in that it is formed from the wolfberry exosome fibrin gel for myocardial repair as described above, and its shape can fit the heart;

[0012] In this cardiac patch, wolfberry exosomes are encapsulated in fibrin.

[0013] According to the fourth aspect of the present invention, the present invention provides the use of the above-mentioned wolfberry exosome fibrin gel for myocardial repair, the precursor of the above-mentioned wolfberry exosome fibrin gel for myocardial repair, or the above-mentioned wolfberry exosome fibrin gel cardiac patch for myocardial repair in the preparation of drugs for myocardial repair.

[0014] According to the fifth aspect of the present invention, the present invention provides the use of the above-mentioned wolfberry exosome fibrin gel for myocardial repair, the precursor of the above-mentioned wolfberry exosome fibrin gel for myocardial repair, or the above-mentioned wolfberry exosome fibrin gel cardiac patch for myocardial repair in the preparation of drugs for treating myocardial infarction.

[0015] As a further preference of the present invention, the drug is specifically a spray-type drug or a coating-type drug.

[0016] Through the above technical solutions conceived by the present invention, compared with the prior art, the present invention constructs a wolfberry exosome fibrin gel using fibrinogen, wolfberry exosomes and thrombin. The corresponding wolfberry exosome fibrin gel and the corresponding cardiac patch can be specifically targeted for alleviating and treating myocardial infarction. The porous reticular fibrin gel evenly encapsulates wolfberry exosomes in its pores and sprays them on the heart surface to form a uniform cardiac patch layer, or the cardiac patch pre-prepared using a heart mold is attached to the heart surface, which can inhibit cardiomyocyte apoptosis, inhibit myocardial fibrosis, improve cardiac function, and thus improve the survival rate after myocardial infarction.

[0017] The present invention uses fibrin gel with natural biocompatibility and biodegradability. The porous reticular fibrin can evenly encapsulate wolfberry exosomes in the pores of the scaffold to form a cardiac patch layer. During the coagulation process, the gel patch naturally forms a curvature that conforms to the cardiac surface, widely covering the surface of the infarcted area, maximizing the possibility of contact between wolfberry exosomes and necrotic tissue. As the fibrin decomposes and is absorbed, the wolfberry exosomes encapsulated therein are slowly released. It can not only target the myocardium for slow drug release and exert the drug effect persistently, but also provide mechanical support to the heart, improve cardiac function, inhibit cardiomyocyte apoptosis and myocardial fibrosis, and increase the survival rate after myocardial infarction. This wolfberry exosome cardiac patch can simultaneously have pharmacological and mechanical therapeutic effects, has good prospects for treating cardiomyocyte apoptosis, excessive cardiac fibrosis, and improving cardiac function after myocardial infarction, and has the advantages of high targeting and high safety.

[0018] In the previous research of the research group where the present invention belongs, wolfberry exosomes were extracted from wolfberry. As natural nanoparticles, wolfberry exosomes have characteristics such as high biocompatibility, biodegradability, and low toxicity. At the same time, fibrin gel has been widely used clinically in intraoperative hemostasis, vascular suture, etc. As a biological scaffold material, it also has characteristics such as biodegradability and tissue compatibility, and can provide sufficient support to bear the mechanical tension of myocardial tissue. It can not only act as a carrier for transporting cells and proteins, but also provide the extracellular matrix, providing a three-dimensional environment for cardiomyocyte adhesion, migration, proliferation, and differentiation. The present invention not only combines the respective functional characteristics of wolfberry exosomes and fibrin gel, but also utilizes the coagulation and structural characteristics of fibrin to enable wolfberry exosomes to target the infarcted area and exert a therapeutic effect persistently, and also solves the disadvantage of poor tissue repair effect of single fibrin gel. Therefore, the fibrin gel cardiac patch encapsulating wolfberry exosomes prepared by the present invention has a powerful ability to target the myocardium for slow drug release and delivery. As the fibrin decomposes and is absorbed, the wolfberry exosomes encapsulated therein are slowly released, exerting the drug effect persistently and achieving precise and efficient treatment of myocardial infarction.

[0019] Specifically, the present invention can achieve the following beneficial effects:

[0020] (1) Wolfberry is the mature fruit of Lycium barbarum L., a plant of the Solanaceae family, and plays an important role in lipid-lowering, blood sugar-lowering, blood pressure-lowering, anti-inflammatory, etc. The present invention is the first to extract wolfberry exosomes for application in the tissue repair after myocardial infarction, and it is found that wolfberry exosomes can exert the ability to inhibit cardiomyocyte apoptosis and anti-myocardial fibrosis.

[0021] (2) When the fibrin mixture obtained by the present invention is sprayed onto the surface of the heart, a three-dimensional patch conforming to the geometric space of the left ventricle can be formed within a short time (within 10 s), which can deform with the change of the dynamic mechanical environment of the myocardium, thereby weakening the thinning of the ventricular wall and adverse remodeling, providing mechanical support for the infarcted weak myocardial area, preventing cardiac rupture, and at the same time providing a three-dimensional environment for the adhesion, migration, proliferation and differentiation of myocardial cells.

[0022] (3) The reticular fibrin gel with a three-dimensional porous matrix evenly wraps the wolfberry exosomes in its pores and adheres to the heart surface to form a uniform cardiac patch layer. On the one hand, it can increase the adhesion force of the wolfberry exosomes on the tissue surface and make them play a role persistently and evenly. On the other hand, the fibrin gel cardiac patch encapsulating wolfberry exosomes prepared by the present invention has a strong targeted myocardial drug sustained-release delivery ability. As the fibrin decomposes and is absorbed, the wolfberry exosomes encapsulated therein are slowly released, effectively prolonging the circulation and retention time of the wolfberry exosomes at the infarcted site, and the drug effect is more persistent.

[0023] The present invention not only provides a strategy for promoting myocardial infarction recovery and regeneration with higher therapeutic effects and lower systemic toxicity, but also provides an exosome sustained-release delivery platform based on fibrin gel.

