CN118806687A - Soluble antibacterial microneedle patch capable of delivering apoptotic vesicles in adipose tissue, preparation method and application thereof - Google Patents

Abstract

The present invention discloses a soluble antibacterial microneedle patch capable of delivering apoptotic vesicles of adipose tissue, a preparation method and application thereof, and belongs to the technical field of medical biomaterials. The soluble antibacterial microneedle patch of the present invention comprises a microneedle matrix and an antibacterial substrate; the microneedle matrix comprises apoptotic vesicles of adipose tissue and methacryloyl hyaluronic acid hydrogel; the substrate comprises methacryloyl hyaluronic acid hydrogel and methacryloyl polylysine hydrogel. The present invention also discloses a preparation method of the soluble antibacterial microneedle patch, and discloses the application of the soluble antibacterial microneedle patch in the preparation of an external preparation for wound treatment. The present invention utilizes apoptotic vesicles of adipose tissue and an antibacterial substrate to form a microneedle patch, which can realize painless and non-invasive drug administration, inhibit the colonization, growth and proliferation of bacteria on the wound, realize the slow release of apoptotic vesicles of adipose tissue at the diseased site, and promote the healing of infected wounds and high-quality skin regeneration.

Description

Soluble antibacterial microneedle patch capable of delivering apoptotic vesicles in adipose tissue, preparation method thereof and application thereof

Technical Field

The present invention belongs to the technical field of medical biomaterials, and in particular relates to a soluble antibacterial microneedle patch capable of delivering apoptotic vesicles of adipose tissue, and a preparation method and application thereof.

Background Art

The high morbidity and mortality of acute and chronic wound infections place a huge burden on the global healthcare system. Although the treatment strategies for infected wounds have been standardized, the diagnosis and treatment of wound infections still face huge challenges due to biofilm formation, delayed healing, and drug resistance. For diabetics, wound infections can also cause difficult-to-heal diabetic foot ulcers. Therefore, it is of great significance to develop a new treatment strategy to promote the healing of infected wounds.

Transplantation of adipose tissue or its components is a high-potential wound treatment strategy. Some studies have used adipose tissue to treat skin wounds and achieved good therapeutic effects, but its underlying mechanism of action is still unclear. The theory that cells exert therapeutic effects through paracrine release of growth factors, cytokines, and extracellular vesicles (such as exosomes) after transplantation has been widely recognized. However, recent studies have found that transplanted cells undergo extensive apoptosis in a short period of time, and the process of apoptosis may play a direct role in the regenerative ability of transplanted cells. The vesicles formed during apoptosis are called apoptotic vesicles.

Apoptotic vesicles are double-layer vesicles containing a variety of proteins, lipids and regulatory nucleic acids. The membrane shell of the exosome layer can prevent the degradation of the contents. Apoptotic vesicles derived from adipose tissue can be extracted directly from adipose tissue obtained by liposuction in clinical practice. They have a wide range of sources, a simple extraction process, and nano-scale particle size suitable for minimally invasive drug delivery. Apoptotic vesicles not only have the advantages of other extracellular vesicles such as exosomes, which do not need to consider cell activity, have no self-replication and avoid the risk of tumorigenesis, and have low immunogenicity, but also, compared with exosomes produced by living cells, apoptotic cells have a higher efficiency in producing apoptotic vesicles, and the cell apoptosis process can be completely controlled through standardized operations. They are more suitable for engineering mass production and clinical transformation applications than exosomes.

The construction of apoptotic vesicle drug delivery system is an important carrier for its clinical transformation. In existing studies, apoptotic vesicles are mainly injected into the lesion site through multi-point and multiple traditional injections. This operation method is complicated, the patient tolerance is poor, and professional personnel are required to operate it. The pinholes produced during the injection process are also prone to cause secondary trauma and secondary infection to the patient. In addition, the bacterial biofilm on the infected wound surface will have an unpredictable effect on the efficacy of the vesicles.

