Macrophage targeting functional extracellular vesicle as well as preparation method and application thereof

Abstract

The invention relates to macrophage targeting functionalized extracellular vesicles and a preparation method and application thereof, and relates to the technical field of biological medicines.The preparation method comprises the following steps that COFs particles loaded with an ATP citrate lyase inhibitor are prepared from a covalent organic framework material and the ATP citrate lyase inhibitor through a one-pot method; the COFs particles loaded with the ATP citrate lyase inhibitor and the apoptosis body membrane are subjected to an ultrasonic and thin film extrusion method, and the macrophage targeting functional extracellular vesicle is prepared. The macrophage targeting functional extracellular vesicle provided by the invention has the advantages that the macrophage targeting functional extracellular vesicle can target the macrophage and accurately release drugs to regulate and control the polarization state of the macrophage, and an effective means is provided for constructing an anti-inflammatory treatment product. Meanwhile, the constructed functionalized extracellular vesicles can reprogram the polarization state of macrophages, regulate the local immune microenvironment, promote the formation of neovascularization of diabetic skin wounds and promote wound healing.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a macrophage-targeted functionalized extracellular vesicle and a preparation method and application thereof. Background Art

[0002] Diabetes is a chronic metabolic disease prevalent worldwide with high incidence and mortality. Slow wound healing is a common complication of diabetes, which seriously affects people's normal life. Chronic wound infection in diabetes is the main cause of non-traumatic amputation, and macrophages play a key role in all stages of tissue repair. Under high blood sugar conditions, the polarization state of macrophages changes and continuously transforms to the pro-inflammatory M1 phenotype, hindering wound healing. Therefore, it is crucial for wound healing to be able to target macrophages to change their polarization state and accelerate angiogenesis.

[0003] Extracellular vesicles (EVs) are a general term for heterogeneous membrane structures enclosed by lipid bilayers secreted by cells. According to their size and source, they can be divided into three subtypes: exosomes (50-200nm), microvesicles (100-1000nm), and apoptotic bodies (50-4000nm). As a small membrane vesicle, EVs play an important role in intercellular communication. They affect the functions of other cells through their surface proteins, intracellular bioinformation molecules such as proteins, nucleic acids, etc., and lipids and polysaccharides they transport. Apoptotic bodies are extracellular vesicles released during apoptosis, mainly referring to bubble-like bodies formed by the shrinkage and invagination of the cell membrane. Apoptotic bodies are then phagocytosed by macrophages, parenchymal cells or tumor cells and degraded in phagosomes. In recent years, the research on apoptotic bodies has been gradually heating up. They can be produced by genetic engineering to carry targeted or therapeutic molecules as drug carriers with better safety and will not trigger immune responses. They have broad prospects in biomedical applications, especially in immunomodulation, vascular protection, tissue regeneration and other fields. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a macrophage-targeted functionalized extracellular vesicle and a preparation method and application thereof.

[0005] In a first aspect, the present invention provides a method for preparing macrophage-targeted functionalized extracellular vesicles, the preparation method comprising the following steps:

[0006] The covalent organic framework material and the ATP citrate lyase inhibitor were prepared by a one-pot method to prepare COFs particles loaded with the ATP citrate lyase inhibitor;

[0007] The COFs particles loaded with ATP citrate lyase inhibitor and apoptotic body membrane are subjected to ultrasound plus thin film extrusion method to obtain the macrophage-targeted functionalized extracellular vesicles.

[0008] Furthermore, the covalent organic framework material is prepared using DVA (2,5-divinylterephthalaldehyde) and TPB (1,3,5-tris(4-aminophenyl)benzene) as raw materials; and the ATP citrate lyase inhibitor includes SB 204990.

[0009] Furthermore, the molar ratio of the DVA to the TPB is (3-9):(2:3).

[0010] Furthermore, the apoptotic body membrane is derived from macrophages, and the macrophages include RAW264.7.

[0011] Furthermore, the method for preparing the apoptotic body membrane comprises the following steps:

[0012] RAW264.7 cells were used to induce apoptosis by adding 1 μM staurosporine, and apoptotic bodies were obtained by differential centrifugation.

