CXCR1 engineered exosome of targeted cardiac ischemia-reperfusion injury area as well as construction method and application of CXCR1 engineered exosome

Abstract

The invention discloses a CXCR1 engineered exosome targeting a cardiac ischemia reperfusion injury area as well as a construction method and application of the CXCR1 engineered exosome, and belongs to the technical field of biomedical engineering. The engineered exosome is loaded with a PDE4 inhibitor, can overexpress a CXCR1-lamp2b fusion protein, and can be chemotactic to a chemotactic factor IL-8 part through a chemotactic factor receptor CXCR1, so that the engineered exosome can be precisely targeted to a neutrophil aggregation part of ischemic myocardium; and the effect of relieving the heart ischemia-reperfusion injury is achieved through the carried PDE4 inhibitor. By utilizing the engineered exosome disclosed by the invention, inhibition on PDE4 subtypes of other organs can be avoided while myocardial ischemia reperfusion injury is relieved, side effects are reduced to the greatest extent, and efficient delivery of a PDE4 inhibitor is realized.

Description

Technical Field

[0001] The present invention relates to the field of biomedical engineering technology, and in particular to a CXCR1 engineered exosome targeting a cardiac ischemia-reperfusion injury region, and a construction method and application thereof. Background Art

[0002] Acute myocardial infarction (AMI) is a common type of fatal cardiovascular disease. Its incidence has remained high in recent years and has a trend of younger patients. The pathophysiological mechanism of AMI is that coronary artery occlusion leads to insufficient oxygen supply to cardiac tissue. The key to the treatment of AMI is to open the infarction-related coronary artery early and perform reperfusion therapy. However, after reperfusion therapy restores blood perfusion to ischemic myocardial tissue, ischemic damage to myocardial tissue not only cannot be alleviated, but is further aggravated. This phenomenon is called myocardial ischemia / reperfusion injury. Excessive activation of inflammatory response is the main pathophysiological process of cardiac ischemia-reperfusion injury, and neutrophils play the role of "vanguard" in this process. Excessive recruitment of leukocytes into the affected myocardium occurs in the initial stage after reperfusion, and neutrophils are the main invaders within the first 24 hours. Moderate neutrophil recruitment is conducive to post-infarction healing, while excessive infiltration is harmful. Therefore, reducing excessive neutrophil infiltration can limit reperfusion injury while maintaining beneficial healing processes.

[0003] Therapeutic strategies that inhibit excessive recruitment of neutrophils are key to reducing the inflammatory cascade of ischemia-reperfusion injury. Directly removing neutrophils and reducing the infiltration of circulating neutrophils into the myocardium may be a feasible strategy. Among them, neutrophils play an important role in maintaining homeostasis by phagocytizing cell fragments and clearing bacterial infections. Removing neutrophils may cause homeostasis imbalance in other organs in homeostasis, resulting in a series of side effects. Therefore, the strategy of reducing the infiltration of circulating neutrophils into the myocardium may be the best choice to reduce cardiac ischemia-reperfusion injury.

[0004] PDE4 is a family of cAMP (CAMP) hydrolases, and there are four subtypes of AD. Recent studies have shown that inhibiting PDE4B will reduce neutrophil-endothelial cell adhesion, and therefore can effectively improve inflammatory cell infiltration in mice and reduce I / R injury, but there is currently no PDE4B subtype inhibitor. Although the application of PDE4 pan-inhibitors can reduce the infiltration of neutrophils and the release of proinflammatory cytokines, that is, reduce the recruitment and infiltration of neutrophils after ischemia-reperfusion injury. However, the existing PDE4 pan-inhibitors can only non-selectively inhibit the various subtypes of PDE4 in the body, lacking targeting. Clinically, PDE4 pan-inhibitors will inhibit gastrointestinal PDE4D and induce gastrointestinal adverse events, which is a common side effect that limits its clinical use. In addition, due to the lack of targeting, PDE4 pan-inhibitors cannot achieve the ideal therapeutic effect by using them to reduce cardiac ischemia-reperfusion injury.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide a CXCR1 engineered exosome targeting the cardiac ischemia-reperfusion injury area and a construction method and application thereof. By constructing CXCR1 engineered exosomes, it is possible to avoid the inhibition of PDE4 subtypes in other organs while alleviating myocardial ischemia-reperfusion injury, minimize side effects, and achieve efficient delivery of PDE4 inhibitors.

