Intermediate material of platelet membrane fusion exosome

Abstract

The invention provides an intermediate material of a platelet membrane fused exosome. The intermediate material is of a structure that the exosome is coated with the platelet membrane. The serum-derived exosome induced by ischemia pretreatment provided by the invention has a strong myocardial protection effect. According to the intermediate material of the platelet membrane fused exosome, the platelet membrane and the serum-derived exosome are extracted from blood, the approach is easy to obtain, and the extraction method is simple and convenient. The preparation of the intermediate material preferably adopts a liposome extrusion fusion technology, which is a physical method, and compared with the existing exosome preparation method, the preparation method is simpler and more convenient, and the loss is smaller.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a material containing exosomes and its applications. Background Technology

[0002] Myocardial infarction (MI) is a process of myocardial cell death and myocardial dysfunction caused by persistent ischemia and hypoxia of the heart. Post-myocardial infarction heart failure is a major cause of mortality and morbidity worldwide. [1] .

[0003] Exosomes are vesicle-like bodies (30-150 nm in diameter) enclosed in a lipid bilayer and secreted by most cells and body fluids in the body. The advantages of exosomes lie in their small molecular weight, which facilitates their crossing of biological barriers; the lipid layer protects their contents from microenvironmental damage; they are non-tumorigenic and non-viral; and they can carry DNA, miRNA, or specific combinations of therapeutic agents to specific target cells, making them ideal nanoscale biological carriers. Exosome therapy is a safe and highly effective cell-free therapy. [2,3] Exosome therapy avoids the potential tumorigenicity and ethical controversies of stem cell therapy and can be preserved long-term through freeze-drying, offering superior stability. Exosome therapy is used to inhibit tumor growth; in the treatment of neurological diseases, exosomes can cross the blood-brain barrier, promoting nerve repair and clearing abnormal protein deposits. The therapeutic potential of exosomes for repair after myocardial infarction has also been extensively studied. Natural exosomes lack targeting specificity; after intravenous injection, they preferentially accumulate in blood-rich tissues such as the liver, kidneys, and lungs, and are easily cleared by the immune system, resulting in very low accumulation in myocardial tissue and poor bioavailability. While existing genetic engineering techniques can achieve reproducible and stable targeting, they are technically complex and carry the risk of immunogenicity.

[0004] Cell membrane biomimetic nanomaterials technology is an emerging pharmaceutical technology that utilizes cell membranes to coat nanoparticles or exosomes to achieve purposes such as immune evasion and targeted drug delivery, showing great potential in the treatment of myocardial infarction. [4] Platelet membranes are unique cell membranes containing specific membrane proteins on their surface that recognize sites of injury, which can facilitate the targeting of nanoparticles to sites of cardiac damage. [5] Taking advantage of the platelet membrane's properties, platelet membrane-coated nanoparticles or fused with exosome membranes have been extensively studied in the treatment of atherosclerosis, myocardial infarction, and other conditions. [6] For example, platelet membrane fusion with macrophage-derived exosomes for targeted therapy of atherosclerosis delivers miR-199a-5p and targets HOXA1, effectively inhibiting foam cell formation, and the membrane fusion process does not have an adverse effect on the contents of the exosomes.

[0005] References [1]Vaduganathan M, Mensah GA, Turco JV, Fuster V, Roth GA. The GlobalBurden of Cardiovascular Diseases and Risk: A Compass for Future Health. J AmColl Cardiol. 2022. 80(25): 2361-2371。

[0006] [2]Yang N, Hou YB, Cui TH, Yu JM, He SF, Zhu HJ. Ischemic-Preconditioning Induced Serum Exosomal miR-133a-3p Improved Post-MyocardialInfarction Repair via Targeting LTBP1 and PPP2CA. Int J Nanomedicine. 2024.19: 9035-9053。

[0007] [3]Li J, Sun S, Zhu D, et al. Inhalable Stem Cell Exosomes PromoteHeart Repair After Myocardial Infarction. Circulation. 2024. 150(9): 710-723。 [4]Yu T, Xu Q, Chen X, et al. Biomimetic nanomaterials in myocardialinfarction treatment: Harnessing bionic strategies for advanced therapeutics.Mater Today Bio. 2024. 25: 100957。

[0008] [5]Dash P, Piras AM, Dash M. Cell membrane coated nanocarriers - anefficient biomimetic platform for targeted therapy. J Control Release. 2020.327: 546-570。