[0013] The apoptotic bodies are subjected to hypotonic and ultrasonic treatment to remove the vesicle contents, thereby obtaining apoptotic body membranes;

[0014] Among them, the time for staurosporine to induce apoptosis is 3-5h.

[0015] Furthermore, in terms of mg / ml, the mass-to-volume ratio of the covalent organic framework material to the ATP citrate lyase inhibitor is 1:(0.5-1), and the weight ratio of the COFs particles loaded with the ATP citrate lyase inhibitor to the apoptotic body membrane is 1:(2-4).

[0016] Furthermore, the working condition parameters of the one-pot method include: rotating mixing at room temperature for 8-12 hours, and drying under high vacuum for 24 hours; the working condition parameters of the ultrasound plus thin film extrusion method include: intermittent ultrasound in a water bath for 5 minutes, then loading the above-mentioned nanoparticles into an extruder, passing through a polycarbonate filter membrane with a diameter of 1000nm, and repeatedly extruding 10 to 15 times.

[0017] In a second aspect, the present invention provides a macrophage-targeted functionalized extracellular vesicle, wherein the macrophage-targeted functionalized extracellular vesicle is prepared by the method for preparing the macrophage-targeted functionalized extracellular vesicle according to any one of the first aspects.

[0018] Furthermore, the particle size of the macrophage-targeted functionalized extracellular vesicle is 600-800 nm.

[0019] In a third aspect, the present invention provides the use of the macrophage-targeted functionalized extracellular vesicles described in any one of the first aspect and the second aspect as a drug carrier and in the preparation of a drug for treating diabetic wound healing diseases.

[0020] The above technical solution provided by the embodiment of the present invention has at least the following advantages compared with the prior art:

[0021] The embodiment of the present invention provides a macrophage-targeted functionalized extracellular vesicle and a preparation method and application thereof. The macrophage-targeted functionalized extracellular vesicle provided by the present invention has the function of targeting macrophages and accurately releasing drugs to regulate the polarization state of macrophages, providing an effective means for constructing anti-inflammatory therapeutic products. At the same time, the constructed functionalized extracellular vesicles can reprogram the polarization state of macrophages, regulate the local immune microenvironment, promote the formation of new blood vessels in diabetic skin wounds, and promote wound healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0024] Figure 1 This is a graph showing the experimental results of exploring the time of apoptosis induced by staurosporine in the present invention (bar=100 μm).

[0025] Figure 2 This is a diagram showing the secretion of apoptotic bodies 3 hours after staurosporine-induced apoptosis under a light microscope in the present invention (bar=50 μm).

[0026] Figure 3 Characterization diagram of apoptotic body membrane in the present invention; wherein, (a) scanning electron micrograph of apoptotic body membrane, bar=500nm; (b) particle size distribution of apoptotic body membrane; (c) fluorescence micrograph of apoptotic markers on the surface of apoptotic bodies (blue fluorescence is Hoechst, green fluorescence is Annexin V, bar=50μm).

[0027] Figure 4 This is a transmission electron microscope image of the COF in the present invention, bar=1μm.

[0028] Figure 5 This is a scanning electron microscope image of ABm@COF in the present invention, bar=500nm.

[0029] Figure 6 This is a graph showing the ABm@COF cytotoxicity test results at different times in the present invention.

[0030] Figure 7 This is a diagram showing the polarization state of macrophages regulated by SB 204990 in the present invention.

[0031] Figure 8 This is the uptake of nanoparticles by macrophages in the present invention, bar=20 μm.

[0032] Fig. 9 This is the specific engulfment of macrophages in the present invention, bar=100 μm.

[0033] Fig.10 This is a diagram showing the results of a diabetic wound healing experiment in the present invention. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0036] In a first aspect, the present invention provides a method for preparing macrophage-targeted functionalized extracellular vesicles, the preparation method comprising the following steps:

[0037] The covalent organic framework material and the ATP citrate lyase inhibitor were prepared by a one-pot method to prepare COFs particles loaded with the ATP citrate lyase inhibitor;

[0038] The COFs particles loaded with ATP citrate lyase inhibitor and apoptotic body membrane are subjected to ultrasound plus thin film extrusion method to obtain the macrophage-targeted functionalized extracellular vesicles.

