A method for preparing and applying anti-atherosclerotic hybrid exosomes

Abstract

This invention belongs to the fields of pharmaceutical formulation and biomedicine, and relates to an anti-atherosclerotic hybrid exosome, its preparation method, and its application. A hybrid composed of M2 macrophage exosomes and liposomes is used as a carrier, with the carrier loaded with a ferroptosis inhibitor and atorvastatin. The drug loading and encapsulation efficiency of the ferroptosis inhibitor are 1.55–2.57% and 11.99–49.86%, respectively; the drug loading and encapsulation efficiency of atorvastatin are 1.48–12.66% and 46.4–77.31%, respectively. The hybrid exosome provided by this invention has inflammatory targeting ability and higher stability, and can avoid the first-pass effect of the liver and the clearance of the circulatory system through intravenous injection. Simultaneously, this hybrid exosome has effects such as inhibiting ferroptosis, promoting cholesterol efflux, promoting cell burial, anti-inflammation, and regulating the immune microenvironment, enabling multi-faceted treatment of atherosclerosis, thus achieving better therapeutic effects.

Description

Technical Field

[0001] This invention belongs to the fields of pharmaceutical preparations and biomedicine, and relates to a method for preparing and applying anti-atherosclerotic hybrid exosomes. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Statins have long been a commonly used clinical treatment for atherosclerosis, primarily administered orally. However, the first-pass effect from the liver and the clearance effect from the circulatory system significantly limit their therapeutic efficacy. Furthermore, ferroptosis, a contributing factor to atherosclerosis, has few targeted therapies, further restricting multi-faceted treatment options. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a method for preparing and applying anti-atherosclerotic hybrid exosomes. The anti-atherosclerotic hybrid exosomes provided by the present invention have inflammatory targeting capabilities and higher stability, and can avoid the first-pass effect of the liver and the clearance effect of the circulatory system through intravenous injection. At the same time, the anti-atherosclerotic hybrid exosomes provided by the present invention can treat atherosclerosis from multiple angles, thereby having a better therapeutic effect on atherosclerosis.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows:

[0006] On one hand, an anti-atherosclerotic hybrid exosome uses a hybrid composed of M2 macrophage exosomes and liposomes as a carrier, wherein the carrier is loaded with Ferrostatin-1 and atorvastatin; the loading and encapsulation efficiency of Ferrostatin-1 are 1.55-2.57% and 11.99-49.86%, respectively; the loading and encapsulation efficiency of atorvastatin are 1.48-12.66% and 46.4-77.31%, respectively.

[0007] Ferrostatin-1 is a ferroptosis inhibitor, and its main reason for not currently being used to treat atherosclerosis is its rapid metabolism in the body, making it difficult to reach atherosclerotic (AS) plaque areas and exert its effect. This invention uses liposomes to load Ferrostatin-1 and atorvastatin to achieve multi-faceted treatment of atherosclerosis, including inhibition of ferroptosis. However, studies have found that loading Ferrostatin-1 and atorvastatin solely with liposomes results in poor stability and an inability to accumulate in AS plaque areas, thus hindering the treatment of atherosclerosis.

[0008] Therefore, this invention uses a hybrid of M2 macrophage exosomes and liposomes as a carrier, which not only has inflammation-targeting ability and higher stability, efficiently delivering Ferrostatin-1 and atorvastatin to the atherosclerotic plaque area, but also retains the general capabilities of macrophages to exert anti-inflammatory effects. In addition, the carried proteins can promote M2 polarization of macrophages and regulate the immune microenvironment. After atorvastatin is taken up, it effectively regulates cholesterol efflux from endothelial cells and macrophages at the plaque site, while also exerting a certain degree of anti-inflammatory effect and reducing ROS production in target cells. Furthermore, it can effectively promote the phagocytosis of apoptotic cells by macrophages at the plaque site, reducing the accumulation of apoptotic cells. Ferrostatin-1 can effectively inhibit ferroptosis of target cells, reduce the production of lipid peroxides, reduce the consumption of glutathione (GSH), and also promote the phagocytosis of macrophages. The combined effect of these multiple factors makes this nanoplatform have a better therapeutic effect on atherosclerosis.

