ZIF-8 loaded M2 macrophage outer vesicle hydrogel as well as preparation method and application thereof

Abstract

The invention discloses ZIF-8 loaded M2 macrophage outer vesicle hydrogel as well as a preparation method and application thereof. The preparation method comprises the following steps: inducing M0 macrophages through IL-4 to obtain M2 type macrophages; collecting cell supernate, and separating by adopting an ultracentrifugation method to obtain the M2 type macrophage extracellular vesicle EV; purifying the obtained M2-type macrophage extracellular vesicle EV, mixing the purified M2-type macrophage extracellular vesicle EV with ZIF-8 nanoparticles, and carrying out electroporation treatment to obtain a ZIF-loaded exosome vesicle ZIF (at) EV; performing ultrasonic stirring and mixing on a sodium alginate solution and calcium carbonate, adding the obtained ZIF-coated EV and D-(+)-gluconic acid delta-lactone, and fully stirring and mixing to obtain the product. The ZIF-EV hydrogel disclosed by the invention is effective in wound healing, and the ZIF-EV hydrogel can continuously release the ZIF-EV on the wound to promote wound healing.

Description

Technical Field

[0001] The invention belongs to the field of medical wound dressings, and particularly relates to a ZIF-8 loaded M2 macrophage extracellular vesicle hydrogel and a preparation method and application thereof. Background Art

[0002] Wounds are common problems in daily life, ranging from minor skin abrasions to larger surgical incisions. Wounds can have a profound impact on quality of life, often causing pain, discomfort, infection, and risk of scarring. Effective wound management and treatment strategies can accelerate skin healing, reduce infection, reduce the incidence of chronic wounds, and significantly reduce scarring. For burns, postoperative trauma, and chronic ulcers (such as diabetic foot ulcers), the use of reasonable wound repair strategies is essential to improve patients' quality of life, reduce medical costs, and prevent long-term complications. However, the natural wound repair process may be disturbed in some cases, such as chronic trauma or immunosuppressed patients, resulting in a slow or incomplete healing process, increased risk of infection, prolonged recovery time, and may cause lasting functional impairment.

[0003] Traditional wound treatment dressings have certain limitations. For example, traditional gauze dressings often have cotton threads fall off to form foreign bodies, and wound adhesion may occur, which may cause secondary damage to the wound when the dressing is removed. The exudate from the wound is easy to form scabs after maceration, which then leads to the growth and invasion of bacteria and other microorganisms. In addition, traditional gauze dressings generally need to be changed frequently, which is time-consuming and painful. Therefore, the development of new biomaterials has been proposed as an advanced therapy for wound healing. Biomaterials have the characteristics of biocompatibility, improving wound healing rate and reducing infection progression, so they have become good candidates for alternatives to traditional therapies.