[0009] [6]Xie L, Chen J, Hu H, et al. Engineered M2 macrophage-derivedextracellular vesicles with platelet membrane fusion for targeted therapy ofatherosclerosis. Bioact Mater. 2024. 35: 447-460. Summary of the Invention

[0010] To address the shortcomings in this field, the first objective of this invention is to propose an intermediate material for platelet membrane fusion with exosomes. This material utilizes the platelet membrane's ability to specifically target damaged myocardial tissue, thereby solving the problem of the lack of targeting ability of natural exosomes. This allows for the full realization of the powerful therapeutic potential of ischemic preconditioning-induced serum exosomes and promotes the clinical translation of exosome therapy for myocardial infarction repair.

[0011] A second objective of this invention is to propose the application of the intermediate material.

[0012] The technical solution for achieving the above-mentioned objective of this invention is as follows: An intermediate material for platelet membrane fusion exosomes is a structure in which platelet membranes encapsulate exosomes. The preferred method for preparing the exosomes proposed in this invention is as follows: the intermediate material for platelet membrane fusion exosomes is prepared through the following steps: 1) Rats were subjected to ischemic preconditioning, and venous blood was collected via the heart to produce ischemic preconditioning derived serum exosomes (IEVs). 2) Whole blood was collected via the abdominal aorta using an anticoagulant tube, and platelet membranes were prepared by gradient centrifugation. 3) Mix the exosomes obtained in step 1) and the platelet membranes obtained in step 2), and repeatedly extrude them using a liposome extruder to obtain intermediate materials (P-IEVs) for platelet membrane fusion with exosomes.

[0013] In step 1), the ischemic pretreatment procedure for rats is as follows: rats are anesthetized by intraperitoneal injection of pentobarbital, and then the left anterior descending coronary artery 2-4 mm below the left atrium is ligated with sutures. After ischemia for 3-6 minutes, the ligation is loosened to allow reperfusion for 5 minutes. This is repeated three times.

[0014] In step 1), the obtained venous blood is placed on ice for 20-40 minutes, and exosomes are separated and purified by ultracentrifugation. Then, the serum is filtered, and the filtered serum is centrifuged at 100,000g-120,000g. The precipitate obtained is ischemic pretreated serum-derived exosomes (IEVs), and the obtained exosomes are resuspended.

[0015] Further, in step 1), the conditions for the ultracentrifugation method are as follows: first, centrifuge at 3000g for 15min to obtain the supernatant, then centrifuge at 12000g for 30min to obtain the supernatant, then filter the serum using a filter with a pore size of 0.22 μm, and centrifuge the filtered serum twice using a Type 100 Ti rotor centrifuge at 110000g for 70min. The precipitate obtained is ischemic pretreatment serum-derived exosomes (IEVs), and the exosomes are resuspended in PBS.

[0016] In step 2), whole blood can be collected using EDTA-2K anticoagulant tubes.

[0017] In step 2), the gradient centrifugation method is as follows: the whole blood is centrifuged twice under the condition of 200 g × 20 min, the supernatant is collected, prostaglandin E1 is added to prevent platelet activation, and then centrifuged under the condition of 900 g × 20 min to obtain platelet precipitate.

[0018] Red blood cells and white blood cells can be separated by two centrifugations.

[0019] More preferably, in step 2), the obtained platelet precipitate is resuspended in PBS containing 10 mM protease inhibitor to obtain a suspension; the suspension is frozen at -80°C for 15 minutes, thawed at room temperature, and the freeze-thaw cycle is repeated three times to obtain a platelet membrane (PM).

[0020] Through experiments, the inventors verified that the platelet membrane precipitate obtained by repeated freeze-thaw cycles could simultaneously meet the concentration requirements and achieve a purity of 90%. Although repeated freeze-thaw cycles could improve the purity of the platelet membrane, the precipitate concentration was too low to produce intermediate materials for platelet membrane fusion with exosomes.

[0021] In step 3), the exosomes and platelet membranes are respectively prepared into solutions with a concentration of 1 mg / mL. Equal volumes of the solutions are drawn up with a syringe, mixed, and placed in a liposome extruder. The syringe is repeatedly squeezed to make the mixture pass through membranes with pore sizes of 100 nm and 200 nm. After the extrusion is completed, the intermediate material of platelet membrane fusion with exosomes (P-IEVs) is obtained.

