Application of cell membrane-modified engineered hypoxic stem cell exosomes in the preparation of drugs for treating myocardial infarction

Abstract

This invention discloses the application of cell membrane-modified engineered hypoxic stem cell exosomes in the preparation of drugs for treating myocardial infarction. Mesenchymal stem cells are pretreated with hypoxia, and the conditioned medium for these cells is collected. Exosomes are then collected by differential centrifugation, and cell membranes are coated onto the surface of the exosomes to obtain hybrid nanovesicles, i.e., cell membrane-modified engineered hypoxic stem cell exosomes. This invention overcomes the lack of targeting in MSC-Exo therapy and solves the problem that current exosome angiogenesis-promoting functions need further enhancement and that poor targeting leads to unsatisfactory therapeutic effects in myocardial infarction.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to the application of cell membrane-modified engineered hypoxic stem cell exosomes in the preparation of drugs for treating myocardial infarction. Background Technology

[0002] Cardiovascular disease is a global health problem, and its prevalence continues to rise in developing countries. [1,2][1,2][1,2] Among cardiovascular diseases, ischemic heart disease is a leading cause of death and disability. Ischemic heart disease is caused by myocardial infarction (MI) following coronary artery blockage. After the blood vessel is blocked, blood flow and oxygen levels decrease, leading to ischemic necrosis of myocardial cells, decreased myocardial contractility, and ultimately the formation of fibrotic scars in the heart, affecting cardiac function. Myocardial infarction has a very high mortality and disability rate, ranking first among all non-communicable diseases in mortality, even surpassing cancer. Studies have shown that angiogenesis can effectively improve the blood supply to ischemic myocardium, so timely vascular reconstruction is necessary after myocardial infarction to restore cardiac function. Currently, the most commonly used clinical treatment for myocardial infarction is coronary artery bypass surgery. Although this is an effective method of vascular reconstruction, it is highly invasive and carries the potential for serious complications such as bleeding and reperfusion injury. Some drug therapies, such as statins, are also commonly used in the treatment of myocardial infarction; however, the efficacy of these drugs varies from person to person, and their impact on survival rates after myocardial infarction remains controversial. Studies have shown that the tissue regeneration capacity of mesenchymal stem cells (MSCs) provides a new pathway for the formation of new blood vessels in ischemic heart disease.

[0003] Mesenchymal stem cells (MSCs) are adult stem cells with self-renewal and multi-lineage differentiation capabilities. They can be isolated from bone marrow, adipose tissue, umbilical cord blood, peripheral blood, and almost all tissues in adults, and can adhere to cell culture flasks under standard culture conditions. MSCs possess advantages such as self-renewal, multi-lineage differentiation potential, and low immunogenicity upon autologous transplantation, effectively reducing or avoiding immune rejection and regulating the microenvironment in host tissues. Furthermore, they can parasecrete soluble factors that promote angiogenesis, making MSCs an effective tool for treating ischemic heart disease.

[0004] Exosomes are a subtype of extracellular vesicles originating from the inward budding of intracellular lysosomal granules, producing multivesicular bodies. The fusion of the outer membrane of these multivesicular bodies with the cell membrane ultimately leads to the production of exosomes. These vesicles typically range in diameter from 30 to 200 nanometers and are known to transport a range of biological compounds, such as cytokines, mRNA, miRNAs, and proteins. Exosomes regulate many biological processes through the binding of surface ligands to target cells and the delivery of their internal contents, often reflecting the functional properties of their originating cells. Mesenchymal stem cells (MSCs) play a crucial role in the treatment of myocardial infarction due to their potent paracrine capabilities, including promoting angiogenesis, suppressing inflammation, and modulating the immune system. Currently, exosomes derived from MSCs have been shown to have potential applications in several diseases. For example, miR2-21-5p enhances the expression of calcium-treated genes hECT, LTCC, and SERCA2 through PI3K, thereby improving the contractility of cardiac tissue; miR-221 significantly reduces apoptosis of rat ventricular cardiomyocytes by reducing p53 and p53-upregulated PUMA; and miR-302d-3p reduces inflammation, myocardial fibrosis, and apoptosis in infarcted areas by inhibiting NFκB pathway-related MD2 and BCL6 levels.

[0005] Oxygen concentration is considered crucial for the proliferation, differentiation, and paracrine function of mesenchymal stem cells (MSCs). However, under in vitro culture conditions, MSCs are typically exposed to normoxic conditions (21%), which differs significantly from the oxygen concentration found in vivo under natural physiological conditions. Studies have found that placing MSCs in a hypoxic environment of 1–5% enhances their stemness and differentiation potential, making this a promising cell preparation method in the field of tissue regeneration. Furthermore, hypoxic pretreatment has been widely reported to enhance the paracrine activity of stem cells. A recent study extracted exosomes from MSCs cultured in a medium similar to that used for peripheral artery disease (0% FBS, 1% O2) and found that these exosomes contained numerous pro-angiogenic factors beneficial to ischemic tissues. Another study using an infarcted cardiac model found that exosomes derived from MSCs after hypoxic treatment exhibited increased angiogenesis, decreased cardiomyocyte apoptosis, and increased recruitment of cardiac progenitor cells.