[0024] The present invention also optimizes the ratios of fibrinogen, wolfberry exosomes, and thrombin in the fibrin gel encapsulating wolfberries, so that in the mixed system obtained by mixing the fibrinogen solution, wolfberry exosome solution, and thrombin solution, the final concentration of fibrinogen satisfies 3.75 - 7.5 mg / mL, the final concentration of wolfberry exosomes satisfies 0.125 - 0.375×10 9 particles / 200 μL, and the final concentration of thrombin satisfies 31.25 - 125 IU / mL, enabling rapid gel formation and strong adhesion, so that the product can directly cover the surface of the infarcted area after spraying and is not easy to fall off, and a gel patch rich in wolfberry exosomes can be obtained. Description of the Drawings

[0025] Figure 1 is the process flow chart of the preparation of the wolfberry exosome fibrin gel cardiac patch and its application to myocardial infarction.

[0026] Figure 2 is the structural diagram of wolfberry exosomes under transmission electron microscopy.

[0027] Figure 3 is the particle size analysis chart of wolfberry exosomes.

[0028] Figure 4 is the gel formation test of the wolfberry exosome fibrin gel cardiac patch.

[0029] Figure 5 It is the structural diagram of fibrin gel encapsulating wolfberry exosomes under a scanning electron microscope.

[0030] Figure 6 It is the observation of wolfberry exosomes being taken up by cardiomyocytes under a confocal microscope.

[0031] Figure 7 It is the result diagram of fluorescence imaging in live mice.

[0032] Figure 8 It is the result of cell viability and apoptosis detection of cardiomyocytes treated with wolfberry exosome fibrin gel heart patches. Among them, Figure 8 A in it corresponds to the effect on cardiomyocyte viability, Figure 8 B in it corresponds to the protective effect on cardiomyocyte toxicity after doxorubicin intervention. * represents significant difference compared with the PBS group, and # represents significant difference compared with the DOX group.

[0033] Figure 9 It is the effect of wolfberry exosome fibrin gel heart patches on the cardiac function of mice with myocardial infarction model observed by echocardiography. Among them, the red arrow represents the thickness of the anterior wall of the left ventricle, and the white arrow represents the end-systolic diameter of the left ventricle. * represents significant difference compared with the CON group, and # represents significant difference compared with the MI group.

[0034] Figure 10 It is the effect of wolfberry exosome fibrin gel heart patches on the mortality of mice with myocardial infarction model.

[0035] Figure 11 It is the result diagram of H&E staining of mouse myocardial tissue sections.

[0036] Figure 12 It is the effect of wolfberry exosome fibrin gel heart patches on the expression of genes related to cardiomyocyte apoptosis in mice with myocardial infarction model. * represents significant difference compared with the CON group, and # represents significant difference compared with the MI group.

[0037] Figure 13 It is the effect of wolfberry exosome fibrin gel heart patches on the expression of genes related to myocardial fibrosis in mice with myocardial infarction model. * represents significant difference compared with the CON group, and # represents significant difference compared with the MI group.

[0038] Figure 14 It is the difference in the protective effect on cardiomyocyte toxicity between wolfberry exosome fibrin gel heart patches and simple fibrin gel. * represents P < 0.05, ** represents P < 0.01, and *** represents P < 0.001.

[0039] Figure 15It was to observe the difference in anti-apoptotic effects between the Lycium barbarum exosome fibrin gel cardiac patch and simple fibrin gel by TUNEL staining.

[0040] In the figure, EXO represents Lycium barbarum exosomes, EXOS represents the Lycium barbarum exosome fibrin gel cardiac patch, DOX represents doxorubicin treatment, DS represents doxorubicin + simple fibrin gel treatment, DEXO represents doxorubicin + simple Lycium barbarum exosome solution treatment, DEXOS represents doxorubicin + Lycium barbarum exosome fibrin gel treatment, CON represents the control group, and MI represents the myocardial infarction model group. Specific implementation manners

[0041] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. In addition, the technical features involved in the various implementation manners of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0042] Generally speaking, for the Lycium barbarum exosome fibrin gel and its precursor for myocardial repair in the present invention, the preparation process may include the following steps:

[0043] Step 1, extraction and preparation of Lycium barbarum exosomes: Based on the mature fruits of the Solanaceae plant Lycium barbarum L. (purchased from the Ningxia Academy of Agriculture and Forestry Sciences, Yinchuan, Ningxia Hui Autonomous Region), Lycium barbarum exosomes are extracted from fresh Lycium barbarum by a method combining ultracentrifugation and density gradient separation; the specific operation steps can be seen in another patent already applied for by this research group (Chinese Patent Application CN116726090A)

[0044] Step 2, preparation of fibrin pre-gel: Prepare a fibrinogen solution with PBS buffer (for example, 50 - 100 mg of freeze-dried fibrinogen powder can be dispersed in 5 - 10 mL of PBS solution), and prepare a thrombin solution with injectable physiological saline (for example, 25 - 50 mg of freeze-dried thrombin powder can be dispersed in 1 - 2 mL of injectable physiological saline);

[0045] After step 2, the obtained discrete fibrinogen solution, Lycium barbarum exosome solution and thrombin solution together constitute the precursor of the Lycium barbarum exosome fibrin gel for myocardial repair.

[0046] Considering that the mixture will solidify within a short time after the addition of thrombin, the fibrinogen solution and the wolfberry exosome solution can be fully mixed first as the first solution (for example, the fibrinogen solution and the wolfberry exosome solution can be mixed and then shaken evenly, and the shaking time can be 1-2 minutes). The thrombin solution is used as the second solution, and the first solution and the second solution together form the precursor of the wolfberry exosome fibrin gel for myocardial repair.