[0022] Furthermore, the syringe was first squeezed repeatedly to pass the mixture through a polycarbonate membrane with a pore size of 200 nm, and then the syringe was squeezed repeatedly to pass the mixture through a polycarbonate membrane with a pore size of 100 nm, 12 times for each membrane, for a total of 24 times.

[0023] The application of the intermediate material described in this invention in the preparation of drugs that improve myocardial ischemia-reperfusion injury.

[0024] The beneficial effects of this invention are as follows: 1. The ischemic preconditioning-induced serum-derived exosomes proposed in this invention have a strong cardioprotective effect.

[0025] 2. This intermediate material for platelet membrane fusion exosomes is obtained from blood, where both the platelet membrane and serum-derived exosomes are readily available and the extraction method is simple.

[0026] 3. The preparation of this intermediate material preferably adopts liposome extrusion fusion technology, which is a physical method that is simpler and less wasteful than existing exosome preparation methods. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the Avanti liposome extruder.

[0028] Figure 2 Characterization and identification of exosomes for platelet membrane fusion. In the figure, A and B are transmission electron microscopy (TEM) images, C and D are dynamic light scattering (NTA) results, and E and F are Western blotting results.

[0029] Figure 3 Comparison of results for intravenous injection of P-IEVs to improve myocardial ischemia-related injury. Detailed Implementation

[0030] The following examples are used to illustrate the present invention, but should not be used to limit the scope of the invention.

[0031] Adult male SD rats weighing 220-250g were used in the experiment.

[0032] In the embodiments, unless otherwise specified, all methods used are conventional techniques in the art; and all raw materials used are commercially available unless otherwise specified.

[0033] Example 1: Preparation of IEVs This embodiment uses ultracentrifugation technology to prepare ischemic pretreated serum-derived exosomes (IEVs), and the steps are as follows: Adult male SD rats were anesthetized by intraperitoneal injection of 50 mg / kg of 3% pentobarbital. The left anterior descending coronary artery was ligated approximately 3 mm below the left atrium using 6-0 nylon sutures. After 5 minutes of ischemia, the suture was released to allow reperfusion for 5 minutes, and this process was repeated three times. Venous blood was collected via the heart, incubated on ice for 30 minutes, and exosomes were separated and purified by ultracentrifugation at 3000g for 15 minutes and 12000g for 30 minutes.

[0034] The serum was then filtered using a 0.22 μm pore size filter. The filtered serum was then subjected to two cycles of 110,000 g × 70 min using a Type 100 Ti rotor. The resulting precipitate was the IEVs. The exosomes were resuspended in PBS and stored at -80°C.

[0035] Comparative Example 1 This embodiment prepares ischemic preconditioning serum-derived exosomes (IEVs). The basic steps are the same as in Example 1, except that low centrifugation force is used in the centrifugation separation and purification of exosomes. The centrifugation conditions are: 300g × 10 min; 2000g × 10 min; 10,000g × 30 min. The core objective of the first two centrifugation steps is to remove red blood cells, white blood cells, and platelets from the blood. Using low centrifugation force is insufficient to completely precipitate these large particles, leaving residual impurities. Through experimental analysis under different conditions, it was found that the separation of exosomes requires further removal of larger microvesicles (100-1000 nm in diameter) after removing large particle impurities. However, a centrifugation force of 10,000g × 30 min is insufficient to completely precipitate microvesicles, resulting in their contamination with the exosome product.

[0036] Ideally, gradient centrifugation should achieve the effect of "targeted removal of impurities of different sizes" by gradually increasing the centrifugal force. However, the gradient span of low centrifugal force (300g→2000g→10000g) is relatively small. In particular, the difference in centrifugal force between the first two steps (1700×g) and the difference between the last two steps (8000×g) are uneven, which cannot give full play to the advantages of gradient centrifugation.

[0037] After comparing the results of ultracentrifugation and low centrifugation, ultracentrifugation was chosen as the method for separating and purifying exosomes.

[0038] Using ultracentrifugation, we successfully isolated ischemic preconditioning-derived serum exosomes (IEVs). Protein quantification showed that ischemic preconditioning induced an increase in serum exosome protein levels. This study found that ischemic preconditioning-derived exosomes exert a strong cardioprotective effect in the repair of myocardial infarction in both normal and heart failure patients. Although they are targeted, their bioavailability in vivo is low due to the low uptake rate of natural exosomes by cardiomyocytes during circulation and their susceptibility to phagocytosis and degradation by macrophages. Current animal experiments all employ multi-site myocardial injection, which has low clinical operability, hindering the clinical translation of exosome therapy for myocardial infarction repair.