[0009] Furthermore, this invention also demonstrates that the hybrid of M2 macrophage exosomes and liposomes, used as a carrier, exhibits high drug loading and encapsulation efficiency for Ferrostatin-1 and atorvastatin, thereby enabling the obtained anti-atherosclerotic hybrid exosomes to achieve the aforementioned therapeutic effects on atherosclerosis.

[0010] On the other hand, a method for preparing the above-mentioned anti-atherosclerotic hybrid exosomes includes the following steps:

[0011] A thin film was prepared by mixing DPPC (dipalmitoylphosphatidylcholine), Ferrostatin-1, and atorvastatin.

[0012] M2 macrophage exosomes were obtained from M2 polarized cells;

[0013] The exocrine bodies of M2 macrophages were resuspended and mixed with a membrane, and then extruded using a liposome extruder to obtain the final product.

[0014] Thirdly, a pharmaceutical composition comprising the aforementioned anti-atherosclerotic hybrid exosomes and pharmaceutical excipients.

[0015] Fourthly, the use of the above-mentioned anti-atherosclerotic hybrid exosome or pharmaceutical composition in the preparation of anti-atherosclerotic products.

[0016] The beneficial effects of this invention are as follows:

[0017] 1. The anti-atherosclerotic hybrid exosomes provided by this invention contain atorvastatin and Ferrostatin-1, which are used for anti-inflammatory treatment of atherosclerosis, can promote cholesterol excretion, reduce ROS, inhibit ferroptosis, promote cell burial and immune microenvironment remodeling.

[0018] 2. The anti-atherosclerotic hybrid exosomes provided by this invention use M2 macrophage exosomes and liposomes to construct a hybrid as a carrier, which can achieve targeted action on inflammatory sites by means of chemotactic proteins on the surface of exosomes; at the same time, through the proteins and cytokines carried by exosomes, the anti-inflammatory M2 phenotype polarization of macrophages in the microenvironment is realized, thereby reshaping the microenvironment.

[0019] 3. The anti-atherosclerotic hybrid exosomes provided by this invention can promote the phagocytosis and clearance of apoptotic cells by macrophages, thereby alleviating the accumulation of apoptotic cells. Attached Figure Description

[0020] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0021] Figure 1 This is a schematic diagram of the synthesis of EL@TF in an embodiment of the present invention.

[0022] Figure 2 This is a schematic diagram of the particle size distribution of exosomes (replaced by E), L@TF, and EL@TF in an embodiment of the present invention.

[0023] Figure 3 The images shown are transmission electron microscopy (TEM) images of (A) EL@TF, (B) L@TF, and (C) exosomes in the embodiments of the present invention.

[0024] Figure 4 The fluorescence spectra of E, L, and EL in the embodiments of the present invention are shown.

[0025] Figure 5 These are confocal fluorescence images of E, L, and EL in an embodiment of the present invention.

[0026] Figure 6 This is a diagram showing the hemolysis test results of EL@TF in an embodiment of the present invention.

[0027] Figure 7 The particle size variation curves of L@TF and EL@TF in the embodiments of the present invention are shown.

[0028] Figure 8 The figures shown are the cytotoxicity test results of L@TF and EL@TF in the embodiments of the present invention; where the cell line in Figure A is HUVECs cells and the cell line in Figure B is RAW264.7 cells.

[0029] Figure 9 The figures show the uptake results of L@TF and EL@TF by RAW264.7 cells under different treatments in this embodiment of the invention; Figure A is a fluorescence confocal image, and Figure B is the quantitative result of flow cytometry.