[0007] The present invention is achieved in that:

[0008] In a first aspect, the present invention provides an engineered exosome targeting the ischemia-reperfusion injury area of ​​the heart, which is loaded with a PDE4 inhibitor and overexpresses a CXCR1-lamp2b fusion protein.

[0009] In a second aspect, the present invention provides a method for constructing the above-mentioned engineered exosomes, which comprises: mixing targeted engineered exosomes overexpressing CXCR1-lamp2b fusion protein with Cilomilast-DSPE-PEG2000, and obtaining engineered exosomes after incubation.

[0010] In a third aspect, the present invention provides the use of the above-mentioned engineered exosomes or the engineered exosomes obtained by the construction method in the preparation of a drug for reducing cardiac ischemia-reperfusion injury.

[0011] In a fourth aspect, the present invention provides a pharmaceutical composition as described above, comprising the engineered exosomes as described above and pharmaceutically acceptable excipients.

[0012] The present invention has the following beneficial effects:

[0013] The engineered exosomes of the present invention are loaded with PDE4 inhibitors and can overexpress CXCR1-lamp2b fusion protein. The engineered exosomes can chemotactic to the chemokine IL-8 site through the chemokine receptor CXCR1, and then accurately target the neutrophil aggregation site of the ischemic myocardium, and then achieve the effect of reducing cardiac ischemia-reperfusion injury through the carried PDE4 inhibitor. The engineered exosomes of the present invention can achieve the effect of reducing myocardial ischemia-reperfusion injury while avoiding the inhibition of PDE4 subtypes of other organs, minimize side effects, and achieve efficient delivery of PDE4 inhibitors. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0015] Figure 1 It is a technical flow chart of the present invention;

[0016] Figure 2 The physical properties and safety evaluation test results of the engineered exosomes in Experimental Example 1;

[0017] Figure 3 The characterization results of Cilomilast-DSPE-PEG2000 and engineered exosomes in Experimental Example 2;

[0018] Figure 4 The results of the in vitro immunofluorescence experiment in Experimental Example 3;

[0019] Figure 5 The results of animal live imaging in Experimental Example 4;

[0020] Figure 6 These are the cardiac function test results of mice in different experimental groups in Experimental Example 5. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0022] Due to the current lack of PDE4B inhibitors and the lack of targeted PDE4 pan-inhibitors, there are still certain challenges in inhibiting the inflammatory cascade caused by cardiac neutrophil recruitment, so the inventors first thought of loading drugs with exosomes. Exosomes themselves are a type of extracellular vesicle (EV), which are nanoscale, lipid-membrane-wrapped vesicles (about 30-150nm in diameter) secreted by most cells. Due to their unique physical and chemical properties, exosomes have been a "super courier" since their birth. They are not only non-immunogenic and low in toxicity, but also able to penetrate into tissues, diffuse into the blood, and even cross the blood-brain barrier. Exosomes have been shown to be able to naturally transport various cellular metabolic cargoes, such as proteins, lipids, transcription factors, miRNAs, and mRNAs. It is this natural role of exosomes as "natural delivery vehicles" that has led to a lot of interest in the development of their potential therapeutic uses.

[0023] However, when exosomes are used as drug delivery vehicles to deliver sustained therapeutic doses to target tissues, there is also the problem of rapid clearance by the body. Even if injected subcutaneously, most of the injected exosomes will be cleared by the reticuloendothelial system or lymphatic transport. At the same time, abnormal tissue or plasma levels of some exosomes diffused from the injection site will cause safety issues of non-target effects on surrounding organs. Therefore, exosome delivery of drugs also needs to improve targeting to improve efficacy and reduce unexpected side effects.

[0024] Neutrophils have chemotaxis, which is specifically manifested in that they can pass through vascular endothelial cells through molecular interactions with endothelial cells, sense the concentration gradient of chemokines, and migrate to the location where chemokines are released. This is the biological basis of neutrophil-mediated targeted delivery. In order to accurately target the target area, the present invention proposes to construct a targeted engineered exosome based on the above chemotaxis, and the targeted exosome can overexpress CXCR1-lamp2b fusion protein.

[0025] Among them, CXCR1 is a chemokine receptor that can target and bind to chemokines. Since IL-8 is the main cytokine involved in recruiting neutrophils to the site of injury or infection, and is also a ligand of CXCR1, the present invention constructs targeted engineered exosomes of CXCR1 that can overexpress on the membrane surface, so that the targeted engineered exosomes, under the chemotactic effect of CXCR1, chemotaxis and aggregation in the ischemic reperfusion myocardial site where IL-8 is secreted, and distributed in the neutrophil-enriched site in the myocardium, thereby achieving precise targeting of the ischemic reperfusion injury site of the heart.