[0039] Example 2 The intermediate material for preparing platelet membrane fusion exosomes in this embodiment is prepared by the following steps: 1) Preparation of ischemic preconditioning-derived serum exosomes using ultracentrifugation technology Following our previous method: Adult male SD rats (220-250g) were anesthetized by intraperitoneal injection of 3% pentobarbital 50mg / kg. The left anterior descending coronary artery, approximately 3mm below the left atrium, was ligated using 6-0 nylon sutures. After 5 minutes of ischemia, the ligation was released to allow reperfusion for 5 minutes, and this process was repeated three times. The rats underwent ischemic preconditioning, and venous blood was collected via the heart. The blood was incubated on ice for 30 minutes, and exosomes were separated and purified by ultracentrifugation at 3000g for 15 minutes and 12000g for 30 minutes. The serum was then filtered using a 0.22 μm pore size filter. The filtered serum was then centrifuged twice using a Type 100 Ti rotor at 110000g for 70 minutes. The final precipitate was the ischemic preconditioning serum-derived exosomes (IEVs). The exosomes were resuspended in PBS and stored at -80℃.

[0040] 2) Gradient centrifugation technique for preparing platelet membranes Whole blood was collected via the abdominal aorta using EDTA-2K anticoagulant tubes at 200 g for 20 min twice to separate erythrocytes and leukocytes. The supernatant was then collected, and an appropriate amount of prostaglandin E1 was added to prevent platelet activation. The platelets were centrifuged at 900 g for 20 min to obtain platelet pellet. The platelets were resuspended in PBS containing 10 mM protease inhibitor to obtain a suspension. The suspension was then frozen at -80°C for 15 minutes, thawed at room temperature, and repeated three times to obtain the platelet membrane (PM).

[0041] 3) Preparation of intermediate materials for platelet membrane fusion exosomes using a liposome extruder Exosomes and platelet membranes were prepared into solutions with a concentration of 1 mg / mL. 500 μL of each solution was drawn into a 1 mL syringe, mixed, and placed into an Avanti extruder. The syringe was squeezed repeatedly to expel the mixture through a 200 nm carbonate membrane of the Avanti liposome extruder 12 times, then through a 100 nm carbonate membrane, expelling each membrane 12 times, for a total of 24 times. After extrusion, the intermediate material of platelet membrane fusion with exosomes (P-IEVs) was obtained.

[0042] Optimization experiment of extrusion purification operation in Comparative Example 2 The procedure is the same as in Example 2, except that in step 3) when preparing the intermediate material for platelet membrane fusion exosomes, the inventors chose the Avanti liposome extruder, an instrument used for preparing high-purity liposome reagents and which is easy to use. The structure of the liposome extruder is shown below. Figure 1 The carbonate membrane is installed between the two filter supports of the liposome extruder.

[0043] Exosomes and platelets were diluted to 1 mg / ml. 500 μL of each sample were sequentially squeezed back and forth using an Avanti liposome extruder with a carbonated membrane. The extraction of 100 nm and 200 nm carbonated membranes alone was compared: 100 nm alone was difficult to extract due to its small pore size; 200 nm alone resulted in leakage, leading to poor purification. Comparatively, the 100 nm membrane showed better purification results.

[0044] Each 500 μL sample was first squeezed back and forth 12 times using a 200 nm carbonate membrane in an Avanti liposome extruder, then replaced with a 100 nm carbonate membrane, and squeezed back and forth 12 times for a total of 24 times. This allowed for successful extrusion and purification of the product.

[0045] Exosomes were then labeled with the flow cytometry antibody HSP70-FITC, and platelets were labeled with the flow cytometry antibody CD42d-APC. These were diluted with PBS at 1:250 and 1:700, respectively, to 1 ml volumes. The fusion membrane samples were then incubated on ice for 30 min, centrifuged at 120,000 g for 70 min, and finally resuspended in PBS to prepare the final test sample. Flow cytometry analysis confirmed the availability of the target product. Example 3: Preparation and Characterization of Platelet Membrane Fusion Exosomes The intermediate material of platelet membrane fusion exosomes (P-IEVs) prepared by the method in Example 2, and the exosomes (IEVs) prepared in Example 1, were identified by transmission electron microscopy (TEM), dynamic light scattering (NTA), and Western blot. The results are as follows: Figure 2 .