[0030] Figure 10 Figure A shows the targeting results of L@TF and EL@TF in the embodiments of the present invention; Figure B is a fluorescence confocal image and Figure B is the quantitative result of flow cytometry.

[0031] Figure 11 Figure A shows the in vivo targeting results of L@TF and EL@TF in the embodiments of the present invention; Figure A shows the fluorescence image of EL in the aorta, Figure B shows the fluorescence image of L in the aorta, and Figure C shows the fluorescence quantitative results. Detailed Implementation

[0032] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0033] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0034] Given the poor therapeutic effect of existing drugs on atherosclerosis, this invention proposes an anti-atherosclerotic hybrid exosome, its preparation method, and its application.

[0035] In one typical embodiment of the present invention, an anti-atherosclerotic hybrid exosome is provided, using a hybrid composed of M2 macrophage exosomes and liposomes as a carrier, wherein the carrier is loaded with Ferrostatin-1 and atorvastatin; the drug loading and encapsulation efficiency of Ferrostatin-1 are 1.55-2.57% and 11.99-49.86%, respectively; the drug loading and encapsulation efficiency of atorvastatin are 1.48-12.66% and 46.4-77.31%, respectively.

[0036] Another embodiment of the present invention provides a method for preparing the above-mentioned anti-atherosclerotic hybrid exosomes, comprising the following steps:

[0037] DPPC, Ferrostatin-1, and Atorvastatin were mixed to form a thin film;

[0038] M2 macrophage exosomes were obtained from M2 polarized cells;

[0039] The exocrine bodies of M2 macrophages were resuspended and mixed with a membrane, and then extruded using a liposome extruder to obtain the final product.

[0040] In some embodiments, DPPC (dispalmitoylphosphatidylcholine), Ferrostatin-1, and atorvastatin are dissolved in an organic solvent, and then the organic solvent is evaporated to obtain a thin film. Specifically, the organic solvent is methanol and / or dichloromethane, preferably a mixture of methanol and dichloromethane. The volume ratio of methanol to dichloromethane is 1:1.5 to 2.5. Specifically, the temperature at which the organic solvent is evaporated is 40 to 45°C.

[0041] In some embodiments, the total mass ratio of atorvastatin and Ferrostatin-1 to DPPC is 2:5 to 30.

[0042] In some embodiments, the mass ratio of atorvastatin to Ferrostatin-1 is 1:0.5 to 2.

[0043] In some embodiments, RAW264.7 cells were treated with IL-4 to obtain M2 polarized cells. Specifically, the concentration of IL-4 was 15–25 ng / ml; the treatment time was 24–48 h.

[0044] In some embodiments, the process of obtaining M2 macrophage exosomes from M2 polarized cells is as follows: Cell culture medium of M2 polarized cells is collected, cells, dead cells, and cell debris are removed, followed by ultracentrifugation to obtain exosomes and protein precipitates. The precipitates are then resuspended in PBS, and ultracentrifugation is performed to finally obtain the exosome precipitate. Specifically, the centrifugation conditions for removing cells are 250–350 g for 5–15 min; the centrifugation conditions for removing dead cells are 1900–2100 g for 5–15 min; and the centrifugation conditions for removing cell debris are 9000–11000 g for 25–35 min. Specifically, the ultracentrifugation conditions for obtaining exosomes are 119000–121000 g for 65–75 min. Specifically, the ultracentrifugation conditions for obtaining the exosome precipitate are 119000–121000 g for 65–75 min.

[0045] In some embodiments, resuspended M2 macrophage exosomes are added to a container containing a membrane, sonicated at 40–45°C, then sonicated in an ice-water bath, centrifuged to remove free drug, and then extruded using a liposome extruder. Specifically, the sonication time is 15–25 min. Specifically, the sonication in an ice-water bath is 4–6 min. Specifically, the ultrasonic dispersion in an ice-water bath has a range of 35–45 W. Specifically, the centrifugation conditions for removing free drug are: 2500–3500 rpm for 5–15 min.