[0039] The embodiment of the present invention provides a macrophage-targeted functionalized extracellular vesicle. The macrophage-targeted functionalized extracellular vesicle provided by the present invention has the ability to target macrophages and accurately release drugs to regulate the polarization state of macrophages, providing an effective means for constructing anti-inflammatory therapeutic products. At the same time, the constructed functionalized extracellular vesicles can reprogram the polarization state of macrophages, regulate the local immune microenvironment, promote the formation of new blood vessels on the wound surface of diabetic rats, and promote wound healing.

[0040] In some specific embodiments, the covalent organic framework material is prepared using DVA and TPB as raw materials; and the ATP citrate lyase inhibitor includes SB 204990.

[0041] In the present invention, DVA is called 2,5-divinylterephthalaldehyde in Chinese, which is one of the key raw materials for synthesizing COFs. It contains two aldehyde groups, which can undergo Schiff base reaction (or other reversible covalent reaction) with compounds containing functional groups such as amino groups, thereby constructing the skeleton structure of COFs.

[0042] In the present invention, the full Chinese name of TPB is 1,3,5-tris(4-aminophenyl)benzene, and its CAS registration number is 118727-34-7.

[0043] In the present invention, SB 204990 is an effective and specific ATP citrate lyase (ACLY) inhibitor, and its CAS number is 154566-12-8.

[0044] In some specific embodiments, the molar ratio of the DVA to the TPB is (3-9):(2:3).

[0045] In some specific embodiments, the apoptotic body membrane is derived from macrophages, and the macrophages include RAW264.7.

[0046] In the present invention, RAW264.7, the full name of mouse mononuclear macrophage leukemia cells, is a leukemia cell line derived from mice. RAW 264.7 cells have the characteristics of macrophages, such as strong phagocytic ability, antigen presentation and cytokine production, etc. These cells are easy to culture and operate in vitro and show functions similar to primary macrophages.

[0047] In some specific embodiments, the method for preparing the apoptotic body membrane comprises the following process:

[0048] RAW264.7 cells were used to induce apoptosis by adding 1 μM staurosporine, and apoptotic bodies were obtained by differential centrifugation.

[0049] The apoptotic bodies are subjected to hypotonic and ultrasonic treatment to remove the vesicle contents, thereby obtaining apoptotic body membranes;

[0050] Among them, the time for staurosporine to induce apoptosis is 3-5h.

[0051] In some specific embodiments, in terms of mg / ml, the mass-to-volume ratio of the covalent organic framework material to the ATP citrate lyase inhibitor is 1:(0.5-1), and the weight ratio of the COFs particles loaded with the ATP citrate lyase inhibitor to the apoptotic body membrane is 1:(2-4).

[0052] In some specific embodiments, the working condition parameters of the one-pot method include: rotating and mixing at room temperature for 8-12 hours, and drying under high vacuum for 24 hours; the working condition parameters of the ultrasound plus thin film extrusion method include: loading the above-mentioned nanoparticles into an extruder, passing through a polycarbonate filter membrane with a diameter of 1000nm, and repeatedly extruding 10 to 15 times.

[0053] In a second aspect, the present invention provides a macrophage-targeted functionalized extracellular vesicle, wherein the macrophage-targeted functionalized extracellular vesicle is prepared by the method for preparing the macrophage-targeted functionalized extracellular vesicle according to any one of the first aspects.

[0054] In some specific embodiments, the particle size of the macrophage-targeting functionalized extracellular vesicle is 600-800 nm.

[0055] In a third aspect, the present invention provides the use of the macrophage-targeted functionalized extracellular vesicles described in any one of the first aspect and the second aspect as a drug carrier and in the preparation of a drug for treating diabetic wound healing diseases.

[0056] It should be noted that the components and raw materials involved in the macrophage-targeted functionalized extracellular vesicles and the preparation method thereof provided in the embodiments of the present invention, unless otherwise specified or specifically described, can be directly commercially available products or homemade using existing public preparation methods; at the same time, the steps and parameters involved, unless otherwise specified or specifically described, can be carried out according to existing processes or directly using existing equipment, and the present invention document will not describe them one by one.