[0046] Figure 2 Characterization and identification of exosomes for platelet membrane fusion. Transmission electron microscopy (TEM) showed that both IEVs and P-IEVs had a double membrane structure. In image A, the outer membrane of IEVs consisted of a thick bilayer of phospholipids and proteins, with an internal "cavity". In image B, P-IEVs showed a thin outer membrane enclosing the exosome vesicles. Figure 2 Figures C and D represent dynamic light scattering (NTA). In figure C, the average particle size of IEVs is 184.5 nm, with a main peak at 101.2 nm; in figure D, the average particle size of P-IEVs is 126.0 nm, with a main peak at 78.3 nm. Western blotting results in figures E and F show that the expression of platelet marker proteins in the P-IEVs group is significantly higher than that in the IEVs group.

[0047] The above results indicate that the platelet membrane successfully coated IEVs.

[0048] Application Examples This embodiment investigates the effect of P-IEVs on improving myocardial ischemia-related injury through intravenous injection of P-IEVs and IEVs in animal experiments.

[0049] A rat MIRI model was established. Rats were intraperitoneally injected with an anesthetic (sodium pentobarbital) and connected to a ventilator via endotracheal intubation. A left 4th intercostal incision was made to access the thoracic cavity and expose the heart. A 6-0 silk suture was passed through the superficial layer of the myocardium at the lower edge of the left atrial appendage, and a short PE-10 tube was placed between the blood vessel and the suture, ligating it to create ischemia (marked by ST segment elevation and whitening of the apex on ECG). The ligation was maintained for 30-45 minutes. The suture was cut, the PE tube was removed, and blood flow was restored (the myocardium regained its pink color). The layers were sutured, the tube was removed, and the rats were kept warm and resuscitated.

[0050] Within 10 minutes after reperfusion, 1 mg / mL P-IEVs or 300 μL of IEVs were administered via the tail vein. The MIRI group received an equal volume of PBS. Two hours after reperfusion, TTC staining was performed to detect the infarct area (IS / AAR) to assess the acute injury. Echocardiography and Masson staining were performed 28 days after reperfusion to evaluate the long-term repair of myocardial injury.

[0051] Cardiomyocytes were seeded in 96-well and 12-well plates. Within 10 minutes after the start of oxygenation, 200 μL of PBS, P-IEVs or IEVs were added. Cell viability was analyzed using a CCK-8 cell viability assay kit after OGD / R.

[0052] See Figure 3Intravenous injection of P-IEVs improved myocardial ischemia-reperfusion injury. Figure A shows TTC staining (MIRI represents myocardial ischemia-reperfusion injury, and Sham represents the sham-operated group). The results showed that both IEVs and P-IEVs reduced the myocardial infarction area after MIRI, and P-IEVs had a significantly stronger ability to reduce IS / AAR than IEVs (P<0.05). Figure B shows the echocardiographic results of cardiac function 28 days after MIRI. The results showed that the LVEF and LVFS of rats in the P-IEVs group were significantly higher than those in the MIRI and IEVs groups (P<0.01). Figure C shows the degree of myocardial fibrosis after MIRI as detected by Masson staining. The results showed that the myocardial fibrosis area in the P-IEVs group was significantly lower than that in the IR and IEVs groups (P<0.01). Figure D shows the cell viability of each group as detected by CCK-8. The results indicate that the cell viability of the P-IEVs group was significantly higher than that of the IEVs and IR groups, suggesting that platelet membrane fusion technology can significantly improve the ability of exosomes to resist hypoxic damage to cardiomyocytes (P<0.01).

[0053] The above results indicate that intravenously injected P-IEVs significantly improve myocardial ischemia-related injury compared to IEVs. Our research group successfully prepared a novel drug intermediate, platelet membrane fusion exosome intermediate (P-IEVs), using liposome extrusion-fusion technology. In vitro experiments showed that the uptake of P-IEVs in hypoxia-reoxygenation-injured cardiomyocytes and H9C2 cells was significantly higher than in the non-fusion group. After intravenous injection, the aggregation of P-IEVs in damaged myocardial tissue was significantly higher than in the IEVs group, and its ability to improve myocardial ischemia-related injury was also significantly higher. Therefore, using liposome extrusion-fusion technology, our research group obtained platelet membrane fusion exosome intermediate (P-IEVs). Preliminary animal experiments showed that, when injected via the tail vein, P-IEVs exhibited a stronger cardioprotective effect compared to IEVs.