[0046] In some embodiments, during the extrusion process using a liposome extruder, the material is physically extruded through a 190–210 nm porous membrane and a 90–100 nm porous membrane for several cycles. Specifically, the porous membrane material is polycarbonate. Specifically, the number of cycles for the 190–210 nm porous membrane is 8–10; the number of cycles for the 90–100 nm porous membrane is 4–6.

[0047] A third embodiment of the present invention provides a pharmaceutical composition comprising the above-described anti-atherosclerotic hybrid exosomes and pharmaceutical excipients.

[0048] In some embodiments, the pharmaceutical composition is administered via intravenous injection.

[0049] In some embodiments, the pharmaceutical excipients are water, buffer solutions, preservatives, solubilizers, etc.

[0050] A fourth embodiment of the present invention provides the use of the above-described anti-atherosclerotic hybrid exosome or pharmaceutical composition in the preparation of an anti-atherosclerotic product.

[0051] Specifically, the product is a medicine.

[0052] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0053] Experimental materials: dipalmitoylphosphatidylcholine (DPPC, Anaiji), Ferrostatin-1 (Shanghai Bide), atorvastatin (Luon), dichloromethane (Tianjin Fuyu), methanol (Tianjin Fuyu).

[0054] Example 1:

[0055] 12 mg DPPC, 0.8 mg Ferrostatin-1, and 1.6 mg atorvastatin were added to a round-bottom flask, and 2 ml of a 2:1 mixture of dichloromethane and methanol was added to dissolve them. The mixture was then rotary evaporated at 40°C for 30 min to remove the organic solvent and form a thin film. RAW264.7 cells were treated with IL-4 (20 ng / ml) for 48 h to obtain M2 polarized cells. The cell culture medium was collected, centrifuged at 300 g for 10 min to remove cells, centrifuged at 2000 g for 10 min to remove dead cells, and centrifuged at 10000 g for 30 min to remove cell debris. The supernatant was filtered through a 0.22 μm filter membrane and centrifuged at 120000 g for 70 min to obtain exosomes and protein precipitates. The precipitates were then resuspended in PBS and centrifuged at 120000 g for 70 min to obtain the final exosome precipitate. 0.24 mg protein equivalent of exosomes were resuspended in 5 ml PBS and added to a round-bottom flask for film formation. After hydration by sonication at 42°C for 20 min, the mixture was dispersed by sonication at 40 W probe in an ice-water bath for 5 min. Free drug was removed by centrifugation at 3000 rpm for 10 min. The EL@TF was then physically extruded sequentially through 200 nm and 100 nm polycarbonate porous membranes for 9 and 5 cycles, respectively, using a microliposome extruder to obtain EL@TF (synthesis schematic diagram can be seen). Figure 1 The drug loadings of Fer-1 and atorvastatin were 2.05±0.15% and 7.10±0.29%, respectively, with encapsulation rates of 35.78±3.32% and 73.45±2.58%, respectively.

[0056] Example 2:

[0057] 12 mg DPPC, 0.8 mg Ferrostatin-1, and 1.6 mg atorvastatin were added to a round-bottom flask and dissolved in 2 ml of a 2:1 mixture of dichloromethane and methanol. The mixture was then rotary evaporated at 40°C for 30 min to remove the organic solvent and form a thin film. The film was resuspended in 5 ml PBS and added to the round-bottom flask containing the film. After hydration by sonication at 42°C for 20 min, the film was dispersed by sonication at 40 W for 5 min in an ice-water bath. The free drug was removed by centrifugation at 3000 rpm for 10 min. The film was then physically extruded through 200 nm and 100 nm polycarbonate porous membranes for 9 and 5 cycles, respectively, to obtain L@TF.