[0047] Step 3: Gel formation: Aspirate the fibrinogen solution, wolfberry exosomes, and thrombin solution respectively and mix them well (either quickly shake and mix the fibrinogen solution, wolfberry exosome solution, and thrombin solution, or quickly shake and mix the first solution and the second solution), then the wolfberry exosome fibrin gel for myocardial repair can be formed. The wolfberry exosome fibrin gel can be dropped or sprayed onto the infarcted area of the myocardial tissue to be treated, or it can be dropped or sprayed onto the heart mold. The gel forms an arc that fits the surface of the heart during the coagulation process, thus forming a fibrin gel heart patch that wraps the wolfberry exosomes, and the shape of the patch fits the heart.

[0048] The following are specific examples (except as otherwise specified in the following examples, all are carried out at room temperature of 25±2°C):

[0049] Example 1:

[0050] As Figure 1 shown, the preparation process of the fibrin gel heart patch for treating myocardial infarction and wrapping wolfberry exosomes provided by the embodiment of the present invention includes the following steps:

[0051] Step 1, extraction and preparation of wolfberry exosomes (i.e., wolfberry extracellular vesicles): Wolfberry exosomes are extracted from fresh wolfberries by a method combining ultracentrifugation and density gradient separation. The specific operation steps can be seen in another patent applied by this research group (Chinese Patent Application CN116726090A). The wolfberry exosome solution is prepared by resuspending the extracted wolfberry exosomes with PBS solution, and the final concentration of wolfberry exosomes used is 1×10 9 particles / 200μL.

[0052] Step 2, preparation of fibrin pre-gel: Prepare a fibrinogen solution by dissolving 50 mg of freeze-dried fibrinogen powder in 5 mL of PBS buffer, and prepare a thrombin solution by dissolving 25 mg of freeze-dried thrombin powder in 1 mL of injectable physiological saline.

[0053] Step 3: Preparation of fibrin gel cardiac patch encapsulated with wolfberry exosomes: Aspirate fibrinogen solution, wolfberry exosomes, and thrombin solution at a ratio of 5:2:1 (v / v) respectively, and mix well to obtain wolfberry exosome fibrin gel. The wolfberry exosome fibrin gel can be dropped onto the infarcted area of the myocardial tissue to be treated to form a fibrin gel cardiac patch encapsulated with wolfberry exosomes. Of course, the wolfberry exosome fibrin gel can also be dropped onto a heart mold to form a fibrin gel cardiac patch encapsulated with wolfberry exosomes with a cardiac curved surface shape, and its shape can fit the heart during subsequent use.

[0054] Table 1: Formulation composition of wolfberry exosome fibrin gel prepared in Example 1

[0055]

[0056] Example 2:

[0057] Identify the wolfberry exosomes prepared in Step 1 of Example 1, including:

[0058] (1) Observe the morphological structure of exosomes by transmission electron microscopy

[0059] Freeze the wolfberry exosomes in a -80 °C ultra-low temperature refrigerator, take them out and thaw. Aspirate 10 μL of wolfberry exosome sample and precipitate it on a copper mesh for 1 - 2 min, then aspirate 10 μL of uranyl acetate and precipitate it on the copper mesh for 1 - 2 min, and dry it at room temperature for 5 min. Observe it under a transmission electron microscope at 100 kV, and the results are as Figure 2 shown. The wolfberry exosomes have a saucer-like structure.

[0060] (2) Particle size analysis of wolfberry exosomes

[0061] Freeze the wolfberry exosomes in a -80 °C ultra-low temperature refrigerator, take them out and thaw in a 25 °C water bath, and place them on ice for use. Add 10 mL of 1×PBS solution to the wolfberry exosome suspension to obtain an exosome dilution. After filtering the sample dilution through a 0.22 μm microporous filter membrane, inject it into a nanoparticle tracking analyzer for analysis. The results are as Figure 3 shown. The particle size analysis shows a single peak and a smooth curve, without extra peaks, and the particle size peak is 126.3 nm. Generally speaking, the proportion of particles with a particle size of 0 - 200 nm is >80%.

[0062] Example 3:

[0063] The present invention provides the gelation property and structure determination of a fibrin gel cardiac patch encapsulated with wolfberry exosomes, including:

[0064] (1) Prepare the Lycium barbarum exosome fibrin gel according to the method provided in Example 1. Spray the gel onto the finger joints. After standing for a few seconds, it can be observed that there is a transparent solidified gel at the finger joints. The gel can form a film at the 7th second after spraying. Continuously observe, as Figure 4 shown.

[0065] (2) Observe the structure of Lycium barbarum exosomes wrapped by fibrin gel under a scanning electron microscope. As Figure 5 shown, the reticular fibrin gel with a three-dimensional porous matrix uniformly wraps the Lycium barbarum exosomes (as indicated by the red arrow in Figure 5 ) in its pores.

[0066] Example 4:

[0067] The test of the targeting affinity of the fibrin gel heart patch encapsulating Lycium barbarum exosomes provided in the embodiments of the present invention with cardiomyocytes and myocardial tissue includes the following steps:

[0068] (1) The uptake of Lycium barbarum exosomes and the fibrin gel heart patch encapsulating Lycium barbarum exosomes by cardiomyocytes:

[0069] Pre-stain exosomes with PKH26: Add 10 μL of PKH26 to 1 mL of Lycium barbarum exosomes or the fibrin gel heart patch encapsulating Lycium barbarum exosomes, incubate at room temperature for 45 min, centrifuge at 150,000 g for 90 min. After centrifugation, discard the supernatant, resuspend the precipitate with PBS, and aliquot 300 μL per tube. (Keep completely dark throughout the process)

[0070] Phalloidin stain the cytoskeleton: When the cell density is 50%-60%, treat the cells with PKH26-pre-stained Lycium barbarum exosomes or the fibrin gel heart patch encapsulating Lycium barbarum exosomes for 12 h. After the intervention, aspirate the culture medium, wash the cells 2 times with pre-warmed PBS; fix the cells with 4% paraformaldehyde without methanol for 10 min (methanol will damage actin); wash the cells 2-3 times with PBS, 30 s each time; permeabilize the cells with a permeabilization buffer (0.5% Triton X-100 dissolved in PBS), treat at room temperature for 5 min; wash the cells 2-3 times with PBS, 30 s each time; take 200 μL of phalloidin working solution, completely cover the cells at the bottom of the dish, incubate at room temperature in the dark for 30 min, wash the cells 2-3 times with PBS, 30 s each time, and the washing time can be appropriately extended.