[0026] Lamp2b is a member of the lysosome-associated membrane protein (LAMP) family, which is mainly localized in lysosomes and endosomes in cells and is also expressed in large quantities on the surface of exosomes. In the present invention, CXCR1 is fused with lamp2b, and lamp2b is used to display CXCR1 on the surface of exosomes.

[0027] In order to achieve the therapeutic effect, the surface of the targeted engineered exosomes of the present invention is also loaded with a PDE4 inhibitor. By performing CXCR1 gene editing on the exosomes, CXCR1 can be expressed on the surface of the exosomes, accurately targeting the site of cardiac ischemia-reperfusion injury, and then the PDE4 inhibitor loaded on the surface of the engineered exosomes is efficiently delivered to the cardiac ischemia-reperfusion area.

[0028] However, during the construction process, the inventors found that not all existing PDE4 inhibitors can achieve good therapeutic effects, mainly because not all existing PDE4 inhibitors can be well loaded on the surface of the above-mentioned targeted engineered exosomes. In order to solve this problem, the present invention first uses DSPE-PEG2000 that can be inserted into the phospholipid bilayer of exosomes as a loading material, but it is found that it cannot carry drugs well. On this basis, the inventors modified the DSPE-PEG2000 material with groups, and then found that among the many PDE4 inhibitors, only Cilomilast can be well carried by DSPE-PEG2000 and successfully inserted into the phospholipid bilayer of exosomes.

[0029] Specifically, the modification of the DSPE-PEG2000 material in the present invention is to modify the amino groups of DSPE-PEG2000, and then mix it with Cilomilast to generate Cilomilast-DSPE-PEG2000 after reaction.

[0030] The engineered exosomes of the present invention are composed of targeted engineered exosomes and Cilomilast loaded on their surface. The membrane surface of the targeted engineered exosomes can overexpress CXCR1. Through the combination of targeted engineered exosomes and Cilomilast, the effects of safely, efficiently and targetedly inhibiting neutrophils in the ischemia-reperfusion area of ​​the heart and alleviating ischemia-reperfusion injury of the heart can be achieved.

[0031] Based on the above-mentioned engineered exosomes, the present invention also provides a method for constructing the engineered exosomes, such as Figure 1 The above construction method includes the following steps:

[0032] S1. Construct a CXCR1-lamp2b fusion expression vector, transfect the fusion expression vector into mesenchymal stem cells, collect the supernatant of the cultured cells, and then purify and extract to obtain targeted engineered exosomes.

[0033] In the present invention, exosomes are derived from mesenchymal stem cells. Compared with exosomes from other sources, exosomes from mesenchymal stem cells have low immunogenicity, reducing the risk of immune rejection during transplantation or treatment, and other advantages.

[0034] For the preparation of the above-mentioned targeted engineered exosomes, conventional technical means in the art can be used, as long as the method can obtain engineered exosomes that overexpress CXCR1 on their surface.

[0035] Optionally, the above preparation method may be:

[0036] (1) The gene fragment of CXCR1-lamp2b is synthesized by a gene synthesis company and introduced into an expression vector to obtain a recombinant vector into which the gene fragment of CXCR1-lamp2b is inserted;

[0037] (2) The above recombinant vector is transferred into competent cells, plasmid is extracted, and then the extracted plasmid is transfected into mesenchymal stem cells for transfection, the supernatant of the cultured cells is collected, impurities are removed, and further concentrated and purified.

[0038] Among them, CXCR1 and lamp2b are existing gene fragments, which can be searched and obtained from common databases. The transfection method can be electroporation, lentiviral infection and liposome transfection, and those skilled in the art can select according to actual conditions.

[0039] S2. Preparation of Cilomilast-DSPE-PEG2000

[0040] The NH2-modified DSPE-PEG2000 and Cilomilast were added at a feed ratio of 1:1, and DSPE-PEG2000 and Cilomilast were reacted, and the addition ratio of the two was 1 mol:1 mol.

[0041] S3. The targeted engineered exosomes obtained in S1 and S2 were mixed with Cilomilast-DSPE-PEG2000, and the engineered exosomes loaded with Cilomilast were obtained after incubation.