[0058] Example 3:

[0059] The particle size of EL@TF obtained in Example 1 and L@TF obtained in Example 2 was measured, as follows: Figure 2 As shown, the particle size increased by approximately 29 nm compared to L@TF and EL@TF, indicating successful exosome incorporation. Figure 3 Transmission electron microscopy provides more direct evidence that exosomes were successfully incorporated into liposome membranes to form uniform EL@TF biomimetic nanoparticles (hybrid exosomes).

[0060] Example 4:

[0061] Dissolve 12 mg DPPC and 0.12 mg DiO in 2 ml of a mixed solvent of dichloromethane and methanol (dichloromethane to methanol ratio 2:1). Rotate at 40°C for 30 min to remove the organic solvent and form a thin film. Follow the steps shown in Example 1 to obtain DiO-labeled L. Add 2.4 μg of dye Dil to 0.24 mg protein equivalent exosome PBS solution, and remove the free dye by ultrafiltration to obtain Dil-labeled E. For EL, add the Dil-labeled exosome PBS solution to a round-bottom flask and follow the steps shown in Example 1 to obtain fluorescently labeled EL. Scan the fluorescence emission spectra of the obtained fluorescently labeled E, L, and EL solutions in the wavelength range of 470-650 nm at an excitation wavelength of 450 nm. Figure 4 As shown, the fluorescence emission peak of EL at 560nm proves the successful fusion of EL. Figure 5 The confocal fluorescence images also demonstrate the successful EL fusion.

[0062] Example 5:

[0063] Add 3 ml of rat blood to a 5 ml centrifuge tube and centrifuge at 3000 rpm for 10 min. After centrifugation, discard the supernatant, add 3 ml of physiological saline to the precipitate, and resuspend the precipitate by pipetting 5 to 6 times. Centrifuge at 3000 rpm for 10 min. Repeat the above operation 3 to 4 times. When the supernatant is colorless, the resulting precipitate is the hematocrit red blood cells. Pipette 1 ml of hematocrit red blood cells into a 50 ml volumetric flask, dilute with physiological saline, and bring to a final volume to obtain a 2% red blood cell suspension. Prepare a blank hybridization exosome solution with a concentration of 5 mg / ml. Add the corresponding volume of reagent to each centrifuge tube to obtain blank hybridization exosome solutions with final concentrations of 0.1, 0.25, 0.5, 0.75, and 1 mg / ml, respectively. All centrifuge tubes were incubated in a 37°C water bath for 1 hour, centrifuged at 3000 rpm for 10 minutes, and the supernatant was aspirated with a pipette. The absorbance of each group was measured at 541 nm using a UV spectrophotometer, and the hemolysis rate was calculated. The results are as follows: Figure 6 As shown, the hemolysis rate of EL@TF was less than 5% at the set concentration, indicating good blood compatibility.

[0064] Example 6:

[0065] L@TF and EL@TF were placed in PBS (pH 7.4) containing 10% plasma and stored at 4°C. The particle size of each experimental group was measured on days 0, 1, 3, 5, and 7. The results are as follows: Figure 7 As shown, the particle size of the formulation did not change significantly over seven days, indicating that the formulation has good stability.

[0066] Example 7: RAW264.7 and HUVECs cells were seeded in 96-well plates and cultured overnight. The cells were then co-cultured with different concentrations of reagents for 24 hours. After washing with PBS, 100 μl of 10% CCK-8 solution was added to each well, and the plates were incubated at 37°C for 2 hours. The absorbance at 450 nm was recorded using a microplate reader. The results are as follows: Figure 8 As shown, the cell survival rate was over 80%, indicating good safety.

[0067] Example 8:

[0068] RAW264.7 was used as the test cell line. RAW264.7 cells were incubated in DMEM medium containing 10% fetal bovine serum (FBS), penicillin (100 U / mL), and streptomycin (100 μg / mL). All cells were cultured at 37°C in a 5% CO2 atmosphere.

[0069] The L@TF and EL@TF encapsulated with C6 were prepared as shown in Example 1, except that the drug was replaced with C6.