[0071] DAPI stain the cell nucleus: After washing, add DAPI staining solution and mix well, place at room temperature for 3-5 min; aspirate the DAPI staining solution, and wash with PBS 2-3 times, 3-5 minutes each time.

[0072] After staining the exosomes and cells, observe and take pictures under a confocal microscope. The results are as Figure 6As shown in the figure, first, the cells were divided into a PBS group, a Lycium barbarum exosome intervention group (EXO), and a fibrin gel cardiac patch intervention group containing Lycium barbarum exosomes (EXOS). Compared with the PBS group, a large number of Lycium barbarum exosomes with red fluorescence were targeted and aggregated in the myocardial cytoplasm, indicating that the Lycium barbarum exosomes and the Lycium barbarum exosomes encapsulated in the fibrin gel cardiac patch could be successfully taken up by cardiomyocytes.

[0073] (2) Fluorescence imaging of live mice:

[0074] DIR-prelabeled exosomes: Add 10 μL of PKH26 to 1 mL of Lycium barbarum exosomes or fibrin gel cardiac patches containing Lycium barbarum exosomes, incubate at room temperature for 45 min, ultracentrifuge at 150,000 g for 90 min. After centrifugation, discard the supernatant, resuspend the precipitate with PBS, and aliquot 300 μL per tube. (Keep completely dark throughout the process)

[0075] Inoculate the labeled exosomes by the methods of in-situ cardiac dropwise addition of exosomes (EXO drop), in-situ cardiac intramuscular injection of exosomes (EXO injection), and in-situ cardiac dropwise addition of fibrin gel cardiac patches containing Lycium barbarum exosomes (EXOS drop), and perform live imaging at 12 h and 24 h after intervention. After conventional gas anesthesia, place the mice on the imaging dark box platform. The software controls the opening and lowering of the platform to a suitable field of view, and automatically turns on the illumination lamp to take the first background image. Next, automatically turn off the illumination lamp and take the fluorescence signal emitted from the mice under the condition of no external light source. After superimposing the background images of bright field and dark field, the position and intensity of the fluorescence signal in the animal body can be visually displayed, and the imaging operation is completed. Use the small animal live imaging image processing software to calculate and analyze the fluorescence area and fluorescence intensity.

[0076] As Figure 7 shown, compared with the groups of simple dropwise addition of exosomes and intramuscular injection of exosomes, the fluorescence intensity of the fibrin gel cardiac patch intervention group containing Lycium barbarum exosomes was significantly higher at 12 h and 24 h after intervention. It is suggested that compared with simple dropwise addition / intramuscular injection of Lycium barbarum exosomes, the fibrin gel cardiac patch containing Lycium barbarum exosomes plays a role at the target site for a longer time and has a more lasting drug effect.

[0077] Example 5:

[0078] The influence of the fibrin gel cardiac patch containing Lycium barbarum exosomes provided by the embodiment of the present invention on cardiomyocytes includes the following steps:

[0079] (1) Influence on cardiomyocyte viability

[0080] Mouse cardiomyocytes that had grown to 80 - 90% were digested with trypsin and then grouped for treatment, namely: PBS group, Lycium barbarum exosome treatment group (EXO group), fibrin gel treatment group encapsulating Lycium barbarum exosomes (EXOS). 1000 cells were seeded into each well of a 96 - well plate, with 100 μL of medium containing the corresponding intervention in each well. The culture plate was placed in an incubator at 37°C and 5% CO 2 2. Incubate for 24 h: Add 10 μL of CCK - 8 solution to each well. Use wells with the corresponding amount of cell culture medium and CCK - 8 solution but without cells as blank controls. Continue to incubate in the cell culture incubator for 1.5 h. Observe the color change of the medium and then measure the absorbance at 450 nm using an enzyme - linked immunosorbent assay (ELISA) reader. Calculate the inhibition rate of the drug on the cells according to the formula.

[0081] The results are as Figure 8 shown in A of

[0082] . The viability of cardiomyocytes was not significantly affected after intervention with Lycium barbarum exosomes and fibrin gel encapsulating Lycium barbarum exosomes, indicating that Lycium barbarum exosomes and fibrin gel encapsulating Lycium barbarum exosomes have no obvious toxicity to cardiomyocytes.

[0083] (2) Protective effect on the toxicity of cardiomyocytes after doxorubicin intervention Mouse cardiomyocytes in the logarithmic growth phase were digested with trypsin and then grouped for treatment for 24 h, namely: PBS group, doxorubicin treatment group (DOX group), doxorubicin + Lycium barbarum exosome treatment group (DEXO), doxorubicin + fibrin gel treatment group encapsulating Lycium barbarum exosomes (DEXOS). Add 10 μL of CCK - 8 solution to each well. Use wells with the corresponding amount of cell culture medium and CCK - 8 solution but without cells as blank controls. Continue to incubate in the cell culture incubator for 1.5 h. Observe the color change of the medium and then measure the absorbance at 450 nm using an ELISA reader. Calculate the inhibition rate of the drug on the cells according to the formula.

[0084] The results are as Figure 8 shown in B of

[0085] . Compared with the PBS group, doxorubicin treatment significantly inhibited the proliferation viability of cells. Compared with the DOX group, the DEXO group and the DEXOS group significantly increased the proliferation viability of cardiomyocytes. And compared with the cells in the DEXO group, the fibrin gel encapsulating Lycium barbarum exosomes had a more significant promoting effect on the proliferation viability of cardiomyocytes after treatment. It shows that, compared with the simple Lycium barbarum exosome solution, the fibrin gel encapsulating Lycium barbarum exosomes has a more significant protective effect on the proliferation viability of cardiomyocytes.