[0006] Therefore, hypoxia pretreatment of mesenchymal stem cells (MSCs) can enhance the paracrine angiogenesis activity of MSCs. However, the effective delivery of exosomes in vivo is a limiting factor for their clinical application. It is noteworthy that after exosomes are injected into the body via the tail vein, they rapidly accumulate in organs such as the liver, spleen, and kidneys, and are quickly excreted through the urinary system or phagocytosed by reticuloendothelial cells. Their distribution in cardiac tissue is very low. Therefore, to improve the therapeutic effect of exosomes, it is necessary to prolong their retention time in myocardial tissue and enhance their targeting specificity to the myocardium. Summary of the Invention

[0007] One objective of this invention is to provide a cell membrane-modified engineered hypoxic stem cell exosome (hybrid nanovesicle), its preparation method, and its application. This invention aims to address the current problems of exosomes, such as the need for further enhancement of their angiogenesis-promoting function and poor targeting, which lead to unsatisfactory therapeutic effects in myocardial infarction.

[0008] The cell membrane-modified engineered hypoxic stem cell exosomes provided by this invention include hypoxic-treated mesenchymal stem cell exosomes and the cell membrane covering their surface.

[0009] The mass ratio of the cell membrane to the exosomes is 25-75:50-200;

[0010] The cell membrane-modified hypoxic stem cell exosomes have a particle size of 120-160 nm.

[0011] The cell membrane is a membrane of at least one of the following: red blood cells, macrophages, platelets, stem cells, and tumor cells.

[0012] The cell membrane-modified engineered hypoxic stem cell exosomes were prepared by a method comprising the following steps: Mesenchymal stem cells were pretreated with hypoxia, and the conditioned medium of the mesenchymal stem cells was collected. Exosomes were collected by differential centrifugation, and cell membranes were coated on the surface of the exosomes to obtain hybrid nanovesicles, namely cell membrane-modified engineered hypoxic stem cell exosomes.

[0013] Taking the macrophage membrane as an example, the intrinsic surface protein LAF-1 of the macrophage membrane (MM) can bind to vascular cell adhesion molecules (ICAM-1, VCAM-1). Vascular cell adhesion molecules are significantly increased in damaged endothelial cells after myocardial infarction. Therefore, the macrophage membrane (MM) can achieve targeting of damaged myocardium.

[0014] The above method specifically includes the following steps: 1) Hypoxia pretreatment of mesenchymal stem cells When mesenchymal stem cells are cultured and the cells reach 80-90% confluence, the original culture medium is discarded, the cells are washed with PBS, and replaced with serum-free DMEM / F12 culture medium. The cells are then grown in a cell culture incubator under 0.1%-8% oxygen conditions, and the conditioned medium is collected. 2) Preparation of exosomes The obtained conditioned medium was centrifuged for the first time to remove cell debris and collect the supernatant. It was then centrifuged for the second time to remove larger vesicles and apoptotic bodies and collect the supernatant again. The supernatant was then centrifuged at high speed and discarded. The precipitate was resuspended in PBS, centrifuged, and the supernatant was discarded. The precipitate was then collected as exosomes (Exo). 3) Preparation of hybrid nanovesicles, i.e., cell membrane-modified engineered hypoxic stem cell exosomes. Exosomes are mixed with cell membranes, vortexed, and extruded to form hybrid nanovesicles.

[0015] In step 1) of the above method, the growth time can be 2-4 days, specifically 2 days; In step 2) of the above method, the conditions for the first centrifugation can be: centrifugation at 500 g for 10-15 min; The conditions for the second centrifugation can be: centrifugation at 10,000 g for 30-40 min; The conditions for ultracentrifugation can be: 100,000 g for 70-90 min; After resuspending, centrifugation can be performed at 100,000 g for 2-3 hours. Step 2) of the above method further includes resuspending the obtained precipitate in an ultracentrifuge tube with 200-300 µl of PBS to obtain an Exo suspension, and storing it at -80℃ for later use.

[0016] In step 3) of the above method, the mass ratio of cell membrane to exosomes can be 25-75:50-200; The cell membrane solution was mixed with the exosome solution; The extrusion molding operation is as follows: the mixed exosomes and cell membranes are added to the two syringes on both sides of the Avanti micro extruder and extruded back and forth at least 30 times; The extruder is loaded with a polycarbonate porous membrane with a pore size of 0.4 µm in the middle.

[0017] The resulting hybrid nanovesicles can have a particle size of 120-160 nm, specifically 150 nm.

[0018] The application of the aforementioned hybrid nanovesicles, namely cell membrane-modified engineered hypoxic stem cell exosomes, in the preparation of drugs for treating myocardial infarction also falls within the scope of protection of this invention.