[0042] In some embodiments, the incubation conditions are: incubation temperature is 2-4°C, and incubation time is: 24h-36h. To further purify Cilomilast-Exo, the obtained Cilomilast-Exo solution is ultracentrifuged at 100,000g for 2h at 4°C, then washed once with PBS buffer (10mM, pH 7.4) and resuspended in 2ml PBS buffer (10mM, pH 7.4).

[0043] In some embodiments, the ratio of Cilomilast-DSPE-PEG2000 to engineered exosomes overexpressing CXCR1-lamp2b fusion protein is 5 (mg): 1×10 11 (piece)~5(mg):2×10 11 (indivual).

[0044] Through the above construction method, CXCR1 engineered exosomes loaded with Cilomilast can be obtained. Since the CXCR1 gene is edited on the exosomes, it can be chemotactic to the site where the chemokine IL-8 is enriched through the chemokine receptor CXCR1, thereby solving the defects of the existing exosome drug delivery technology, so that the modified exosomes can be targeted to the cardiac ischemia-reperfusion area. At the same time, the CXCR1 engineered exosomes can overcome the current situation of the lack of PDE4B subtype inhibition through the Cilomilast carried, reduce the adhesion of neutrophils, and reduce the inflammatory cascade. Experiments have shown that the CXCR1 engineered exosomes prepared in the present invention can achieve the reduction of myocardial ischemia-reperfusion injury while avoiding the inhibition of PDE4 subtypes of other organs, minimize side effects, and achieve efficient delivery of Cilomilast through gene editing and drug loading.

[0045] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0046] Example 1

[0047] This example is a method for constructing engineered exosomes, and the specific steps are as follows:

[0048] Mouse Cxcr1 and lamb2b genes were combined to construct a fusion gene, and the PCR-amplified gene fragment was cloned into a plasmid. Mesenchymal stem cells (1.5×10 6 ) were inoculated in a 6 cm culture dish for 12 h, and then the constructed fusion gene plasmid was transfected into mesenchymal stem cells using the lipo3000 kit for 24 h. The culture medium was replaced with serum-free medium, and the supernatant of the transfected mesenchymal stem cells was collected and exosomes were collected by ultra-high speed centrifugation.

[0049] DSPE-PEG2000 and Cilomilast modified with NH2 were added at a ratio of 1:1, and DSPE-PEG2000 and Cilomilast were reacted at a ratio of 1 mol:1 mol. The obtained targeted engineered exosomes were mixed with Cilomilast-DSPE-PEG2000, and engineered exosomes loaded with Cilomilast were obtained after incubation. The incubation temperature was 2°C to 4°C, and the incubation time was 24h to 36h.

[0050] To further purify Cilomilast-Exo, the resulting Cilomilast-Exo solution was ultracentrifuged at 100,000 g for 2 h at 4°C, then washed once with PBS buffer (10 mM, pH 7.4) and resuspended in 2 ml PBS buffer (10 mM, pH 7.4).

[0051] Comparative Example 1

[0052] The construction method is basically the same as that of Example 1, except that the PDE4 inhibitor used in this experiment is roflumilast.

[0053] Comparative Example 2

[0054] The construction method is basically the same as that of Example 1, except that the PDE4 inhibitor used in this experiment is Rolipram.

[0055] Although the PDE4 inhibitors selected in Comparative Examples 1 and 2 are also commercial PDE4 pan-inhibitors, these inhibitors cannot be carried by DSPE-PEG2000 and successfully inserted into the phospholipid bilayer of exosomes.

[0056] Experimental Example 1

[0057] This experiment is to evaluate the physical properties and safety of the engineered exosomes prepared in Example 1, including HE staining of various organs and the most suitable storage conditions for engineered exosomes. The specific experimental steps are as follows:

[0058] (1) HE staining of various organs: After the mouse model of cardiac ischemia-reperfusion injury (I / R) was established, engineered MSC exosomes were injected into the model group through the tail vein. After 24 hours, the liver, lung, kidney and other organs of healthy mice and mice injected with engineered exosomes after modeling were taken out, fixed with 4% paraformaldehyde for 16 hours, and then paraffin sections were made and stained with the Bio-Tech HE staining kit. The test results are shown in Figure 2. Figure 2 As shown in A: There is no significant difference in the liver, lung, and kidney sections of the two, indicating the safety of the engineered exosomes to various organs.