[0085] Example 6:

[0086] The effects of the fibrin gel heart patch encapsulating Lycium barbarum exosomes provided in the embodiments of the present invention on the cardiac function and survival rate of myocardial infarction model mice include the following steps:

[0087] (1) Preparation of a myocardial infarction mouse model and intervention with a fibrin gel cardiac patch encapsulating wolfberry exosomes

[0088] All mice were divided into a control group (CON), a myocardial infarction model group (MI), and a myocardial infarction model + fibrin gel cardiac patch encapsulating wolfberry exosomes group (MI + EXOS).

[0089] All surgical instruments were sterilized by high-temperature steam for 30 min before use. The experimental mice were fasted for 12 h before surgery. After anesthesia, the mice were placed in the supine position (preferably the right lateral position), and the surgical area on the left chest was fixed. The hair was removed and the area was disinfected. After tracheal intubation, the ventilator was connected. An incision about 1.5 cm was made at the 3rd and 4th intercostal spaces of the heart, and the subcutaneous tissue, pectoralis major muscle, and serratus anterior muscle were bluntly separated (gently separated with the tip of a round and blunt forceps, taking care not to cut the blood vessels or other easily bleeding parts); the heart was gently squeezed at the 4th intercostal space to expose it outside the thoracic cavity. A 6-0 suture was quickly inserted 2 mm below the lower edge of the left atrial appendage and exited at the connection between the pulmonary artery conus and the apex of the heart on the right side of the heart. The left anterior descending branch was ligated under visual inspection to completely block the blood flow through the left anterior descending branch. After ligation, the apex of the heart turned white, indicating successful ligation.

[0090] After ligation, the mice in the MI group were not treated. The mice in the MI + EXOS group were immediately dropped with a fibrin gel cardiac patch encapsulating wolfberry exosomes onto the myocardial ligation site. After checking for no bleeding, the thoracic cavity was closed. The left thumb, index finger, and middle finger were used to gently squeeze the thoracic cavity to expel the air inside. The serratus minor muscle was quickly repositioned above the 3rd - 4th intercostal spaces, and the pectoralis major muscle was covered over the serratus minor muscle. The chest was closed. The surgical area was disinfected after surgery, the respiratory equipment was removed, and the mice were placed on a 37 °C constant temperature electric heater, and the state of the mice was closely observed.

[0091] The mice in the CON group only underwent thoracotomy, without coronary artery ligation or other interventions.

[0092] (2) Echocardiography was performed to detect cardiac function 4 h and 14 days after mouse model establishment

[0093] One day before echocardiography, the chest area of the mice was shaved to fully expose the heart position; on the day of echocardiography, a small animal echocardiograph was used to detect the cardiac function indexes of the mice. The probe direction was adjusted, and the long axis and short axis cross-sections of the left ventricle beside the sternum were recorded respectively, and the M-mode echocardiogram cross-sectional images were recorded.

[0094] The results were as Figure 9 shown. Echocardiography showed that compared with the mice in the CON group, the left ventricular ejection fraction, left ventricular fractional shortening, systolic and diastolic functions of the mice in the MI group decreased, the left ventricular wall thickness became significantly thinner, while after intervention with the fibrin gel cardiac patch encapsulating wolfberry exosomes, the ejection ability, fractional shortening, and systolic and diastolic functions of the mice's hearts were significantly improved.

[0095] (3) Record the death of mice every day starting from the 0th day of myocardial infarction modeling, and draw a survival curve. The results are as Figure 10 shown. Compared with the control group, the survival rate of mice in the MI group decreased significantly, while the survival rate of mice increased significantly after intervention with fibrin gel cardiac patches encapsulating wolfberry exosomes. It is indicated that the intervention with fibrin gel cardiac patches encapsulating wolfberry exosomes can effectively improve the survival rate of mice with myocardial infarction and reduce the risk of death after myocardial infarction.

[0096] Example 7:

[0097] The effects of the fibrin gel cardiac patch encapsulating wolfberry exosomes provided in the embodiments of the present invention on myocardial repair and cardiomyocyte apoptosis in mice with myocardial infarction model include the following steps:

[0098] All mice were divided into a control group (CON), a myocardial infarction model group (MI), and a myocardial infarction model + fibrin gel cardiac patch group encapsulating wolfberry exosomes (MI + EXOS). The mice were treated according to the protocol of Example 6. After 14 days, the mice were sacrificed, and the heart tissue sections of each group of mice were stained with H&E, and the morphological changes of cardiomyocytes were observed under the microscope.

[0099] The results are as Figure 11 shown. In the CON group, the myocardial arrangement was neat, the cytoplasm was rich and uniform, and the stroma was normal; in the MI group, some myocardial cell nuclei were lost, the cardiomyocytes showed vacuolar degeneration, the myocardial tissue was disordered in the infarct area, and the cardiomyocytes in the infarct area disappeared, replaced by fibrous scar tissue; in the MI + EXOS group, the morphology of cardiomyocytes was improved compared with the MI group, the cell arrangement was more normal, the vacuolar degeneration was reduced, the number of cardiomyocytes increased, and angiogenesis occurred.

[0100] Furthermore, qRT-PCR was used to detect the expression of apoptosis-related genes Casp3, Casp7, Bax, and Bcl2 in the myocardial tissue of mice. The results are as Figure 12 shown. Compared with the control group, the expression of apoptosis-related genes Casp3, Casp7, and Bax in the MI group of mice increased significantly, while the expression of apoptosis-related genes in the intervention group with fibrin gel cardiac patches encapsulating wolfberry exosomes decreased significantly; and the expression of anti-apoptosis-related gene Bcl2 in the MI group of mice decreased significantly, while the intervention group with fibrin gel cardiac patches encapsulating wolfberry exosomes significantly increased the expression of anti-apoptosis-related gene Bcl2 in the infarcted area. It is indicated that the intervention with fibrin gel cardiac patches encapsulating wolfberry exosomes can significantly inhibit cardiomyocyte apoptosis caused by myocardial infarction.