[0019] The present invention also provides a drug for treating myocardial infarction.

[0020] The drug for treating myocardial infarction includes the aforementioned hybrid nanovesicles, namely cell membrane-modified engineered hypoxic stem cell exosomes.

[0021] This invention pre-treats mesenchymal stem cells with hypoxia, collects the conditioned medium from the mesenchymal stem cells, collects exosomes by differential centrifugation, and coats the exosomes with a cell membrane capable of targeting ischemic sites to prepare hybrid nanovesicles. First, the properties of the exosomes are characterized; then, in vitro cell experiments are used to study the effects of hybrid nanovesicles on endothelial cell survival and function; finally, the effect of hybrid nanovesicles on restoring cardiac function in mice with myocardial infarction is investigated.

[0022] This invention overcomes the deficiency of MSC-Exo therapy in lack of targeting and solves the problem that the current exosome angiogenesis function needs to be further improved and the targeting is poor, resulting in unsatisfactory treatment effects in myocardial infarction. Attached Figure Description

[0023] Figure 1 This is a transmission electron microscope (TEM) image of Exo in Embodiment 2 of the present invention.

[0024] Figure 2 This is a particle size and potential diagram of Exo in Example 3 of the present invention.

[0025] Figure 3 This is a diagram illustrating how Exo promotes endothelial cell migration in Example 4 of the present invention.

[0026] Figure 4 This is a diagram illustrating how Exo promotes endothelial cell tube formation in Example 5 of the present invention.

[0027] Figure 5 This is a TEM image of the hybrid nanovesicles in Example 7 of the present invention.

[0028] Figure 6 This is a diagram showing the particle size and potential of the hybrid nanovesicles in Example 8 of the present invention.

[0029] Figure 7 This is a diagram of myocardial cell uptake of hybrid nanovesicles in Example 9 of the present invention.

[0030] Figure 8 This is a diagram illustrating how hybrid nanovesicles promote endothelial cell migration in Example 10 of the present invention.

[0031] Figure 9 This is a diagram illustrating how hybrid nanovesicles promote endothelial cell tube formation in Example 11 of this invention.

[0032] Figure 10 This is a distribution diagram of the hybrid nanovesicles in the heart of a mouse with myocardial infarction after being injected into the body in Example 12 of the present invention.

[0033] Figure 11 This is an echocardiogram of mice with myocardial infarction treated with hybrid nanovesicles on day 15 in Example 13 of this invention.

[0034] Figure 12 This is an echocardiogram of mice with myocardial infarction treated with hybrid nanovesicles on day 28 in Example 13 of this invention.

[0035] Figure 13 This is a diagram illustrating how hybrid nanovesicles promote angiogenesis in mice with myocardial infarction in Example 14 of this invention.

[0036] Figure 14 The image shows the H&E staining of the major organs of mice with myocardial infarction by hybrid nanovesicles in Example 15 of this invention. Detailed Implementation

[0037] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0038] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0039] Example 1: Preparation of exosomes (Exo) Culture mesenchymal stem cells until they reach 80-90% confluence. Discard the original culture medium, wash the cells with PBS, and replace with serum-free DMEM / F12 medium. Incubate the cells in cell culture incubators under 21% oxygen and 5% oxygen conditions for two days, respectively. Then collect the conditioned medium under the corresponding conditions and store it at -80°C for later use.

[0040] After collecting a certain amount of conditioned medium, the two conditioned mediums mentioned above were taken out of the -80℃ freezer and placed in a 4℃ freezer overnight to thaw. Before the experiment, the conditioned medium was first filtered through a syringe filter and then used for the preparation of exosomes.

[0041] A certain amount of the two conditioned media were centrifuged at 500 g for 15 min to remove cell debris. The supernatant was collected and centrifuged at 10,000 g for 40 min to remove larger vesicles and apoptotic bodies. The supernatant was collected again, transferred to an ultracentrifuge tube, and centrifuged at 100,000 g for 80 min. The supernatant was discarded, and the precipitate was resuspended in sterile PBS and centrifuged at 100,000 g for 3 h. The supernatant was discarded, and the final precipitate was the exosomes (Exo) used in this study. 250 µl of PBS was added to the ultracentrifuge tube to resuspend the Exo suspension. The concentration was measured using a BCA kit and stored at -80℃ for later use.

[0042] Example 2: Preparation of exosome TEM samples First, place a 200-mesh copper grid with a carbon support film on a sealing film. Take 15µl of Exo (after resuspending) extracted by differential centrifugation and drop it onto the copper grid. Let it stand at room temperature for 4 hours to allow the sample on the copper grid to dry. Then, add 2% phosphotungstic acid staining solution to the copper grid containing the sample and stain for 15 min. Blot off the excess staining solution with filter paper and observe its morphology under a transmission electron microscope.