Patent CN 116440060 A discloses a method for preparing a microneedle loaded with stem cell exosomes to promote rapid regional epithelialization. However, the preparation of existing stem cell exosome microneedle patches is limited by the source of stem cells and the extraction efficiency of cell exosomes, making it difficult to achieve mass production. Patent CN 115381767A discloses a microneedle patch for preventing or treating skin infections and a method for preparing the same. The microneedles of the patent contain modified natural bactericides and nano-tigecycline, and the substrate contains an antibacterial agent. Tigecycline is an ultra-broad-spectrum antibiotic commonly used to treat a variety of microbial infections caused by multidrug-resistant Gram-positive and Gram-negative pathogens. Although it can quickly inhibit bacteria as a microneedle tip, if its long-term use causes bacterial antibiotic resistance, it will cause long-term and irreversible damage to patients with chronic infections. Furthermore, the patent adjusts the ratio of sucrose and sodium chloride as a drug dissolution medium. Whether it is used as a needle tip or a substrate, if the osmotic pressure does not match that of the skin wound, it will affect the wound edge cell microenvironment, thereby affecting wound healing and cell regeneration.

Therefore, there is an urgent need to develop a drug delivery carrier with a wide source of raw materials, which is painless, non-invasive and antibacterial, so that the apoptotic vesicles can be effectively controlled and released at the infected wound surface, while inhibiting the growth of bacteria on the wound surface, thereby promoting the healing of purulent infected wounds and high-quality skin regeneration.

Summary of the invention

One of the purposes of the present invention is to provide a soluble antibacterial microneedle patch that can deliver apoptotic vesicles from adipose tissue, which can achieve painless and non-invasive drug administration, inhibit the colonization, growth and proliferation of bacteria on the wound surface, achieve the slow release of apoptotic vesicles from adipose tissue at the diseased site, and promote the healing of infected wounds and high-quality skin regeneration.

The second purpose of the present invention is to provide a method for preparing the soluble antibacterial microneedle patch.

The third object of the present invention is to provide an application of the soluble antibacterial microneedle patch.

To achieve the above purpose, the technical solution adopted by the present invention is as follows:

The present invention discloses a soluble antibacterial microneedle patch capable of delivering apoptotic vesicles of adipose tissue (ApoEVs-AT), comprising a microneedle matrix and an antibacterial substrate; the microneedle matrix comprises apoptotic vesicles of adipose tissue and methacryloyl hyaluronic acid hydrogel; the substrate comprises methacryloyl hyaluronic acid hydrogel and methacryloyl polylysine hydrogel.

In some embodiments of the present invention, the mass ratio of the methacrylylated hyaluronic acid hydrogel to the methacrylylated polylysine hydrogel in the substrate is 3-10:3-5, preferably 5:4.

In some embodiments of the present invention, the content of apoptotic vesicles in adipose tissue in each microneedle patch is 320-480 μg/piece.

In some embodiments of the present invention, the diameter of the microneedle base is 250-300 μm, the needle height is 600-800 μm, and the needle tip distance is 550-700 μm; preferably, the patch size and the number of microneedles are adapted to the wound size.

The present invention discloses a method for preparing a soluble antibacterial microneedle patch capable of delivering apoptotic vesicles in adipose tissue, comprising the following steps:

Step 1. Prepare bio-ink: mix adipose tissue apoptotic vesicles with methacrylated hyaluronic acid hydrogel solution to form bio-ink;

Step 2. preparing a mixed antibacterial hydrogel solution of methacrylated hyaluronic acid and methacrylated polylysine water (HAMA/PLMA);

Step 3. dripping the bio-ink into a mold, removing bubbles, heating, and concentrating;

Preferably, after concentration, the bio-ink is added dropwise again, heated, and concentrated;

Step 4. adding the mixed antibacterial hydrogel solution dropwise into the mold, heating and concentrating to prepare an antibacterial substrate;

Step 5. UV light curing and demolding to obtain a soluble antibacterial microneedle patch capable of delivering apoptotic vesicles in adipose tissue.

The preparation method of the mold of the present invention is a prior art. As some embodiments of the present invention, the preparation method of the mold includes the following steps: 3D printing to construct a resin microneedle positive mold, and molding to prepare a polydimethylsiloxane mold; the specifications of the resin microneedle positive mold can be adjusted according to the specifications of the final required soluble antibacterial microneedle patch (ApoEVs-AT@MN) that can deliver apoptotic vesicles in adipose tissue, the wound area, and the epidermal thickness of different host species.

In some embodiments of the present invention, the content of apoptotic vesicles of adipose tissue in the bio-ink is 400-600 μg/ml; preferably 500 μg/ml;

The concentration of the methacrylated hyaluronic acid is 3 to 10% w/v, preferably 5 to 10% w/v, and more preferably 5% w/v.