[0004] Wound repair involves a precisely coordinated multi-stage biological process, in which specific cell types and molecular signals play a central role in each stage. Macrophages play a key role in regulating inflammation and wound healing, and changes in macrophage polarity can promote or inhibit the inflammatory stage of wound repair. M1 macrophages promote inflammatory responses and clear pathogens and dead cells in wounds. M2 macrophages, on the other hand, promote tissue repair and remodeling. Extracellular vesicles (EVs) from M2 macrophages play an important role in regulating inflammatory responses, promoting wound healing and tissue repair in wound healing. The mechanism of action of EVs in wound repair is mainly reflected in three aspects: (1) Promoting cell proliferation and migration: EVs produced by M2 macrophages contain cytokines that promote cell proliferation and migration, such as VEGF, TGF-β and PDGF. These cytokines can directly act on endothelial cells and fibroblasts near the wound to promote angiogenesis and rapid tissue repair. (2) Regulating inflammatory response: Anti-inflammatory miRNAs and proteins in EVs can inhibit excessive inflammatory response, reduce tissue damage caused by inflammation, and create a more favorable environment for wound healing. (3) Promoting immune regulation: EVs of M2 macrophages can also regulate the response of immune cells, including promoting the anti-inflammatory phenotype conversion of T cells and enhancing the polarization of other phenotype macrophages to M2, which together promote the transition of wounds from the inflammatory stage to the healing stage. Summary of the invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a ZIF-8 loaded M2 macrophage extracellular vesicle hydrogel and a preparation method thereof in view of the deficiencies in the prior art, so as to achieve effective intervention in the wound healing process by continuously releasing EVs with biological activity and ZIF-8 with antibacterial activity.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows: A method for preparing a ZIF-8 loaded M2 macrophage extracellular vesicle hydrogel comprises the following steps: (1) Preparation of extracellular vesicles from M2 macrophages: M0 macrophages were induced with IL-4 to obtain M2 macrophages; the cell supernatant was collected and separated by ultracentrifugation to obtain M2 macrophage extracellular vesicles EVs; (2) Preparation of exosome vesicles loaded with ZIF: The M2 macrophage extracellular vesicles EV obtained in step (1) are purified, mixed with ZIF-8 nanoparticles and subjected to electroporation to obtain ZIF-loaded exosome vesicles ZIF@EV; (3) Preparation of ZIF@EV hydrogel: After the sodium alginate solution and calcium carbonate are mixed by ultrasonic stirring, the ZIF@EV and D-(+)-glucono delta-lactone obtained in step (2) are added, and the mixture is thoroughly stirred and mixed to obtain the product.

[0007] In the present invention, the zeolite imidazolate framework material (ZIF-8) is a porous MOF, which is a type of metal organic framework formed by the coordination of zinc ions (Zn2+) and 2-methylimidazole (2-MiM). The ZIF has a complete inner cavity and regular porous sites on the surface, and has good biocompatibility and sensitivity to acidic environments. ZIF will not destroy the activity of biological macromolecules such as proteins and nucleic acids, but can provide a certain degree of protection for these biological macromolecules. It remains stable under physiological conditions and disintegrates under acidic conditions, and is an ideal carrier for drug transportation and sustained release. It also has an antibacterial effect and can reduce wound infection.

[0008] In the present invention, alginate hydrogel is a multifunctional biomaterial with biocompatibility and biodegradability. It can control the release of active ingredients in acute and chronic wound healing, and is increasingly used in wound treatment. Its dual functions of providing physical protection and controlling the sustained release of therapeutic molecules are of great significance for promoting effective wound healing. The hydrogel system combining therapeutic EVs and antibacterial nanomaterial ZIF-8 can not only provide an optimized healing environment for damaged tissues, but also effectively intervene in the wound healing process by continuously releasing bioactive EVs and antibacterial ZIF-8.

[0009] Specifically, in step (1), M0 macrophages are added to an IL-4 solution with a final concentration of 15-25 ng / mL and incubated for 20-25 h to obtain M2 macrophages with elevated surface marker CD206, the cell supernatant of the M2 macrophages is discarded and the cells are washed several times with PBS, followed by addition of 5-15% Hyclone defined FBS (DFBS) DMEM medium, and the cell supernatant is collected after further incubation for 45-50 h.

[0010] Specifically, in step (1), the collected cell supernatant is subjected to ultracentrifugation to separate M2 macrophage extracellular vesicles EVs, the centrifugal force is controlled at 500-100,000 g, and the centrifugation time is 5-120 min.

[0011] Specifically, in step (1), the cell supernatant is discarded after centrifugation, the obtained extracellular vesicles are resuspended with PBS, and sterilized by passing through a 0.22 μm filter; finally, the sterilized extracellular vesicles are placed in a centrifuge tube and stored in an environment below -80°C for later use.

[0012] Specifically, in step (2), the purified M2 macrophage extracellular vesicles EV are mixed with ZIF-8 nanoparticles in a mass ratio of 1:(4-6), and then electroporated in an electroporation cell at 1000-1200 kV for 4-10 milliseconds.