[0101] Example 8:

[0102] The effects of the fibrin gel cardiac patch encapsulating wolfberry exosomes provided in the embodiments of the present invention on myocardial fibrosis in mice with myocardial infarction model include the following steps:

[0103] All mice were divided into a control group (CON), a myocardial infarction model group (MI), and a myocardial infarction model + a fibrin gel cardiac patch group encapsulating wolfberry exosomes (MI+EXOS). The mice were treated according to the protocol of Example 6, and the mice were killed 14 days later to collect the myocardial tissues of the mice, and the expression of myocardial fibrosis-related genes Acta2, FN1, Col1a1, and Col3a1 was detected by qRT-PCR.

[0104] The results are as follows Figure 13 As shown in the figure, compared with the control group, the expression of myocardial fibrosis-related genes in the MI group mice was significantly increased, while the expression of myocardial fibrosis-related genes in the fibrin gel heart patch intervention group encapsulating wolfberry exosomes was significantly reduced. This indicates that the fibrin gel heart patch intervention encapsulating wolfberry exosomes can significantly improve excessive myocardial fibrosis caused by myocardial infarction.

[0105] Embodiment 9:

[0106] This example discusses the screening of the optimal ratio of fibrinogen, wolfberry exosomes, and thrombin. The difference between this example and Example 1 is that the ratios of the added fibrinogen solution, wolfberry exosome solution, and thrombin solution are different.

[0107] In this embodiment, the fibrinogen solution, the wolfberry exosomes, and the thrombin solution were respectively sucked in the volume ratio shown in the following table (the original concentrations of the three solutions remained unchanged, as shown in Table 1), and the corresponding wolfberry fibrin suspension was obtained after sufficient mixing. The obtained mixed solution was subjected to gelation time and adhesion test experiments, and the specific experimental data are as follows:

[0108] Table 2: Gelation performance corresponding to different ratios of fibrinogen solution, wolfberry exosome solution, and thrombin solution

[0109]

[0110]

[0111] The above results show that the volume ratio of fibrinogen solution: wolfberry exosome solution: thrombin solution can be (6-3): (1-3): (1-4), and the wolfberry fibrin suspension obtained at this time has a gelation time of no more than 20s, a fast gelation speed, and an adhesion force of no less than 175g / cm 2, the adhesion effect is better (of course, according to different gelation times and actual requirements for adhesion, the specific volume ratio can also be flexibly selected, that is, the final concentrations of fibrinogen, exosomes from Lycium barbarum, and thrombin in the fibrinogen suspension of Lycium barbarum); and the volume mixing ratio of 5:2:1 used in Example 1 has the best comprehensive effect on gelation time and adhesion, and can prepare a gel patch with rapid gelation, good colloidal adhesion, and rich exosomes from Lycium barbarum encapsulated therein.

[0112] Comparative Example 1:

[0113] Preparation of pure fibrin gel

[0114] The difference between this comparative example and Example 1 is that the step of adding exosomes from Lycium barbarum is not required. Specifically, it is divided into two steps:

[0115] Step 1, preparation of fibrin pre-gel: Prepare a fibrinogen solution by dissolving 50 mg of freeze-dried fibrinogen powder in 5 mL of PBS buffer, and prepare a thrombin solution by dissolving 25 mg of freeze-dried thrombin powder in 1 mL of injectable normal saline.

[0116] Step 2, preparation of pure fibrin gel cardiac patch: Aspirate the fibrinogen solution and thrombin solution at a ratio of 7:1 (v / v) respectively, and mix well to obtain pure fibrin gel.

[0117] Performance test:

[0118] (1) Discuss the effects of the mixtures prepared in Comparative Example 1 and Example 1 on the viability of mouse cardiomyocytes after doxorubicin intervention

[0119] Mouse cardiomyocytes in the logarithmic growth phase were digested with trypsin and then grouped and treated for 24 h, which were: PBS group, doxorubicin treatment group (DOX group), doxorubicin + pure fibrin gel treatment group (DS group), doxorubicin + fibrin gel encapsulating exosomes from Lycium barbarum treatment group (DEXOS). Add 10 μL of CCK-8 solution to each well, use the wells with the corresponding amount of cell culture medium and CCK-8 solution but without cells as blank controls, continue to incubate in a cell culture incubator for 1.5 h, observe the color change of the culture medium, and then measure the absorbance at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader. Calculate the inhibition rate of the drug on cells according to the formula.

[0120] The results are as Figure 14As shown, compared with the PBS group, the viability of cells was significantly inhibited after doxorubicin treatment. Compared with the cells in the DOX group, treatment with pure fibrin gel alone had no significant effect on cell viability. Compared with the pure fibrin gel treatment group, the DEXOS group significantly increased the proliferative viability of cardiomyocytes. The above results indicate that, compared with pure fibrin gel, fibrin gel encapsulating wolfberry exosomes has a significant protective effect on cardiomyocyte viability.

[0121] (2) Observe the protective effect of the mixtures prepared in Comparative Example 1 and Example 1 on cardiomyocyte apoptosis by TUNEL staining

[0122] Culture adherent cells on a coverslip chamber and treat them according to the following groups: PBS group, doxorubicin treatment group (DOX group), doxorubicin + pure fibrin gel treatment group (DS group), doxorubicin + fibrin gel encapsulating wolfberry exosomes treatment group (DEXOS group). After 24 hours of culture, add an appropriate amount of 4% paraformaldehyde solution (dissolved in PBS) to each coverslip chamber for fixation and incubate at room temperature for 20 min; remove the fixative, add PBS and wash 3 times, 5 min each time; immerse each sample in the permeabilization solution and incubate at room temperature for 5 min for permeabilization treatment; immerse and wash the sample with PBS 2 - 3 times; add 50 μL of Equilibration Buffer to each sample to completely cover the area to be tested and incubate at room temperature for 10 min; try to remove the balanced Equilibration Buffer as much as possible, then add 56 μL of TdT incubation buffer to each tissue sample and incubate at 37 °C for 1 h; pay attention not to let the slides dry and keep the slides away from light; after nuclear staining with DAPI, add an anti-fluorescence quenching mounting medium to cover the slides; immediately analyze the samples under a fluorescence microscope. DAPI can stain both apoptotic and non-apoptotic cells blue, and only apoptotic cell nuclei have red fluorescence localized by the incorporation of TMR-5-dUTP.