[0043] Figure 1 This is a transmission electron microscope (TEM) image of Exo.

[0044] As can be seen from the figure, the exosomes prepared under both conditions are cup-shaped structures.

[0045] Example 3: Measurement of exosome particle size and potential A certain amount of 150 µg / ml Exo was taken and the particle size and potential of the nanoparticles were measured using a Malvern Zetasizer Nano ZS (DLS) dynamic light scattering particle size analyzer.

[0046] Figure 2 The diagram shows the particle size and potential of Exosomes. The average particle size of the exosomes was measured to be 105.7 nm, and the average potential was -11.2 mV.

[0047] Example 4: The process by which Exo promotes endothelial cell migration To further evaluate the potential of collected normoxic exosomes (21% Exo) and hypoxic exosomes (hy Exo) from MSCs to promote endothelial cell angiogenesis, we conducted an endothelial cell migration assay. 1640 basal medium was used as the control group, while 21% Exo and hy Exo, collected for 2 days at a concentration of 150 µg / ml, were used as the experimental groups. The endothelial cell migration assay was performed according to the following steps: 1. Digest endothelial cells, centrifuge at 1000 rpm for 5 min, and resuspend the endothelial cells with different exosomes as set in the experiment, adjusting the endothelial cell density to 2.5 × 10⁻⁶. 5 / ml; 2. Take 0.25 ml of cell suspension from each experimental group and add it to the upper chamber of the Trans well. Add 600 µl of 1640 complete culture medium to each well below the chamber and incubate at 37°C, 5% CO2, 21% O2 for 40 h. 3. Discard the culture medium in the lower and inner chambers; 4. Wash with PBS, fix with 4% paraformaldehyde for 20 min; wipe away the cells in the upper layer of the chamber with a cotton swab; Stain with 5.1% crystal violet for 35 min, then place the chamber on a glass slide; 6. Take pictures with an inverted fluorescence microscope and quantify the number of migrating cells using ImageJ.

[0048] Figure 3 A diagram illustrating how Exo promotes endothelial cell migration. (Example:) Figure 3 As shown, compared with the control group (endothelial cells only, no exosomes) and the 21% Exo group (exosomes extracted from mesenchymal stem cells after two days of normoxic pretreatment), the hy Exo group (exosomes extracted from mesenchymal stem cells after two days of hypoxic pretreatment) significantly promoted the migration of endothelial cells.

[0049] Example 5: The process by which Exo promotes endothelial cell tubulation To further evaluate the angiogenesis-promoting potential of collected MSCs normoxan exosomes (21% Exo) and hypoxic exosomes (hy Exo), we performed an endothelial cell tube formation assay. 1640 basal medium was used as the control group, while 21% Exo and hy Exo, collected for 2 days at a concentration of 150 µg / ml, were used as the experimental groups. The endothelial cell tube formation assay was performed as follows: The yellow pipette tip and 48-well plates were pre-chilled at 4°C overnight. Matrigel was removed from -20°C, placed in an ice bath, and then placed in a 4°C refrigerator overnight to thaw. The next day, the 48-well plates were placed on an ice bath. Using the chilled yellow pipette tip, 150 µl of Matrigel was evenly applied to the bottom of each well. The plates were then placed in a 4°C refrigerator for 20 min to allow the Matrigel to spread. Finally, the Matrigel was incubated at 37°C for 75 min to cure.

[0050] The endothelial cells were then washed with PBS, digested with trypsin, centrifuged at 1000 rpm for 5 min, and resuspended with different exosomes as set in the experiment. The endothelial cell density was adjusted to 2.5 × 10⁻⁶ cells / mL. 5 / mL, 0.3 ml of cell suspension was added to each experimental group into a 48-well plate; then it was placed in a 37℃, 5% CO2, 21% O2 cell culture incubator for 5 h and washed with PBS.

[0051] Then proceed with the following steps for staining, observation, and quantification: 1. Add an appropriate amount of 4% paraformaldehyde and fix at 37℃ for 30 min; 2. Wash with PBS, then permeate with 0.5% Triton X-100 solution for 20 min; 3. Wash with PBS, add fluorescein isothiocyanate (FITC) labeled phalloidin, and incubate at room temperature in the dark for 1 hour; 4. Wash with PBS, observe the endothelial cell tube formation using a confocal microscope and take pictures; 5. Use ImageJ to measure the number of tubes and vascular nodes for quantitative analysis.

[0052] Figure 4 Diagram showing how Exo promotes endothelial cell tube formation. Experimental results are as follows. Figure 4 As shown in the figure. Compared with the control group and the 21% Exo group, the hy Exo group significantly promoted endothelial cell tube formation.

[0053] Exosomes alone have poor therapeutic effects. This invention coats the surface of exosomes with a macrophage membrane that can target ischemic injury sites, enabling the exosomes to target the ischemic sites and release their contents more quickly, promoting angiogenesis in the ischemic sites.