[0013] Specifically, in step (2), the ZIF-loaded exosome vesicles ZIF@EV obtained by electroporation were washed several times with PBS, then incubated for 2 to 4 hours, and finally resuspended in PBS for later use.

[0014] Specifically, in step (3), the concentration of the sodium alginate solution is 1-3 wt%, the mixed mass volume ratio of calcium carbonate and sodium alginate solution is 10-50 mg / mL, and ultrasonic stirring is performed for 20-40 min to complete the fusion and obtain a mixed slurry.

[0015] Specifically, in step (3), the mass volume ratio of the added mass of ZIF@EV to the sodium alginate solution is 0.1-20 mg / mL; the mass volume ratio of the added amount of D-(+)-glucono δ-lactone to the sodium alginate solution is 1-10 mg / mL.

[0016] Furthermore, the ZIF-8 loaded M2 macrophage extracellular vesicle hydrogel prepared by the above preparation method is also within the protection scope of the present invention.

[0017] Furthermore, the present invention also claims to protect the use of the above-mentioned ZIF-8 loaded M2 macrophage extracellular vesicle hydrogel in the preparation of wound healing dressings.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The present invention loads the nanomaterial ZIF-8 into M2 macrophage EV and integrates it into sodium alginate hydrogel to successfully construct ZIF@EV hydrogel. We verified the effectiveness of ZIF@EV hydrogel in wound healing through mouse experiments. ZIF@EV hydrogel can continuously release ZIF@EV on the wound surface to promote wound healing. ZIF@EV can promote endothelial cell migration, enhance angiogenesis, regulate macrophages to the M2 phenotype, promote the wound immune microenvironment from the inflammatory stage to the proliferation stage to promote wound healing, and ZIF@EV hydrogel has antibacterial properties to reduce wound infection. This hydrogel can be directly applied to wounds, has good biocompatibility, is biodegradable, and has a simple preparation method and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0020] Figure 1 is a TEM image of ZIF@EV prepared in the present invention, scale bar = 100 nm.

[0021] Figure 2 This is the NTA distribution of the M2 macrophage extracellular vesicles EV prepared by the present invention.

[0022] Figure 3 These are images of ZIF@EV hydrogel after (left) and before (right) gelation.

[0023] Figure 4 The degradation curve of ZIF@EV hydrogel (left) and the release curve of ZIF@EV by ZIF@EV hydrogel (right).

[0024] Figure 5 Representative images of HUVEC transwell migration assay, scale bar = 200 μm.

[0025] Figure 6 Representative images of HUVEC migration at 12 and 24 h after cell scratch assay. Scale bar = 200 μm.

[0026] Figure 7 The lumen formation of HUVECs treated with different materials (A) and the paths, nodes and blood vessel contours of 3h and 6h vascular images were outlined using ImageJ (B).

[0027] Figure 8 The morphological changes of macrophages after EV stimulation and the polarization of macrophage M2 phenotype in the present invention. Bright field image of macrophages, scale bar = 50 μm (A); CD206 + Flow cytometric analysis of macrophages (B); CD80 + Flow cytometric analysis of macrophages (C).

[0028] Fig. 9 These are fluorescence images of skin wounds 6 and 12 hours after treatment with different materials. Red fluorescence represents high density, and blue fluorescence represents low density.

[0029] Fig.10 Representative wound images after treatment with the present invention.

[0030] Fig.11 The antibacterial effect of ZIF@EV hydrogel on Staphylococcus aureus and Escherichia coli. DETAILED DESCRIPTION

[0031] The present invention can be better understood with reference to the following examples.

[0032] 1. Preparation of EVs (1) Take macrophages that are in good growth condition and have grown to 70-80% in number and add IL-4 at a final concentration of 20 ng / mL. Then incubate in a cell culture incubator at 37°C and 5% CO2 for 24 hours to obtain M2 macrophages with elevated surface marker CD206. Discard the supernatant of M2 macrophages and wash the cells three times with PBS. Then add DMEM medium containing 10% DFBS and incubate in a cell culture incubator at 37°C and 5% CO2 for 48 hours before collecting the cell supernatant.