[0070] RAW264.7 cells were loaded at a rate of 2 × 10⁻⁶. 5 Cells were seeded at a density of [number] cells / well in 12-well plates and induced for 24 h with PBS, 1 μg / ml LPS, or 1 μg / ml LPS + 20 μg / ml ox-LDL, respectively. Cells were then treated with L@C6 solution or EL@C6 for 4 h, respectively. Cells were collected, washed with PBS, and analyzed by flow cytometry. Figure 9 As shown, L@C6 showed significant uptake fluorescence in macrophages under three different conditions, indicating that L@C6 does not have the function of long-term circulation and immune escape in vivo. Uninduced macrophages uptake EL@C6 less than L@C6, which may be due to the CD47 protein expressed on the surface of EL. In addition, since the CCR2 protein on the surface of EL can bind to MCP-1 secreted by inflammatory macrophages and foam cells, induced macrophages can effectively take up EL@C6, which is conducive to the accumulation of the preparation.

[0071] Example 9:

[0072] HUVECs were used as the test cell line. HUVECs were incubated in DMEM / F12 medium containing 10% fetal bovine serum (FBS), penicillin (100 U / mL), and streptomycin (100 μg / mL). All cells were cultured at 37°C in a 5% CO2 atmosphere.

[0073] HUVECs cells were used at 10 5 Cells were seeded at a density of 100 cells / well in 12-well plates and induced for 24 h with PBS at 1 μg / ml psi. Cells were then treated with L@C6 solution or EL@C6 for 1 and 4 h, respectively. Cells were collected, washed with PBS, and analyzed by flow cytometry. Results are shown below. Figure 10 As shown, activated inflammatory endothelial cells increased uptake of EL@C6, which was due to the expression of integrin β-1, LFA1, CD44, and CCR2 on the M2 exosome membrane, which bound to VCAM-1, ICAM-1, and E-selectin on inflammatory endothelial cells and MCP-1 at the site of inflammation, demonstrating a targeting effect on the site of inflammation.

[0074] Example 10:

[0075] ApoE- / - atherosclerotic mice were used as experimental animals. Liposomes and hybrid exosomes loaded with DiD fluorescent dye were prepared according to the method described in Example 4. Mice with atherosclerosis were randomly divided into two groups of three, and were injected via tail vein into DiD-L and DiD-EL, respectively.

[0076] Ten hours after injection, mice were anesthetized with ready-to-use 1.25% aphthylamine via intraperitoneal injection. The mice were then perfused with PBS until the outflow became clear, at which point it was replaced with 4% paraformaldehyde. Once the mice's tails were raised, the aorta, heart, liver, spleen, lungs, and kidneys were dissected. The aorta was then imaged using a small animal in vivo imaging system, and quantitative fluorescence analysis was performed. Results are as follows: Figure 11 As shown, Did-EL accumulated significantly more in aortic lesions than Did-L (p<0.01), indicating that EL has good in vivo plaque targeting, which is attributed to the inflammatory chemotactic proteins on the surface of EL.

[0077] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of them. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An anti-atherosclerotic hybrid exosome, characterized in that, A hybrid of M2 macrophage exosomes and liposomes was used as a carrier to load Ferrostatin-1 and atorvastatin. The loading and encapsulation efficiency of Ferrostatin-1 were 1.55–2.57% and 11.99–49.86%, respectively; the loading and encapsulation efficiency of atorvastatin were 1.48–12.66% and 46.4–77.31%, respectively. The method for preparing the anti-atherosclerotic hybrid exosomes includes the following steps: DPPC, Ferrostatin-1, and atorvastatin were dissolved in an organic solvent, and then the organic solvent was evaporated to obtain a thin film; the organic solvent was methanol and dichloromethane. M2 macrophage exosomes were obtained from M2 polarized cells; The resuspended M2 macrophage exosomes were added to a container containing a membrane, sonicated at 40-45 °C, then sonicated in an ice-water bath, centrifuged to remove free drugs, and then extruded using a liposome extruder. During the extrusion process, the extruder physically extruded the cells through porous membranes of 190-210 nm and 90-100 nm for several cycles.