[0054] Example 6: Preparation of hybrid nanovesicles using macrophage membranes as an example Before preparing hybrid nanovesicles, macrophage cell membranes were first extracted.

[0055] 1. After the cells have grown to confluence, discard the original culture medium and wash with PBS; 2. Add an appropriate amount of culture medium, scrape off the adherent cells with a cell scraper, collect them into a 15 ml centrifuge tube, and centrifuge at 1000 rpm for 5 min. 3. Resuspend the cell pellet in pre-cooled 1 / 4 X PBS and freeze at -80°C for later use; 4. After removing the frozen macrophages, place them in an ice box to thaw for 2 hours; 5. After dissolving, use a pipette to transfer all the macrophages into a 50 ml centrifuge tube; 6. Adjust the cell disruptor to 60% power and run for 25 minutes; 7. Then, aliquot the broken cell fragments into 1.5 ml centrifuge tubes, pre-cool the centrifuge to 4°C, centrifuge at 3200 g for 15 minutes; 8. Take the supernatant, aliquot it into new 1.5 ml centrifuge tubes, centrifuge at 15000 g for 40 minutes at 4°C, discard the supernatant, resuspend all precipitates in PBS, determine the concentration using a BCA kit, and store at -80°C for later use.

[0056] Preparation of hybrid vesicles: Take out the pre-prepared exosomes and macrophage membranes. Take 0.75 ml of exosomes with a concentration of 150 µg / ml and another 0.3 ml of macrophage membrane with a concentration of 120 µg / ml. Mix the two in a centrifuge tube and vortex several times. Then add them to the syringes on both sides of the Avanti micro extruder. Load the middle of the extruder with a polycarbonate porous membrane with a pore size of 0.4 µm. Extrude back and forth at least 30 times to obtain hybrid nanovesicles.

[0057] Example 7: Preparation of TEM samples of hybrid nanovesicles Please refer to Example 2 for details.

[0058] Figure 5 This is a TEM image of hybrid nanovesicles. (By...) Figure 5 It can be seen that the fused (M-Exo) has a clear bilayer membrane structure, proving that the preparation of hybrid nanovesicles was successful.

[0059] Example 8: Measurement of Hybrid Nanovesicle Particle Size and Potential Please refer to Example 3 for details.

[0060] Figure 6 This is a diagram showing the particle size and potential of hybrid nanovesicles.

[0061] Before fusion, the particle size of individual exosomes was around 90 nm, and the particle size of individual macrophage membranes was around 100 nm. After fusion, the particle size of M-Exo was around 150 nm. Furthermore, the potential of M-Exo after fusion was between that of exosomes and macrophage membranes, indicating successful membrane fusion.

[0062] Example 9: Uptake of hybrid nanovesicle materials by cardiomyocytes We used a fluorescent labeling method under confocal microscopy to detect the targeting of M-Exo to H9C2 cells. The specific steps are as follows: 1. To prepare M-Exo with two fluorescent labels, 750 µl of 150 µg / ml Exo was added to Dio staining solution and stained for 20 min. Then, 325 µl of 120 µg / ml MM was added to Did staining solution and stained for 20 min. After vortexing and mixing, the mixture was extruded through a polycarbonate porous membrane for approximately 30 cycles to obtain M-Exo with two fluorescent labels. 2. Plating. Wash H9C2 cells with PBS, digest with trypsin, centrifuge at 1000 rpm for 5 min, resuspend in DMEM medium, and adjust the H9C2 cell density to 5.5 × 10⁻⁶ cells / cm². 4 1.5 ml of cell suspension was added to each confocal dish and incubated at 37°C, 5% CO2, 21% O2 for 48 h. After washing with PBS, M-Exo labeled with two different fluorescent markers was added to the confocal dish and incubated at 37°C, 5% CO2, 21% O2 for another 7 h. The cells were then washed with PBS, and an appropriate amount of DAPI staining solution was added to each well. The cells were incubated in the dark for 8 min. The uptake of M-Exo by H9C2 cells was then detected under a confocal microscope.

[0063] Figure 7Image of hybrid nanovesicles taken up by cardiomyocytes.

[0064] like Figure 7 As shown, individual exosomes were stained with green fluorescence, individual macrophage membranes were stained with red fluorescence, and the nuclei of H9C2 cells were stained with DAPI. After the red and green merge, the result is yellow, and most of the merged image is yellow, which further proves the success of membrane fusion, that is, the successful preparation of hybrid nanovesicles.

[0065] Example 10: The process by which hybrid nanovesicles (M-Exo) promote endothelial cell migration For specific instructions, please refer to Example 4.

[0066] MM-21% Exo consists of 21% Exo encased in a macrophage membrane. Mhy Exo consists of hy Exo encased in a macrophage membrane.