[0033] (2) The collected cell supernatant was used to separate the M2 macrophage extracellular vesicles (EVs) by ultracentrifugation. At 4°C, centrifuge at 500g, 10,000g, and 100,000g for 5 min, 30 min, and 120 min, respectively.

[0034] (3) After centrifugation, discard the cell supernatant, resuspend the obtained extracellular vesicles with 100 μL PBS, and sterilize them by passing them through a 0.22 μm filter.

[0035] (4) Place the sterilized extracellular vesicles in a centrifuge tube and store them at -80°C for subsequent experimental use.

[0036] 2. Preparation of exosome vesicles loaded with ZIF (1) First, 10 μg of purified M2 macrophage-derived EVs were mixed with 50 μg of ZIF nanoparticles.

[0037] (2) The mixture was placed in a 4 mm electroporation cell (using an X-Porator H1 device) and electroporated at 1000 kV for 5 ms.

[0038] (3) The solution containing ZIF@EV was washed twice with PBS. Subsequently, the treated ZIF@EV product was incubated at 37°C for 3 h to promote the potential interaction between ZIF and M2 macrophage EVs and ensure the stable loading of ZIF. After the incubation, the final purified ZIF@EV product was resuspended in PBS. Figure 1 EV and ZIF nanoparticles were electroporated to obtain ZIF@EV. TEM results showed that ZIF@EV still exhibited a typical teacup-shaped structure and did not change the configuration of EV. We then used Nanoparticle Tracking Analysis (NTA) to show that the diameter of the EV particles we obtained was in the range of 100nm-400nm, concentrated around 180nm ( Figure 2 ), which is consistent with the size range of extracellular vesicles.

[0039] 3. Preparation of ZIF@EV hydrogel 1 mL of 2% sodium alginate solution and 30 mg of calcium carbonate were mixed by ultrasonic stirring for 30 min. After the fusion was completed, 8 mg of ZIF@EV and 5 mg of D-(+)-glucono δ-lactone were added and gently stirred for 3 min to obtain ZIF@EV hydrogel. Figure 3 shown.

[0040] 4. Degradation performance of ZIF@EV hydrogel (1) Weigh the prepared hydrogel sample and record the mass as Wi.

[0041] (2) Place the sample in PBS at pH 7.4 and leave it at room temperature. Take the sample out of PBS every day, wipe it gently with filter paper to remove the PBS on the sample surface, and record the sample weight Wc.

[0042] (3) Degradation rate = (Wi-Wc) / Wi×100%. Figure 4 (Left) shows that the hydrogel degradation experiment shows that the hydrogel is gradually degraded within 12 days, which provides an ideal time window for animal treatment and ensures that the therapeutic material can naturally degrade and release the loaded therapeutic factors without the need for surgical removal.

[0043] 5. Performance of ZIF@EV hydrogel in releasing extracellular vesicles (1) DID solution with a final concentration of 1 μM was added to ZIF@EV for fluorescent labeling. The labeled ZIF@EV was centrifuged at 100,000 g for 2 h to remove excess DID.

[0044] (2) DID-labeled ZIF@EV, gluconolactone, and SA@CaCO3 were mixed to form ZIF@EV hydrogel.

[0045] (3) The ZIF@EV hydrogel was incubated with PBS (pH = 7.4) at 37 °C in the dark for different periods of time.

[0046] (4) The fluorescence in the supernatant was measured and quantified using a fluorescence spectrophotometer to analyze the EVs released from ZIF@EV hydrogel at different time points.

[0047] like Figure 4(Right), the release study of ZIF@EV hydrogel (right) shows that ZIF@EV can be gradually released from the hydrogel. The rapid release on the first day can release about 60% of ZIF@EV, and then the release rate slows down. On the fifth day, ZIF@EV can be completely released from the hydrogel. This provides a basis for ensuring that the therapeutic factor has sufficient concentration in the wound or lesion area. This controlled release property, coupled with the therapeutic potential of ZIF@EV itself, makes ZIF@EV hydrogel an extremely attractive new therapeutic material.