In some embodiments of the present invention, in the mixed antibacterial hydrogel solution, the concentration of methacrylated hyaluronic acid hydrogel is 3 to 10% w/v; preferably 5 to 10% w/v, more preferably 5% w/v;

The concentration of or/and methacryloyl polylysine hydrogel is 3-5% (w/v), preferably 4%.

In some embodiments of the present invention, in step 3, the bio-ink is dripped into the mold, and bubbles are removed under vacuum conditions;

Preferably, after vacuum degassing, heating and concentrating in an oven at 35-37° C. for 5-6 hours;

Preferably, after the second drop of the bio-ink, the mixture is heated and concentrated in an oven at 35 to 37° C. for 5 to 6 hours;

Preferably, 300 to 600 μl of the bio-ink is added dropwise each time, more preferably 400 μl.

In some embodiments of the present invention, in step 4, 300 to 400 μl of the mixed antibacterial hydrogel solution is added dropwise;

Preferably, after the antibacterial hydrogel solution is added dropwise, the mixture is heated and concentrated in an oven at 35 to 37° C. for 10 to 18 hours, more preferably for 12 hours, to prepare the antibacterial substrate.

The present invention discloses an application of a soluble antibacterial microneedle patch capable of delivering apoptotic vesicles of adipose tissue, wherein the application includes application in preparing an external preparation for wound treatment;

Preferably, the wound surface includes acute and/or chronic infected suppurative wound surface;

Preferably, the wound surface includes difficult-to-heal infected wound surfaces accompanied by various underlying diseases.

The "% (w/v)" mentioned in the present invention represents the mass volume concentration. For example, if the concentration of methacrylated hyaluronic acid hydrogel is 5% (w/v), it means that 100 ml of aqueous solution contains 5 g of methacrylated hyaluronic acid hydrogel.

The method for preparing the apoptotic vesicles of adipose tissue described in the present invention is a prior art. The apoptotic vesicles of adipose tissue can be extracted from adipose tissue of various sources such as rats, pigs, and humans, or from liposuction waste fluid.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention is scientifically designed and ingeniously conceived. It utilizes adipose tissue apoptotic vesicles and an antibacterial substrate to form a microneedle patch, which can achieve painless and non-invasive drug delivery, inhibit the colonization, growth and proliferation of bacteria on the wound surface, and achieve the slow release of adipose tissue apoptotic vesicles at the diseased site.

The microneedle matrix in the soluble antibacterial microneedle patch of the present invention can painlessly and non-invasively deliver the apoptotic vesicles of adipose tissue to the infected wound in a controllable, accurate and efficient manner, which can effectively promote the healing of infected suppurative wounds and high-quality skin regeneration, including the regeneration of skin appendages such as hair follicles, sebaceous glands, and sweat glands, and reduce the formation of scars; at the same time, the antibacterial substrate can effectively inhibit the growth and reproduction of bacteria on the infected wound. The synergistic effect of the two can significantly promote the healing of infected wounds and high-quality skin regeneration on the basis of anti-infection treatment of infected suppurative wounds.

The microneedle matrix of the present invention is composed of a bio-ink prepared from apoptotic vesicles of adipose tissue and methacryloyl hyaluronic acid hydrogel. After piercing the skin, it quickly absorbs interstitial fluid into its grid, realizing the characteristic that the material swells but does not dissolve, and produces micropores in the gel that can deliver vesicles. Under the penetration and diffusion of tissue fluid, apoptotic vesicles of adipose tissue are delivered into the body through the micropores of the hydrogel. The slow release of apoptotic vesicles of adipose tissue in the diseased part is achieved, and the efficiency of the action of apoptotic vesicles of adipose tissue is improved. Hydrogel can play a certain sustained release role, but a simple hydrogel dressing cannot penetrate the epidermis and cannot deliver the effective ingredients to the dermis. The present invention adopts the drug-carrying method of hydrogel microneedles, which ensures that the effective ingredients can be efficiently delivered to the dermis while playing a sustained release role for the internal active transmitter.

At the same time, the tiny holes formed by microneedle puncture can heal automatically within a few hours without causing bleeding and trauma, thus achieving painless and non-invasive drug delivery. In addition, the antibacterial substrate is prepared from a mixed solution of methacryloyl hyaluronic acid and methacryloyl polylysine hydrogel, which can directly act on bacterial biofilms, inhibit the colonization, growth and proliferation of bacteria on the wound surface, and then assist the microneedle matrix in promoting the healing of infected wounds.