[0067] Figure 8 This diagram illustrates how hybrid nanovesicles promote endothelial cell migration. Based on... Figure 8 This study demonstrated that coating the surface of exosomes with a macrophage membrane does not affect their original function. The hypoxia group (hy 1% Exo, Mhy Exo) was still greater than the normoxic group (21% Exo, MM-21% Exo) and the control group, significantly promoting endothelial cell migration.

[0068] Example 11: The process by which hybrid nanovesicles promote endothelial cell tubulation The experimental groups are as described in Example 10, and the steps are as described in Example 5.

[0069] Figure 9 A diagram illustrating how hybrid nanovesicles (M-Exo) promote endothelial cell tubing. Based on... Figure 9 This study demonstrated that coating exosomes with a macrophage membrane did not affect their original function; the hypoxia group (hy Exo, Mhy Exo) still showed higher levels of exosomes than the normoxic group (21% Exo, MM-21% Exo) and the control group. It also significantly promoted endothelial cell tube formation.

[0070] Example 12: The ability of hybrid nanovesicles to target damaged blood vessels To verify the targeting ability of the hybrid nanovesicles provided in this embodiment of the invention to damaged blood vessels, a small animal in vivo imaging system was used for evaluation. Eight-week-old male C57 mice were selected, and myocardial infarction was induced in the mice after a one-day preoperative fast. The surgical procedure is as follows: 1. Preoperative preparation: After anesthetizing the mice with isoflurane gas, the mice's limbs were fixed to the operating table with tape. A 1.5 cm incision was made in the lower third of the mouse's neck along the midline of the anterior neck. The trachea was bluntly dissected, taking care not to damage the thyroid arteries on either side of the trachea. The trachea in the mouse's neck was incised, and the tube was intubated and connected to an anesthesia ventilator for assisted breathing. The ventilator (Hallowell EMC, Microvent 1) was set to a frequency of 115 breaths / minute and a tidal volume of 1.2 ml. The tube was then secured to the operating table with tape. The isoflurane dosage was adjusted to 1.2% to maintain the mice's anesthesia. Hair removal cream was used to remove hair from the mouse's chest, and the area was disinfected with iodine and medical alcohol.

[0071] 2. Thoracotomy and Ligation: Using forceps, bluntly separate the fascia at the junction of the pectoralis major and serratus anterior muscles. After locating the ribs, make an arc-shaped incision at the second rib. Bluntly insert the probe into the thoracic cavity and use a thoracotomy instrument to open the thoracic cavity and expand the field of vision. Use sterile cotton balls to push aside the lungs to fully expose the heart. Carefully tear open the cardiac capsule with forceps to expose the anterior wall of the left ventricle. Ligate the left anterior descending branch of the coronary artery with 7-0 sutures at a point 2.5 mm below the origin of the left coronary artery and from the left atrial appendage. The ligation depth is approximately 0.6 mm and the width is 1.5 mm. After ligation, if the myocardium below the ligation site changes from red to light white or becomes cyanotic, and the ventricular wall mobility decreases or disappears, it indicates that the mouse myocardial infarction model has been successfully established.

[0072] 3. Chest closure and suturing: Observe for active bleeding in the pleural cavity. If present, remove the accumulated blood with a cotton swab. After the mouse's circulation stabilizes, ligate and close the pleural cavity with 5-0 sutures, suture the skin, and disinfect with povidone-iodine. Postoperatively, place the mouse on a heating pad and continue intubation and mechanical ventilation, observing the mouse's condition. Separately, prepare fluorescently labeled M-Exo: Take 750 µl of 150 µg / ml Exo, add Dio staining solution, and stain for 20 min. Then, take 325 µl of 120 µg / ml MM. Vortex mix the two and extrude through a polycarbonate porous membrane for 32 cycles to obtain fluorescently labeled M-Exo.

[0073] To verify the targeting of M-Exo to myocardial infarction, we used small animal in vivo imaging to evaluate the distribution of M-Exo. The experimental animals were divided into two groups (n=3 per group): the Sham group (normal mice) and the MI group (mice with myocardial infarction). Immediately after model establishment, mice were injected with fluorescently labeled M-Exo. Twelve hours after injection, heart tissue was harvested from the mice, and the fluorescence intensity of the hearts in both groups was observed using a small animal in vivo imaging system. Statistical analysis of the fluorescence intensity was performed using Live Image software.

[0074] Figure 10This image shows the distribution of hybrid nanovesicles in the heart of two groups of mice after intravenous injection via the tail vein. Figure 10 It can be seen that the average fluorescence intensity of the MI group was greater than that of the Sham group, showing a significant difference. This indicates that hybrid nanovesicles can target ischemic sites in myocardial infarction.