[0048] 6. Transewell migration assay (1) Select HUVECs with good growth status and suspend the cells in serum-free ECM.

[0049] (2) The selected HUVECs were inoculated into the upper chamber of the Transwell, with 100 μL per well containing 1×10 4 cells.

[0050] (3) Add 500 μL of serum-free ECM to the upper chamber of the Transwell, and add PBS, ZIF, EV, and ZIF@EV for treatment, respectively, and incubate in a cell culture incubator at 37°C and 5% CO2 for 24 h.

[0051] (4) After the incubation, use a cotton swab to carefully remove excess cells in the upper chamber and place the Transwell chamber in 4% paraformaldehyde for fixation for 30 min.

[0052] (5) Stain it with 0.5% crystal violet for 15 min and carefully wash it with PBS five times.

[0053] (6) Measure the absorbance of each well at 450 nm using a microplate reader and capture the image of the stained cells using an optical microscope.

[0054] like Figure 5 As shown, HUVECs treated with EV and ZIF@EV exhibited stronger migration ability.

[0055] 7. Cell scratch test (1) Select HUVECs with good growth status, inoculate them into 6-well plates, add ECM complete medium and incubate overnight until the cell density grows to 70%-80%.

[0056] (2) Use a 1 mL pipette tip to draw a straight line vertically on the bottom of a 6-well plate, and draw three straight lines in each well.

[0057] (3) Wash the cells three times with PBS to remove the scratched cells, add 1 mL of serum-free ECM, and add PBS and EV for treatment respectively, and incubate in a cell culture incubator at 37 °C, 5% CO2 for 24 h.

[0058] (4) At three time points, 0 h, 12 h, and 24 h, cell migration images were captured using an optical microscope and quantitatively analyzed using ImageJ software.

[0059] like Figure 6 As shown in the figure, the cell scratch assay measured the cell migration rate at specific time points (12 hours and 24 hours), and it was found that the migration rate of HUVEC treated with EV and ZIF@EV was significantly higher than that of the control group, which indicates that our materials can activate the migration mechanism of endothelial cells and provide the necessary cell power for vascular regeneration.

[0060] 8. In vitro angiogenesis assay (1) Place the pipette tips and 96-well plate in a -20°C refrigerator overnight to pre-cool, and place the Matrigel in a 4°C refrigerator overnight to pre-cool.

[0061] (2) Before the experiment, place the pre-cooled matrix gel on ice water and place the pipette tip and 96-well plate on a flat ice pack.

[0062] (3) Then spread the matrix gel evenly in a 96-well plate, with 50 μL added to each well. Be careful during each operation to avoid creating bubbles.

[0063] (4) After laying the matrix gel, place the 96-well plate in a 37°C incubator and incubate for 1 hour to form a gel.

[0064] (5) HUVECs with good growth status were selected and inoculated into a 96-well plate covered with Matrigel. ECM complete medium was added, and 100 μL of medium containing approximately 1×10 4 The cells were treated with PBS, ZIF, EV and ZIF@EV at the same time and incubated in a cell culture incubator at 37°C and 5% CO2.

[0065] (6) After incubation in the incubator for 3 h and 6 h, images of angiogenesis were captured using an optical microscope.

[0066] like Figure 7 , showing that EV and ZIF@EV treatment has a significant effect on promoting endothelial cell differentiation and tubular structure formation, which can accelerate the process of angiogenesis.

[0067] 9. Macrophage polarization assay (1) Select M0 macrophages with good growth status, inoculate them into 6-well plates, add DMEM complete medium and incubate overnight until the cell density grows to 70%-80%.