[0059] (2) Optimal storage conditions for engineered exosomes: The synthesized engineered exosomes were divided into four groups and stored at -80°C, 4°C, -20°C, and room temperature (21°C) for 1 week. Then, their size and concentration were detected using NTA, and the morphology and structure of the exosomes were observed using an electron microscope.

[0060] The experimental results are as follows Figure 2 As shown in B: The size of the engineered exosomes did not change significantly under the four conditions, but the number of exosome particles was the largest at room temperature and 4°C. Therefore, it is recommended that the engineered exosomes be stored at 4°C for a short period of time.

[0061] Experimental Example 2

[0062] This experiment uses nuclear magnetic resonance to characterize the Cilomilast-DSPE-PEG2000 compound, and also uses nanoflow cytometry, WB, etc. to characterize the engineered exosomes. The specific experimental steps are as follows:

[0063] (1) Figure 3 A is the result of H NMR spectrum analysis: Cilomilast-DSPE-PEG2000 was characterized by H NMR: The synthesized compound DSPE-PEG-2000 was analyzed by H NMR spectrum to determine the molecular structure of the synthesized compound. The H NMR result showed the successful coupling of DSPE-PEG-2000 and Cilomilast.

[0064] (2) Figure 3 B is the result of nanoflow cytometry: the fluorescently labeled CXCR1-lamb2b-eGFP plasmid was transfected into mesenchymal stem cells, and the transfected and untransfected mesenchymal stem cell exosomes were collected respectively. After the isolated exosomes were synthesized with Cilomilast-DSPE-PEG2000 to synthesize CXCR1 engineered MSC exosomes, the obtained engineered MSC exosomes were tested for particle size and CXCR1 overexpression by nanoflow cytometry. The nanoflow cytometry results showed that the particle size of the engineered exosomes was generally within 100-200nm, which was consistent with the properties of exosomes. Among them, compared with ordinary mesenchymal stem cell exosomes that were not transfected with plasmids, the fluorescence of overexpressed (OE) exosomes transfected with CXCR1-lamb2b-eGFP plasmids was significantly enhanced, indicating that CXCR1 was successfully overexpressed in engineered exosomes.

[0065] (3) Figure 3C is the result of Western Blot experiment: As mentioned above, the exosomes of mesenchymal stem cells after transfection and those without plasmid transfection were collected respectively, and the isolated exosomes were synthesized with Cilomilast-DSPE-PEG2000 to synthesize CXCR1 engineered MSC exosomes. Then, RIPA (protein lysis buffer), protease inhibitors and phosphatase inhibitors were added to extract the proteins of the engineered exosomes. The overexpression of CXCR1 and the expression of exosome markers CD9, TSG101 and CD63 were verified again by Western Blot experiment.

[0066] (4) Figure 3 D is the result of immunofluorescence detection: CY3-lysine fluorescence was connected to Cilomilast-DSPE-PEG2000 to synthesize fluorescent DSPE-PEG2000-Cilomilast-CY3. After the isolated CXCR1 overexpression exosomes were synthesized with DSPE-PEG2000-Cilomilast-CY3 into engineered MSC exosomes, the engineered exosomes were captured by CD63 immunomagnetic beads, and the presence of CY3 fluorescence was observed by laser confocal microscopy. The experimental results showed that CY3 fluorescence could be successfully observed, indicating that the synthesized compound was successfully coupled with the overexpressed CXCR1 exosomes.

[0067] The above results indicate that DSPE-PEG-2000 was successfully synthesized with Cilomilast, and that it is feasible to successfully isolate exosomes overexpressing CXCR1, and that the synthesized compounds were successfully coupled with exosomes overexpressing CXCR1.

[0068] Experimental Example 3

[0069] This experiment is an in vitro immunofluorescence experiment to verify that CXCR1 engineered MSC exosomes (PKH26) have a high affinity with neutrophils and can be taken up by neutrophils (Ly6G+).

[0070] Neutrophils were isolated from the femur and tibia of mouse bone marrow by centrifugation at 300 x g for 10 min and suspended at 1 x 10 in RoboSepTM buffer. 8 After adding rat serum to 100 cells / ml, neutrophils were isolated using a neutrophil negative selection kit (Stemcell, 19762RF). The isolated CXCR1 overexpressing engineered exosomes were labeled with PKH26 dye, and after being added to the isolated neutrophils for 24 hours, Ly6G was used to label the neutrophils.