[0057] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually measured according to national standards. If there is no corresponding national standard, then the conditions recommended by the manufacturer are followed.

[0058] Example 1

[0059] This example provides the preparation and characterization of apoptotic body membranes, including the following steps:

[0060] Method for preparing apoptotic bodies

[0061] Raw264.7 cells were cultured at 37°C and 5% CO2. Apoptosis was induced by chemical means. 1 μM staurosporine was added to the culture medium for 3 h. Under a light microscope, spherical protrusions were observed on the cell surface, which gradually fell off. Figure 2 As shown. Gently blow the culture medium and rinse with PBS, take the supernatant and centrifuge at 300g for 10min, keep the supernatant and remove the precipitate. Then collect the precipitate and apoptotic bodies at 1000g for 10min. After washing three times with PBS, resuspend in PBS and perform corresponding characterization tests. Immunofluorescence staining and flow cytometry were used to detect the apoptosis-related marker Annexin V, and the positive rate was about 88.6%.

[0062] Based on the above experiments, this case also conducted an exploratory experiment on the time of apoptosis induced by staurosporine. The experimental results are as follows: Figure 1 shown.

[0063] Preparation of apoptotic body membranes

[0064] The apoptotic body membrane (ABm) was collected by resuspending the apoptotic body in hypotonic lysis buffer and keeping it at 4°C for 1 hour. Ultrasonication was performed for 5 seconds, and the debris was removed by centrifugation at 100g for 10 minutes. The supernatant was collected and centrifuged at 10000g for 10 minutes. The precipitate was collected as the apoptotic body membrane (ABm), and the cell membrane content was removed by rinsing with ddH2O, and the cell membrane was resuspended and stored at 4°C.

[0065] In this example, the cell contents were expelled from the apoptotic body membrane by hypotonic treatment and ultrasound, placed in a hypotonic lysis solution for 1 hour, then ultrasounded for 2 minutes, and washed three times with PBS, and the cell nucleus was labeled with Hochest and the apoptotic body membrane was labeled with Annexin V. As shown in the figure, the amount of Hochest in the apoptotic body is small, which means that the contents are expelled, and the diameter of the apoptotic body membrane is about 800-1000nm.

[0066] Example 2

[0067] In this example, macrophage-targeted functionalized extracellular vesicles (extracellular vesicle-COF composite drug delivery system, abbreviated as ABm@COF) were prepared, which specifically included the following processes:

[0068] Preparation of Covalent Organic Frameworks (COFs)

[0069] Weigh 0.12mM DVA and 0.08mM TPB and place them in a centrifuge tube. Add 8ml ACN (acetonitrile) and sonicate for 1min to completely dissolve it. Add 0.4ml 17M HAc (acetic acid) to the test tube. Shake the mixture vigorously in a vortex mixer for 30s, and centrifuge it for 72h at room temperature. Wash it with Dry THF (dry tetrahydrofuran) and ethanol three times respectively. Vacuum dry it at 60℃ for 24h. The transmission electron microscopy image of COF is as follows Figure 4 As shown, the prepared COF particles are uniform and stable, with a particle size of about 600-800 nm.

[0070] ACLY inhibitor loading

[0071] COF has the advantages of large specific surface area, high porosity and high drug loading capacity. SB 204990 is loaded in the pores of COF by electrostatic adsorption. COF particles are ultrasonically dispersed in the SB 204990 solution. After stirring for 8 to 12 hours, the suspension is ultrasonically applied for 5 minutes, washed with ddH2O three times, and dried under high vacuum for 24 hours.

[0072] Preparation of extracellular vesicle-COF composite drug delivery system

[0073] 1 mg of COF-SB was mixed with 2 mg of apoptotic body membrane, and ultrasonicated for 2 min in an ultrasonicator. The suspension was then centrifuged at 5000 g for 10 min. The precipitate was the extracellular vesicle-COF composite drug delivery system. The nanoparticles were loaded into an extruder and passed through a polycarbonate filter with a diameter of 1000 nm. The extrusion was repeated 10 to 15 times to obtain ABm@COF. The scanning electron microscope image is shown in Figure 2. Figure 5 shown.