[0068] (2) Discard the cell supernatant, wash the cells three times with PBS, add DMEM complete medium containing 5% FBS, and add PBS and EV to each well respectively, and incubate in a cell culture incubator at 37°C and 5% CO2 for 48 h.

[0069] (3) Use optical microscopy to capture the morphological changes of macrophages.

[0070] (4) After 48 h of co-incubation, a macrophage suspension was prepared, and CD206 (0.5 μg / mL) and CD11b (0.4 μg / mL) antibodies were added and incubated at 37 °C in the dark for 30 min.

[0071] (5) Detect the phenotype of macrophages using flow cytometry.

[0072] like Figure 8 ,The results showed that our material can effectively promote the polarization of M1 macrophages to M2 phenotype.

[0073] 10. In vivo ZIF@EV release experiment in mice In order to identify the migration of ZIF@EV from ZIF@EV hydrogel to the wound surface and its interaction with mouse skin tissue cells, DID-labeled free ZIF@EV and ZIF@EV hydrogel were applied to the wound surface of mice, respectively. At 6h and 12h after application, the wound surface was imaged by fluorescence using a small animal in vivo imaging system.

[0074] like Fig. 9 The ZIF@EV hydrogel can still continuously release ZIF@EV at the wound site of Balb / c mice, which provides a guarantee for the in vivo application of this dressing and subsequent in vivo experiments.

[0075] 11. Experiment on promoting wound healing in mice using ZIF@EV hydrogel (1) Balb / C mice were anesthetized with chloral hydrate, their back hair was shaved, and a full-thickness skin wound with a diameter of approximately 9 mm was made on the back using a hole punch.

[0076] (2) The mice were randomly divided into five groups, with three mice in each group: Control (normal saline), hydrogel (Gel) group, gel-type ZIF group, gel-type EV group, and gel-type ZIF@EV group.

[0077] (3) On days 0, 4, 8, and 12 after wound formation, each group applied different reagents to the wound surface to verify the therapeutic effect of ZIF@EV hydrogel.

[0078] (4) A transparent ring with an inner diameter of 9 mm was placed on the wound surface to characterize the size of the wound surface, and a camera was used to record the wound surface area on days 0, 4, 8, and 12.

[0079] like Fig.10 , indicating that ZIF@EV hydrogel can significantly promote wound healing in mice and can effectively promote epidermal reconstruction and collagen deposition.

[0080] 12. Antibacterial test The mice were divided into two groups: Staphylococcus aureus group and Escherichia coli group. 6*10 8 1 ml of Staphylococcus aureus (standard bacteria) and Escherichia coli (standard bacteria) suspensions were applied twice on the wounds of the two groups of mice. After 5 hours, saline, Gel, ZIF, EV and ZIF@EV were applied to the wounds of the mice respectively. On the fourth day after injury, the scab on the wound was removed by aseptic operation, and a 0.5 cm × 0.5 cm filter paper soaked in saline was applied to the wound. After 5 seconds, it was removed and placed in 1 ml of saline. After sufficient shaking, it was diluted 10 times. Then 200 μL of the dilution was taken with a pipette and placed on the agar plate for even coating, and then placed in a 37°C oven. Finally, the colony of the culture medium was observed.

[0081] like Fig.11 The colony counts of Staphylococcus aureus and Escherichia coli in the ZIF group and ZIF@EV hydrogel group were significantly lower than those in the control group, indicating that ZIF and ZIF@EV hydrogels showed significant bactericidal effects on Staphylococcus aureus and Escherichia coli, while the EV group did not show antibacterial effects, indicating the antibacterial properties of the nanomaterial ZIF. This further confirms that ZIF@EV hydrogel is not only effective in promoting angiogenesis and regulating immune responses, but also has excellent antibacterial properties, providing additional anti-infection protection for promoting wound healing.