[0075] Example 13: The ability of hybrid nanovesicles to enhance cardiac function in mice with myocardial infarction To verify the repair ability of the hybrid nanovesicles provided in this embodiment of the invention on damaged hearts, small animal ultrasound was used for evaluation. Following Example 12, a myocardial infarction model was established, and mice were randomly divided into 5 groups (n=5 per group). The experimental groups were: 1. Sham group: no myocardial infarction model; 2. PBS group: injected with 150 µl of sterile PBS; 3. 21% Exo group: exosomes extracted from MSCs cultured under 21% normoxic conditions were prepared; 4. hy Exo group: exosomes extracted from MSCs cultured under hypoxic conditions were prepared; 5. MM-21% Exo group: exosomes extracted from MSCs cultured under 21% normoxic conditions were prepared and coated with a macrophage membrane; 6. Mhy Exo group: exosomes extracted from MSCs cultured under hypoxic conditions were prepared and coated with a macrophage membrane. After the myocardial infarction model was established, each group was treated with different drugs. Small animal ultrasound was used to observe cardiac recovery on days 15 and 28 of treatment.

[0076] Figure 11 Echocardiogram of mice with myocardial infarction treated with hybrid nanovesicles on day 15. Figure 12 Echocardiography of mice with myocardial infarction treated with hybrid nanovesicles on day 28. Figure 11 It was found that after half a month of treatment, the group with hypoxic exosomes encapsulating macrophage membranes increased the left ventricular ejection fraction (EF) and fulminant ejection fraction (FS) in mice with myocardial infarction, indicating that hypoxic exosomes can significantly improve cardiac function in mice with myocardial infarction. Figure 12 It can be seen that after one month of treatment, compared with the PBS group, the other groups all improved the left ventricular ejection fraction (EF) and septal defect (FS) of mice with myocardial infarction to varying degrees. The group with hypoxia exosomes encapsulating macrophage membranes showed the greatest improvement, indicating that hypoxia exosomes can significantly improve cardiac function in mice with myocardial infarction.

[0077] Example 14: The ability of hybrid nanovesicles to promote angiogenesis in mice with myocardial infarction The recovery of cardiac blood flow in mice with myocardial infarction is related to the remodeling of blood vessels in the ischemic area. Therefore, this invention collects cardiac tissue samples from mice treated with exosomes and stains them with CD31, a marker of cardiac endothelial cells, to observe the recovery of cardiac blood flow in ischemic mice after treatment. First, cardiac tissue samples are collected from the mice. After a one-month treatment cycle, the mice with myocardial infarction are euthanized with an excessive amount of ether. The skin is cut along the line connecting the xiphoid process and the midaxillary line, and the muscles are separated before opening the thoracic cavity to expose the heart. The mouse heart is quickly removed. The heart is immersed in embedding medium, rapidly frozen in liquid nitrogen, and then frozen into sections. The section thickness is adjusted to 10µm, and the sections are stored at -80℃. Subsequently, CD31 samples are prepared: 1. Rewarming: Place at room temperature for 30 minutes; 2. Fixation: Place the sections in pre-cooled acetone for 5 min to fix; 3. Permeability: Wash with 0.2% Tween 20 solution for 15 min; 4. Blocking: Incubate with 2% BSA blocking solution at room temperature in the dark for 30 min; 5. Incubation with primary antibody: Add CD31 antibody and incubate overnight at 4°C in the dark; 6. Washing: Wash with PBS for 5 min; 7. Incubation with secondary antibody: Add mouse IgG antibody and incubate at 37°C in the dark for 2.5 h; 8. Washing: Wash with PBS for 5 min; 9. Mounting: Mount the slides with mounting medium containing DAPI; 10. Laser confocal imaging, Image J quantitative analysis.

[0078] Figure 13 Figure showing how hybrid nanovesicles promote angiogenesis in mice with myocardial infarction.

[0079] Depend on Figure 13 The results showed that, compared with normal mouse hearts, the vascular density of mice treated with 21% Exo was significantly reduced, indicating that blood flow in the mice had not been restored. The vascular density of mice treated with hy Exo and MM-21% Exo increased by 15% and 39%, respectively, while the vascular density of mice treated with Mhy Exo increased by 73.1%. This indicates that Mhy Exo can more significantly increase vascular remodeling in ischemic sites to restore cardiac function in mice compared to other treatment groups.