[0123] As Figure 15 shown, compared with the PBS group, the number of apoptotic cardiomyocytes with red fluorescence labeling in the DOX group and the DS group was significantly increased. Compared with the pure fibrin gel treatment group, the number of apoptotic cardiomyocytes in the DEXOS group was significantly reduced. The above results indicate that, compared with pure fibrin gel, fibrin gel encapsulating wolfberry exosomes has an obvious anti-apoptotic effect on cardiomyocytes.

[0124] The above embodiments are only examples. For example, according to actual needs, the film-forming rate of the prepared wolfberry exosome fibrin gel can be adjusted by adjusting the concentration and volume ratio of the fibrinogen solution, wolfberry exosome solution, and thrombin solution. (For example, the concentration of wolfberry exosomes in the wolfberry exosome solution can be greater than or equal to 1.0×10 9particles / 200μL and less than 1.0×10 10 (other concentration values within the range of particles / 200μL); for another example, the above-mentioned embodiments were carried out under the room temperature condition of 25±2°C. Since different temperatures will affect the gel formation time, it can also be flexibly selected. In addition, considering the pericardial cavity volume of the mouse heart and the biological tolerance, the dosage of the heart patch obtained by the present invention can be applied at 5-10 ul / g body weight.

[0125] It should be noted that: for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and retouches can be made, and these improvements and retouches should also be regarded as the protection scope of the present invention.

[0126] It is easy for those skilled in the art to understand that the above are only the preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention should all be included in the protection scope of the present invention.

Claims

1. A use of wolfberry exosome fibrin gel for myocardial repair in the preparation of a myocardial repair drug for inhibiting the expression of Casp3, Casp7, Bax, Acta2, FN1, Col1a1 and Col3a1 genes, characterized in that: The wolfberry exosome fibrin gel for myocardial repair includes fibrinogen, wolfberry exosomes and thrombin, and is obtained by uniformly mixing the fibrinogen solution, the wolfberry exosome solution and the thrombin solution; The fibrinogen solution is obtained by dispersing freeze-dried fibrinogen powder in PBS buffer, the wolfberry exosome solution is obtained by resuspending wolfberry exosomes in PBS solution, and the thrombin solution is obtained by dispersing freeze-dried thrombin powder in physiological saline for injection; In the mixed system obtained by mixing the fibrinogen solution, the wolfberry exosome solution and the thrombin solution, the final concentration of fibrinogen meets 3.75-7.5 mg / mL, and the final concentration of wolfberry exosome meets 0.125-0.375×10 9 particles / 200μL, and the final concentration of thrombin meets the requirement of 31.25-125IU / mL, which can take into account both gelation time and adhesion.

2. Use of a precursor of wolfberry exosome fibrin gel for myocardial repair in the preparation of a myocardial repair drug for inhibiting the expression of Casp3, Casp7, Bax, Acta2, FN1, Col1a1 and Col3a1 genes, characterized in that: The precursor of the wolfberry exosome fibrin gel for myocardial repair includes separate fibrinogen solution, wolfberry exosome solution and thrombin solution; when used, the wolfberry exosome fibrin gel for myocardial repair can be obtained by uniformly mixing the fibrinogen solution, wolfberry exosome solution and thrombin solution; The fibrinogen solution is obtained by dispersing freeze-dried fibrinogen powder in PBS buffer, the wolfberry exosome solution is obtained by resuspending wolfberry exosomes in PBS solution, and the thrombin solution is obtained by dispersing freeze-dried thrombin powder in physiological saline for injection; In the mixed system obtained by mixing the fibrinogen solution, the wolfberry exosome solution and the thrombin solution, the final concentration of fibrinogen meets 3.75-7.5 mg / mL, and the final concentration of wolfberry exosome meets 0.125-0.375×10 9 particles / 200μL, and the final concentration of thrombin meets the requirement of 31.25-125IU / mL, which can take into account both gelation time and adhesion.

3. Use of a precursor of wolfberry exosome fibrin gel for myocardial repair in the preparation of a myocardial repair drug for inhibiting the expression of Casp3, Casp7, Bax, Acta2, FN1, Col1a1 and Col3a1 genes, characterized in that: The precursor of the wolfberry exosome fibrin gel for myocardial repair includes a separate first solution and a second solution, wherein the first solution is obtained by uniformly mixing a fibrinogen solution and a wolfberry exosome solution; the second solution is a thrombin solution; when used, the wolfberry exosome fibrin gel for myocardial repair can be obtained by uniformly mixing the first solution and the second solution; The fibrinogen solution is obtained by dispersing freeze-dried fibrinogen powder in PBS buffer, the wolfberry exosome solution is obtained by resuspending wolfberry exosomes in PBS solution, and the thrombin solution is obtained by dispersing freeze-dried thrombin powder in physiological saline for injection; In the mixed system obtained by mixing the fibrinogen solution, the wolfberry exosome solution and the thrombin solution, the final concentration of fibrinogen meets 3.75-7.5 mg / mL, and the final concentration of wolfberry exosome meets 0.125-0.375×10 9 particles / 200μL, and the final concentration of thrombin meets the requirement of 31.25-125IU / mL, which can take into account both gelation time and adhesion.