[0074] Example 3

[0075] In this example, the performance of the macrophage-targeted functionalized extracellular vesicles (extracellular vesicle-COF composite drug delivery system) obtained in Example 2 was tested.

[0076] 1) Cytotoxicity assay

[0077] First, different concentrations of ABm@COF were added to macrophages. CCK-8 was used to detect the cytotoxicity of nanoparticles. The CCK8 results showed that as the concentration increased, even when the concentration increased to 200μg / mL ABm@COF nanoparticles did not have obvious cytotoxicity. As the culture time increased, high concentrations of ABm@COF only had a partial effect on cell activity when cultured for 72h, proving that ABm@COF has good biocompatibility. Figure 6 shown.

[0078] 2) Extracellular vesicle-COF macrophage phagocytosis experiment

[0079] BMDMs cells were extracted and inoculated into confocal culture dishes. 50 μg / ml Rh B-labeled ABm@COF was added and incubated for 6 hours. The culture medium was removed, and after rinsing with PBS, 4% paraformaldehyde was added for fixation for 1 hour. After fixation, PBS was rinsed three times, and goat serum was added, and the slides were blocked at 37°C in the dark for 30 minutes. The blocking solution was removed and the diluted primary antibody F4 / 80 was added and incubated at 4°C overnight. The slides were rinsed with PBS 3 times, each for 5 minutes. The diluted fluorescent secondary antibody was added and incubated in a dark humid box at 37°C for 1 hour. The slides were rinsed with PBS 3 times, each for 5 minutes. DAPI was added and incubated in the dark for 5 minutes to stain the nucleus of the specimen, and the excess DAPI was washed with PBS. The liquid on the slide was dried with absorbent paper, and the slide was sealed with a sealing solution containing an anti-fluorescence quencher. The images were collected under confocal observation, such as Figure 7 shown.

[0080] 3) Effect of SB 204990 on the polarization state of macrophages

[0081] ATP citrate lyase (ACLY) is an important enzyme that produces acetyl-CoA from citrate. Acetyl-CoA is an important intermediate metabolite of material metabolism and an intermediate hub of sugar, fat, and amino acid metabolism. Excessive nutrient input and inflammation can cause abnormal glucose and lipid metabolism in cells. SB 204990 is an effective inhibitor of ATP citrate lyase (ACLY) that can effectively reprogram macrophages. Raw264.7 cells were treated with different conditions (LG, HG, HG+LPS, HG+TLPS+SB204990), and proteins were extracted and detected by Western blot to detect the protein expression of ACLY, macrophage M1 marker CD86, and macrophage M2 marker CD206. The results showed that compared with the HG+LPS group, the addition of SB204990 could reduce the expression of ACLY, while the expression of pro-inflammatory M1 macrophage CD86 was reduced, and the expression of anti-inflammatory M2 macrophage CD206 was increased, which changed the polarization state of macrophages from pro-inflammatory to anti-inflammatory. Figure 8 shown.

[0082] 4) Macrophage targeting experiment of extracellular vesicle-COF composite nanoparticles

[0083] In order to explore the targeting of ABm@COF to macrophages, we added 25 μg / ml RhB-labeled ABm@COF to different cell lines and cultured them for 6 h. Under fluorescence, it can be observed that Raw264.7 (macrophages) obviously engulfed the nanoparticles, while HcASMC (smooth muscle cells), HFB, and HCF (fibroblasts) basically did not engulf the nanoparticles. Only some fluorescent particles adhered to the cell surface, proving that the synthesized ABm@COF has good macrophage targeting, such as Fig. 9 shown.

[0084] 5) Construction of skin injury model

[0085] Male C57 mice aged 6-8 weeks were selected. After 1 week of adaptive feeding, they were fasted for 12 hours and given an intraperitoneal injection of 50 mg / ml STZ for 5 consecutive days. The model was successfully established when the fasting blood glucose was higher than 11 mmol / L. The diabetic mice were randomly divided into 4 groups (PBS group, ABm group, COF-SB group, and ABm@COF-SB group). After intraperitoneal injection of sodium pentobarbital for anesthesia, the mice were shaved and a circular full-thickness skin wound was created with a skin perforator. The wound healing was observed by taking photos at 0, 3, 7, 10, and 14 days after the opening to verify the effect of ABm@COF-SB on the healing of diabetic wounds in rats. Fig.10 As shown. Functionalized extracellular vesicles have a good wound healing effect.