[0080] Example 15: Biosafety evaluation of hybrid nanovesicles in other organs To evaluate the effects of each treatment group on the major organs of mice and whether it would cause toxicity to the organism, we collected major organs (liver, spleen, lung, and kidney) from mice treated with exosomes for paraffin sectioning and H&E staining. We observed the differences between these organs and those of normal mice and examined the biocompatibility of the exosomes by observing whether they induced inflammatory cell infiltration. Mice with myocardial infarction were euthanized with an excessive amount of ether after a one-month treatment cycle. The liver, spleen, lung, and kidney of the mice were quickly removed. The tissues were first embedded in paraffin. 1. Freshly collected tissue samples were first fixed overnight in 4% paraformaldehyde; 2. Dehydrate the fixed tissues with gradient ethanol concentrations of 30%, 50%, and 70%, respectively, soaking for 45 min and gently shaking. 3. Continue dehydration in 70% ethanol for 1.5 h, 80% ethanol for 1.5 h, 90% ethanol for 1.5 h, 95% ethanol for 45 min, 99% ethanol for 30 min, 99% ethanol for 30 min, xylene I immersion for 30 min, xylene II immersion for 50 min, paraffin I immersion for 1 h, and paraffin II immersion for 1.2 h; 4. Subsequently, the tissue samples were embedded in paraffin and sectioned using a paraffin microtome. The section thickness was adjusted to 10 µm, and the sections were stored at 4°C. The embedded tissues were then stained with H&E. 1. Dewaxing: Immerse in xylene I for 20 min, then in xylene II for 25 min, and air dry; 2. Rehydration: 5 min in anhydrous ethanol I, 8 min in anhydrous ethanol II, 5 min in 95% ethanol, 5 min in 90% ethanol, 8 min in 80% ethanol, and 10 min in tap water. 3. Hematoxylin staining for 5 minutes; 4.1% hydrochloric acid-alcohol differentiation for 1 second; 5. Stain with eosin for 30 seconds; 6. Dehydration: 80% ethanol for 10 s, 90% ethanol for 10 s, 95% ethanol for 10 s, anhydrous ethanol I for 3 min, anhydrous ethanol II for 3 min; 7. Permeability test: Xylene I and Xylene II, 5 min each; 8. After drying, seal with neutral resin; Figure 14 HE staining images of major mouse organs provided in embodiments of the present invention, such as... Figure 14 As shown, there was no obvious inflammatory cell infiltration in any of the organs, indicating that the hybrid nanovesicles prepared in the embodiments of the present invention have good biosafety.

[0081] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Cell membrane-modified engineered hypoxic stem cell exosomes, including hypoxic-treated mesenchymal stem cell exosomes and their surface-coated cell membranes.

2. The cell membrane-modified engineered hypoxic stem cell exosomes according to claim 1, characterized in that, The mass ratio of the cell membrane to the exosomes is 25-75:50-200; The particle size of the cell membrane-modified engineered hypoxic stem cell exosomes is 120-160 nm.

3. The cell membrane-modified engineered hypoxic stem cell exosomes according to claim 1, characterized in that, The cell membrane is a membrane of at least one of the following: red blood cells, macrophages, platelets, stem cells, and tumor cells.

4. A method for preparing cell membrane-modified engineered hypoxic stem cell exosomes as described in claim 1 or 2, comprising the following steps: subjecting mesenchymal stem cells to hypoxia pretreatment, collecting the conditioned medium of mesenchymal stem cells, collecting exosomes by differential centrifugation, coating the exosome surface with a cell membrane to obtain hybrid nanovesicles, i.e., cell membrane-modified engineered hypoxic stem cell exosomes.

5. The method according to claim 4, characterized in that, The method includes the following steps: 1) Hypoxia pretreatment of mesenchymal stem cells When mesenchymal stem cells are cultured and the cells reach 80-90% confluence, the original culture medium is discarded, the cells are washed with PBS, and replaced with serum-free DMEM / F12 culture medium. The cells are then grown in a cell culture incubator under 0.1%-8% oxygen conditions, and the conditioned medium is collected. 2) Preparation of exosomes The obtained conditioned medium was centrifuged for the first time to remove cell debris and collect the supernatant. It was then centrifuged for the second time to remove larger vesicles and apoptotic bodies and collect the supernatant again. The supernatant was then centrifuged at high speed and discarded. The precipitate was resuspended in PBS, centrifuged, and the supernatant was discarded. The precipitate was then collected as exosomes (Exo). 3) Preparation of hybrid nanovesicles, i.e., cell membrane-modified engineered hypoxic stem cell exosomes. Exosomes are mixed with cell membranes, vortexed, and extruded to form hybrid nanovesicles.

6. The method according to claim 4, characterized in that, In step 1), the growth time is 2-4 days; In step 2), the conditions for the first centrifugation are: centrifuge at 500 g for 10-15 min; The conditions for the second centrifugation were: 10,000 g for 30-40 min; The conditions for ultracentrifugation are: 100,000 g for 70-90 min; The resuspended centrifugation conditions were 100,000 g for 2-3 h.

7. The method according to claim 4, characterized in that, The mass ratio of cell membrane to exosomes is 25-75:50-200; The cell membrane solution was mixed with the exosome solution; The extrusion molding operation is as follows: the mixed exosomes and cell membranes are added to the two syringes on both sides of the Avanti micro extruder and extruded back and forth at least 30 times; The extruder is loaded with a polycarbonate porous membrane with a pore size of 0.4 µm in the middle.

8. The use of the cell membrane-modified engineered hypoxic stem cell exosomes as described in claim 1 or 2 in the preparation of a drug for treating myocardial infarction.

9. A medicament for treating myocardial infarction, comprising cell membrane-modified engineered hypoxic stem cell exosomes as described in claim 1 or 2.

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