[0071] Figure 4 Laser confocal microscopy was used to observe the uptake of exosomes by cells. The experiment suggested that neutrophils can successfully take up engineered exosomes.

[0072] Experimental Example 4

[0073] This experiment is an animal experiment of the engineered exosomes prepared in Example 1. The specific experimental steps are as follows:

[0074] First, adult C57BL / 6 mice were divided into four groups, and then the cardiac ischemia-reperfusion injury (I / R) model was constructed in two of the groups. The isolated engineered MSC exosomes were stained with DiR (MCE) dye for 15 minutes, and then the excess dye was washed off. DiR-labeled Cilomilast CXCR1 engineered MSC exosomes and PBS were injected into the tail vein of the experimental group and the control group mice, respectively. The four groups of mice were imaged in vivo at 6h, 12h, 24h, and 72h, respectively, to observe the aggregation site of CXCR1 engineered exosomes.

[0075] Test results such as Figure 5 As shown in the figure, it can be seen that most of the CXCR1 engineered MSC exosomes injected after modeling can accumulate in the heart after myocardial ischemia-reperfusion injury, and a small part accumulates in the liver. In the Sham group, the mice were mainly accumulated in the liver after injection of labeled CXCR1 engineered exosomes.

[0076] Experimental Example 5

[0077] To further confirm the efficacy of the engineered exosomes, this experiment divided adult C57BL / 6 mice into three groups, and then constructed a cardiac ischemia-reperfusion injury (I / R) model in two of the groups of mice. The experimental group mice were injected with Cilomilast-loaded CXCR1 engineered MSC exosomes and PBS into the tail vein, and finally the cardiac function of the three groups was tested.

[0078] like Figure 6 As shown, cardiac ultrasound was performed on mice 7 days after modeling, and compared with the anterior wall activity, EF (ejection fraction), and ES (short axis shortening rate) of mice with simple I / R modeling, the cardiac function of mice injected with CXCR1 engineered MSC exosomes loaded with Cilomilast was significantly restored.

[0079] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An engineered exosome targeting the ischemia-reperfusion injury area of ​​the heart, characterized in that: The engineered exosomes are loaded with a PDE4 inhibitor and overexpress a CXCR1-lamp2b fusion protein.

2. The engineered exosome according to claim 1, characterized in that The PDE4 inhibitor is loaded on the surface of the engineered exosomes via DSPE-PEG2000.

3. The engineered exosome according to claim 2, characterized in that The PDE4 inhibitors include Cilomilast.

4. The engineered exosome according to claim 1, characterized in that The engineered exosomes are derived from mesenchymal stem cells.

5. The method for constructing engineered exosomes according to any one of claims 1 to 4, characterized in that: include: The targeted engineered exosomes overexpressing the CXCR1-lamp2b fusion protein are mixed with Cilomilast-DSPE-PEG2000, and the engineered exosomes are obtained after incubation; Preferably, the incubation conditions are: incubation temperature is 2°C to 4°C, and incubation time is 24h to 36h.

6. The construction method according to claim 5, characterized in that: The preparation method of Cilomilast-DSPE-PEG2000 comprises: modifying the amino group of DSPE-PEG2000, and then mixing it with Cilomilast to generate Cilomilast-DSPE-PEG2000 after reaction; Preferably, the group modification is: amino modification of DSPE-PEG2000; Preferably, the modified DSPE-PEG2000 reacts with Cilomilast, and the addition ratio of the two is 1 mol:1 mol.

7. The construction method according to claim 5, characterized in that: The ratio of Cilomilast-DSPE-PEG2000 to targeted engineered exosomes is 5 (mg): 1×10 11 ~2×10 11 (indivual).

8. The construction method according to claim 5, characterized in that: The method for preparing the targeted engineered exosomes comprises: transfecting a CXCR1-lamp2b fusion expression vector into mesenchymal stem cells, collecting the supernatant of the cultured cells, and then purifying and extracting to obtain the targeted engineered exosomes; Preferably, the transfection includes lentiviral infection and liposome transfection.

9. Use of the engineered exosomes according to any one of claims 1 to 4 or the engineered exosomes obtained by the construction method according to any one of claims 5 to 8 in the preparation of a drug for alleviating cardiac ischemia-reperfusion injury.

10. A pharmaceutical composition, characterized in that The invention comprises the engineered exosomes according to any one of claims 1 to 4 or the engineered exosomes obtained by the construction method according to any one of claims 5 to 8, and pharmaceutically acceptable excipients.

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