[0086] Various embodiments of the present invention may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity, and should not be understood as a rigid limitation on the scope of the present invention; therefore, the range description should be considered to have specifically disclosed all possible sub-ranges and single numerical values ​​within the range. For example, the range description from 1 to 6 should be considered to have specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which apply regardless of the range. In addition, whenever a numerical range is indicated herein, it is meant to include any cited number (fractional or integer) within the indicated range.

[0087] The foregoing is merely a specific embodiment of the present invention, which enables those skilled in the art to understand or implement the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A method for preparing macrophage-targeted functionalized extracellular vesicles, characterized in that: The preparation method comprises the following steps: The covalent organic framework material and the ATP citrate lyase inhibitor were prepared by a one-pot method to prepare COFs particles loaded with the ATP citrate lyase inhibitor; The COFs particles loaded with ATP citrate lyase inhibitor and apoptotic body membrane are subjected to ultrasound plus thin film extrusion method to obtain the macrophage-targeted functionalized extracellular vesicles.

2. The method for preparing macrophage-targeted functionalized extracellular vesicles according to claim 1, characterized in that: The covalent organic framework material is prepared by using 2,5-divinylterephthalaldehyde and 1,3,5-tri(4-aminophenyl)benzene as raw materials; and the ATP citrate lyase inhibitor includes SB 204990.

3. The method for preparing a covalent organic framework material according to claim 2, characterized in that: The molar ratio of the 2,5-divinylterephthalaldehyde to the 1,3,5-tris(4-aminophenyl)benzene is (3-9):2:

3.

4. The method for preparing macrophage-targeted functionalized extracellular vesicles according to claim 1, characterized in that: The apoptotic body membrane is derived from macrophages, and the macrophages include RAW264.

7.

5. The method for preparing macrophage-targeted functionalized extracellular vesicles according to claim 1, characterized in that: The method for preparing the apoptotic body membrane comprises the following steps: RAW264.7 cells were used to induce apoptosis by adding 1 μM staurosporine, and apoptotic bodies were obtained by differential centrifugation. The apoptotic bodies are subjected to hypotonic and ultrasonic treatment to remove the vesicle contents, thereby obtaining apoptotic body membranes; Among them, the time for staurosporine to induce apoptosis is 3-5h.

6. The method for preparing macrophage-targeted functionalized extracellular vesicles according to claim 1, characterized in that: In mg / ml, the mass-to-volume ratio of the covalent organic framework material to the ATP citrate lyase inhibitor is 1:(0.5-1), and the weight ratio of the COFs particles loaded with the ATP citrate lyase inhibitor to the apoptotic body membrane is 1:(2-4).

7. The method for preparing macrophage-targeted functionalized extracellular vesicles according to claim 1, characterized in that: The working condition parameters of the one-pot method include: rotating and mixing at room temperature for 8-12 hours, and drying under high vacuum for 24 hours; the working condition parameters of the ultrasound plus film extrusion method include: intermittent ultrasound in a water bath for 5 minutes, then loading the above-mentioned nanoparticles into an extruder, passing through a polycarbonate filter membrane with a diameter of 1000nm, and repeatedly extruding 10 to 15 times.

8. A macrophage-targeted functionalized extracellular vesicle, characterized in that: The macrophage-targeted functionalized extracellular vesicles are prepared by the preparation method according to any one of claims 1 to 7.

9. The macrophage-targeting functionalized extracellular vesicle according to claim 8, characterized in that: The particle size of the macrophage-targeted functionalized extracellular capsule is 600-800 nm.

10. Use of the macrophage-targeted functionalized extracellular vesicle according to any one of claims 1 to 9 as a drug carrier and in the preparation of a drug for treating diabetic wound healing diseases.

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