4. Use of a wolfberry exosome fibrin gel cardiac patch for myocardial repair in the preparation of a myocardial repair drug for inhibiting the expression of Casp3, Casp7, Bax, Acta2, FN1, Col1a1 and Col3a1 genes, characterized in that: The wolfberry exosome fibrin gel heart patch for myocardial repair is obtained by forming a film of wolfberry exosome fibrin gel for myocardial repair, and its shape can fit the heart; the wolfberry exosome fibrin gel for myocardial repair includes fibrinogen, wolfberry exosomes and thrombin, and is obtained by uniformly mixing a fibrinogen solution, a wolfberry exosome solution and a thrombin solution; The fibrinogen solution is obtained by dispersing freeze-dried fibrinogen powder in PBS buffer, the wolfberry exosome solution is obtained by resuspending wolfberry exosomes in PBS solution, and the thrombin solution is obtained by dispersing freeze-dried thrombin powder in physiological saline for injection; In the mixed system obtained by mixing the fibrinogen solution, the wolfberry exosome solution and the thrombin solution, the final concentration of fibrinogen meets 3.75-7.5 mg / mL, and the final concentration of wolfberry exosome meets 0.125-0.375×10 9 particles / 200μL, the final concentration of thrombin meets the requirement of 31.25-125IU / mL, which can take into account both the gelation time and the adhesion; In this cardiac patch, wolfberry exosomes are encapsulated in fibrin.

5. Use of a wolfberry exosome fibrin gel for myocardial repair in the preparation of a drug for treating myocardial infarction that inhibits the expression of Casp3, Casp7, Bax, Acta2, FN1, Col1a1 and Col3a1 genes, characterized in that: The wolfberry exosome fibrin gel for myocardial repair includes fibrinogen, wolfberry exosomes and thrombin, and is obtained by uniformly mixing the fibrinogen solution, the wolfberry exosome solution and the thrombin solution; The fibrinogen solution is obtained by dispersing freeze-dried fibrinogen powder in PBS buffer, the wolfberry exosome solution is obtained by resuspending wolfberry exosomes in PBS solution, and the thrombin solution is obtained by dispersing freeze-dried thrombin powder in physiological saline for injection; In the mixed system obtained by mixing the fibrinogen solution, the wolfberry exosome solution and the thrombin solution, the final concentration of fibrinogen meets 3.75-7.5 mg / mL, and the final concentration of wolfberry exosome meets 0.125-0.375×10 9 particles / 200μL, and the final concentration of thrombin meets the requirement of 31.25-125IU / mL, which can take into account both gelation time and adhesion.

6. Use of a precursor of wolfberry exosome fibrin gel for myocardial repair in the preparation of a drug for treating myocardial infarction that inhibits the expression of Casp3, Casp7, Bax, Acta2, FN1, Col1a1 and Col3a1 genes, characterized in that: The precursor of the wolfberry exosome fibrin gel for myocardial repair includes separate fibrinogen solution, wolfberry exosome solution and thrombin solution; when used, the wolfberry exosome fibrin gel for myocardial repair can be obtained by uniformly mixing the fibrinogen solution, wolfberry exosome solution and thrombin solution; The fibrinogen solution is obtained by dispersing freeze-dried fibrinogen powder in PBS buffer, the wolfberry exosome solution is obtained by resuspending wolfberry exosomes in PBS solution, and the thrombin solution is obtained by dispersing freeze-dried thrombin powder in physiological saline for injection; In the mixed system obtained by mixing the fibrinogen solution, the wolfberry exosome solution and the thrombin solution, the final concentration of fibrinogen meets 3.75-7.5 mg / mL, and the final concentration of wolfberry exosome meets 0.125-0.375×10 9 particles / 200μL, and the final concentration of thrombin meets the requirement of 31.25-125IU / mL, which can take into account both gelation time and adhesion.

7. Use of a precursor of wolfberry exosome fibrin gel for myocardial repair in the preparation of a drug for treating myocardial infarction that inhibits the expression of Casp3, Casp7, Bax, Acta2, FN1, Col1a1 and Col3a1 genes, characterized in that: The precursor of the wolfberry exosome fibrin gel for myocardial repair includes a separate first solution and a second solution, wherein the first solution is obtained by uniformly mixing a fibrinogen solution and a wolfberry exosome solution; the second solution is a thrombin solution; when used, the wolfberry exosome fibrin gel for myocardial repair can be obtained by uniformly mixing the first solution and the second solution; The fibrinogen solution is obtained by dispersing freeze-dried fibrinogen powder in PBS buffer, the wolfberry exosome solution is obtained by resuspending wolfberry exosomes in PBS solution, and the thrombin solution is obtained by dispersing freeze-dried thrombin powder in physiological saline for injection; In the mixed system obtained by mixing the fibrinogen solution, the wolfberry exosome solution and the thrombin solution, the final concentration of fibrinogen meets 3.75-7.5 mg / mL, and the final concentration of wolfberry exosome meets 0.125-0.375×10 9 particles / 200μL, and the final concentration of thrombin meets the requirement of 31.25-125IU / mL, which can take into account both gelation time and adhesion.

8. Use of a wolfberry exosome fibrin gel cardiac patch for myocardial repair in the preparation of a drug for treating myocardial infarction that inhibits the expression of Casp3, Casp7, Bax, Acta2, FN1, Col1a1 and Col3a1 genes, characterized in that: The wolfberry exosome fibrin gel heart patch for myocardial repair is obtained by forming a film of wolfberry exosome fibrin gel for myocardial repair, and its shape can fit the heart; the wolfberry exosome fibrin gel for myocardial repair includes fibrinogen, wolfberry exosomes and thrombin, and is obtained by uniformly mixing a fibrinogen solution, a wolfberry exosome solution and a thrombin solution; The fibrinogen solution is obtained by dispersing freeze-dried fibrinogen powder in PBS buffer, the wolfberry exosome solution is obtained by resuspending wolfberry exosomes in PBS solution, and the thrombin solution is obtained by dispersing freeze-dried thrombin powder in physiological saline for injection; In the mixed system obtained by mixing the fibrinogen solution, the wolfberry exosome solution and the thrombin solution, the final concentration of fibrinogen meets 3.75-7.5 mg / mL, and the final concentration of wolfberry exosome meets 0.125-0.375×10 9 particles / 200μL, the final concentration of thrombin meets the requirement of 31.25-125IU / mL, which can take into account both the gelation time and the adhesion; In this cardiac patch, wolfberry exosomes are encapsulated in fibrin.

9. The use according to any one of claims 1 to 8, characterized in that: The medicine is specifically a spray-type medicine or a coating-type medicine.

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