2. The anti-atherosclerotic hybrid exosome as described in claim 1, characterized in that, The volume ratio of methanol to dichloromethane is 1:1.5~2.

5.

3. The anti-atherosclerotic hybrid exosome as described in claim 1, characterized in that, The temperature for evaporation of organic solvents is 40~45℃.

4. The anti-atherosclerotic hybrid exosome as described in claim 1, characterized in that, The total mass ratio of atorvastatin and Ferrostatin-1 to DPPC is 2:5~30.

5. The anti-atherosclerotic hybrid exosome as described in claim 1, characterized in that, The mass ratio of atorvastatin to Ferrostatin-1 is 1:0.5~2.

6. The anti-atherosclerotic hybrid exosome as described in claim 1, characterized in that, M2 polarized cells were obtained by treating RAW264.7 cells with IL-4.

7. The anti-atherosclerotic hybrid exosome as described in claim 1, characterized in that, The concentration of IL-4 was 15-25 ng / ml; the treatment time was 24-48 h.

8. The anti-atherosclerotic hybrid exosome as described in claim 1, characterized in that, The process of obtaining M2 macrophage exosomes from M2 polarized cells is as follows: collect the cell culture medium of M2 polarized cells, remove cells, dead cells and cell debris, then centrifuge at high speed to obtain exosomes and protein precipitates, then resuspend the precipitates in PBS, and finally centrifuge at high speed to obtain exosome precipitates.

9. The anti-atherosclerotic hybrid exosome as described in claim 8, characterized in that, Centrifugation conditions for removing cells: 250-350g for 5-15 min; centrifugation conditions for removing dead cells: 1900-2100g for 5-15 min; centrifugation conditions for removing cell debris: 9000-11000g for 25-35 min.

10. The anti-atherosclerotic hybrid exosome as described in claim 8, characterized in that, The centrifugation conditions for obtaining exosomes by ultracentrifugation are 119000~121000g for 65~75min.

11. The anti-atherosclerotic hybrid exosome as described in claim 8, characterized in that, The centrifugation conditions for obtaining exosome precipitates by ultracentrifugation are 119000~121000g for 65~75min.

12. The anti-atherosclerotic hybrid exosome as described in claim 1, characterized in that, The ultrasonic hydration time is 15~25 minutes.

13. The anti-atherosclerotic hybrid exosome as described in claim 1, characterized in that, Disperse ultrasonically in an ice-water bath for 4-6 minutes.

14. The anti-atherosclerotic hybrid exosome as described in claim 1, characterized in that, The power of ultrasonic dispersion in an ice-water bath is 35~45 W.

15. The anti-atherosclerotic hybrid exosome as described in claim 1, characterized in that, The conditions for centrifugation to remove free drug are: 2500~3500 rpm, 5~15 min.

16. The anti-atherosclerotic hybrid exosome as described in claim 1, characterized in that, The porous membrane is made of polycarbonate.

17. The anti-atherosclerotic hybrid exosome as described in claim 1, characterized in that, The number of cycles for porous membranes with a wavelength of 190~210nm is 8~10; the number of cycles for porous membranes with a wavelength of 90~100nm is 4~6.

18. A pharmaceutical composition, characterized in that, Includes the anti-atherosclerotic hybrid exosomes and pharmaceutical excipients as described in any one of claims 1-17.

19. The pharmaceutical composition of claim 18, characterized in that, The drug composition is administered via intravenous injection.

20. The pharmaceutical composition of claim 18, characterized in that, The pharmaceutical excipients are water, buffer solutions, preservatives, or solubilizers.

21. Use of an anti-atherosclerotic hybrid exosome according to any one of claims 1-17 or a pharmaceutical composition according to any one of claims 18-20 in the preparation of an anti-atherosclerotic medicament.

Images