[0054] In practice, P-IEVs extracted and prepared from rats were found to be equally effective in mice, significantly enhancing their ability to improve myocardial ischemia-related injury. This validates the cross-species application of this technology, further suggesting that the same technology could be applied to the treatment of myocardial ischemia-related injury in humans.

[0055] Although the present invention has been described in detail above, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention are within the scope of protection claimed by the present invention.

Claims

1. An intermediate material of a platelet membrane fusion exosome, characterized by, is a structure of platelet membrane-coated exosome.

2. The intermediate material of platelet membrane-fused exosomes according to claim 1, characterized in that, Prepared by the following steps: 1) ischemic preconditioning operation is performed on rats, venous blood is taken from the heart, and ischemic preconditioning serum-derived exosomes are prepared; 2) whole blood is taken from the abdominal aorta with an anticoagulant tube, and platelet membranes are prepared by gradient centrifugation method; 3) the exosomes obtained in step 1) and the platelet membranes obtained in step 2) are mixed, and a liposome extruder is repeatedly extruded to prepare an intermediate material of platelet membrane-fused exosomes.

3. The intermediate material of platelet membrane-fused exosomes of claim 2, wherein, In step 1), the operation of ischemic preconditioning on rats is as follows: pentobarbital is injected into the abdominal cavity of rats for anesthesia, then a suture is used to ligate the left anterior descending coronary artery 2-4 mm below the left atrium, the ligation line is loosened to reperfuse for 5 min after ischemia for 3-6 min, and the operation is repeated three times.

4. The intermediate material of platelet membrane-fused exosomes of claim 2, wherein, In step 1), the obtained venous blood is placed on ice for 20-40 minutes, and the exosomes are separated and purified by ultracentrifugation, then the serum is filtered, and the filtered serum is centrifuged at 100000g-120000g to obtain the precipitate, which is the ischemic preconditioning serum-derived exosomes, and the obtained exosomes are resuspended.

5. The intermediate material of platelet membrane-fused exosomes of claim 3, wherein, In step 1), the conditions of the ultracentrifugation method are as follows: first centrifuge at 3000g for 15 min to take the supernatant, then centrifuge at 12000g for 30 min to take the supernatant, then filter the serum using a filter with a pore size of 0.22 μm, and centrifuge the filtered serum twice using the Type100 Ti rotor of the centrifuge at 110000g for 70 min, to obtain the precipitate, which is the ischemic preconditioning serum-derived exosomes, and resuspend the exosomes with PBS.

6. The intermediate material of platelet membrane-fused exosomes of claim 2, wherein, In step 2), the gradient centrifugation method is as follows: the obtained whole blood is centrifuged twice at 200 g for 20 min, the supernatant is collected, prostaglandin E1 is added to prevent platelet activation, and then the blood is centrifuged at 900 g for 20 min to obtain platelet precipitate.

7. The intermediate material of platelet membrane-fused exosomes of claim 6, wherein, In step 2), the obtained platelet precipitate is resuspended in PBS containing 10 mM protease inhibitor to obtain a suspension; freeze in a-80℃ refrigerator for 2 hours, thaw at room temperature, and repeat the freeze-thawing three times to obtain platelet membranes.

8. The intermediate material of platelet membrane-fused exosomes according to claims 2-7, characterized in that, In step 3), the exosomes and platelet membranes are prepared into solutions with a concentration of 1 mg / mL, respectively, an equal volume of each solution is taken with a syringe, mixed and placed in a liposome extruder, the syringe is repeatedly extruded to make the mixture pass through membranes with pore sizes of 100 nm and 200 nm, and the intermediate material of platelet membrane-fused exosomes is obtained after extrusion.

9. The intermediate material of platelet membrane-fused exosomes of claim 8, wherein, The mixture is repeatedly extruded through a polycarbonate membrane with a pore size of 200 nm, and then repeatedly extruded through a polycarbonate membrane with a pore size of 100 nm, 12 times for each membrane, a total of 24 times.

10. Use of the intermediate material of any one of claims 1-9 in the preparation of a drug for improving myocardial injury after ischemia.

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