[0082] The present invention provides a ZIF-8 loaded M2 macrophage extracellular vesicle hydrogel and its preparation method and application ideas and methods. There are many methods and ways to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A method for preparing a ZIF-8 loaded M2 macrophage extracellular vesicle hydrogel, characterized in that: The steps include: (1) Preparation of extracellular vesicles from M2 macrophages: M0 macrophages were induced with IL-4 to obtain M2 macrophages; the cell supernatant was collected and separated by ultracentrifugation to obtain M2 macrophage extracellular vesicles EVs; (2) Preparation of exosome vesicles loaded with ZIF: The M2 macrophage extracellular vesicles EV obtained in step (1) are purified, mixed with ZIF-8 nanoparticles and subjected to electroporation to obtain ZIF-loaded exosome vesicles ZIF@EV; (3) Preparation of ZIF@EV hydrogel: After the sodium alginate solution and calcium carbonate are mixed by ultrasonic stirring, the ZIF@EV and D-(+)-glucono delta-lactone obtained in step (2) are added, and the mixture is thoroughly stirred and mixed to obtain the product.

2. The method for preparing the ZIF-8 loaded M2 macrophage extracellular vesicle hydrogel according to claim 1, characterized in that: In step (1), the M0 macrophages are added to an IL-4 solution with a final concentration of 15-25 ng / mL and incubated for 20-25 h to obtain M2 macrophages with elevated surface marker CD206. The supernatant of the M2 macrophages is discarded and the cells are washed several times with PBS. Subsequently, a DMEM medium containing 5-15% special grade fetal bovine serum is added, and the cell supernatant is collected after continuing to incubate for 45-50 h.

3. The method for preparing the ZIF-8 loaded M2 macrophage extracellular vesicle hydrogel according to claim 1, characterized in that: In step (1), the collected cell supernatant is subjected to ultracentrifugation to separate the M2 macrophage extracellular vesicles EVs, the centrifugal force is controlled at 500-100,000 g, and the centrifugation time is 5-120 min.

4. The method for preparing the ZIF-8 loaded M2 macrophage extracellular vesicle hydrogel according to claim 1, characterized in that: In step (1), the cell supernatant is discarded after centrifugation, the obtained extracellular vesicles are resuspended with PBS, and sterilized by passing through a 0.22 μm filter; finally, the sterilized extracellular vesicles are placed in a centrifuge tube and stored in an environment below -80°C for future use.

5. The method for preparing the ZIF-8 loaded M2 macrophage extracellular vesicle hydrogel according to claim 1, characterized in that: In step (2), the purified M2 macrophage extracellular vesicles EV are mixed with ZIF-8 nanoparticles in a mass ratio of 1:(4-6), and then electroporated in an electroporation cell at 1000-1200 kV for 4-10 milliseconds.

6. The method for preparing the ZIF-8 loaded M2 macrophage extracellular vesicle hydrogel according to claim 1, characterized in that: In step (2), the ZIF-loaded exosome vesicles ZIF@EV obtained by electroporation were washed several times with PBS, then incubated for 2 to 4 hours, and finally resuspended in PBS for later use.

7. The method for preparing the ZIF-8 loaded M2 macrophage extracellular vesicle hydrogel according to claim 1, characterized in that: In step (3), the concentration of the sodium alginate solution is 1-3 wt%, the mixed mass volume ratio of calcium carbonate and sodium alginate solution is 10-50 mg / mL, and the mixture is ultrasonically stirred and mixed for 20-40 min to complete the fusion and obtain a mixed slurry.

8. The method for preparing the ZIF-8 loaded M2 macrophage extracellular vesicle hydrogel according to claim 7, characterized in that: In step (3), the mass volume ratio of the added mass of ZIF@EV to the sodium alginate solution is 0.1-20 mg / mL; the mass volume ratio of the added amount of D-(+)-gluconoδ-lactone to the sodium alginate solution is 1-10 mg / mL.

9. The ZIF-8 loaded M2 macrophage extracellular vesicle hydrogel prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the ZIF-8 loaded M2 macrophage extracellular vesicle hydrogel according to claim 9 in preparing wound healing dressings.

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