The active transmitter contained in the present invention is apoptotic vesicles of adipose tissue, which not only has the advantages of stem cell exosomes, such as no need to consider cell activity, no self-replication and avoiding the risk of tumor formation, low immunogenicity, etc., but also compared with exosomes produced by living cells, apoptotic cells produce apoptotic vesicles with higher efficiency, and can completely control the apoptotic process through standardized operations, which is more suitable for engineering mass production and clinical transformation applications than exosomes. In addition, the present invention directly induces apoptosis of adipose tissue in vitro and extracts apoptotic vesicles from adipose tissue, without the need to extract and culture a large number of living cells, which greatly reduces the technical cycle and technical cost.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic flow diagram of a method for preparing a soluble antibacterial microneedle patch capable of delivering apoptotic vesicles of adipose tissue according to the present invention;

FIG2 is a schematic diagram of the in vivo action process of the soluble antibacterial microneedle patch capable of delivering apoptotic vesicles of adipose tissue of the present invention;

FIG3 is a comparison of the histological structures of natural adipose tissue and apoptotic adipose tissue;

FIG4 is a comparison of TUNEL staining of native adipose tissue and apoptotic adipose tissue, wherein the left picture is native adipose tissue and the right picture is apoptotic adipose tissue;

FIG5 is a transmission electron microscopy scanning result of apoptotic vesicles in adipose tissue;

FIG6 is a graph showing the immunofluorescence staining results of Annexin V protein expression in apoptotic vesicles of adipose tissue;

FIG7 is a graph showing the particle size distribution of apoptotic vesicles in adipose tissue;

FIG8 is a graph showing the expression of marker proteins of apoptotic vesicles in adipose tissue detected by Western blot;

FIG9 is an appearance diagram of hydrogels with different concentrations;

FIG10 is a graph showing the viscosity of hydrogels at different concentrations;

FIG11 is a graph showing the dynamic modulus investigation results of hydrogels of different concentrations;

FIG12 is a graph showing the results of the growth inhibition of high concentration Staphylococcus aureus by different hydrogels;

FIG13 is a graph showing the bacterial area counts of different hydrogels inhibiting high concentrations of Staphylococcus aureus;

FIG14 is a comparison of bacterial OD values after 0 hour and 24 hours of co-culture of different hydrogels and bacteria;

FIG15 is a general digital image of ApoEVs-AT@MN of the present invention, wherein the right image is an enlarged image of the left image;

FIG16 is a statistical diagram of the needle tip angle of ApoEVs-AT@MN of the present invention;

FIG17 is a three-dimensional reconstruction of ApoEVs-AT@MN prepared by fluorescently labeled methacrylated hyaluronic acid hydrogel;

Figure 18 is a scanning electron microscope image of ApoEVs-AT@MN, wherein the right image is an enlarged image of the left image;

FIG19 is a micrograph of an axial compression experiment of ApoEVs-AT@MN;

FIG20 is a statistical diagram of the mechanical strength results of the axial compression test of ApoEVs-AT@MN;

FIG21 is a three-dimensional reconstruction of DiO-labeled ApoEVs-AT@MN placed under a fluorescence confocal microscope;

FIG22 is a graph showing the protein release curve of ApoEVs-AT@MN;

FIG23 is a graph showing the results of fibroblast uptake of DiO-labeled ApoEVs-AT released from ApoEVs-AT@MN;

FIG24 is a graph showing the results of a fibroblast proliferation test;

Figure 25 is a micrograph of a Transwell assay;

Figure 26 is a statistical diagram of Transwell test results;

FIG27 is a graph showing the expression of genes related to adipogenesis promoted by ApoEVs-AT;

FIG28 is a graph showing the results of endothelial cells taking up DiO-labeled ApoEVs-AT released from ApoEVs-AT@MN;

Figure 29 is a graph showing the results of an endothelial cell proliferation assay;

Figure 30 is a micrograph of the scratch test results;

FIG31 is a statistical diagram of scratch test results;

Figure 32 is a micrograph of a Transwell assay;

Figure 33 is a statistical diagram of Transwell test results;

FIG34 is a micrograph of the tube forming test results;

FIG35 is a statistical diagram of the tube forming test results;

Figure 36 is a timeline diagram of the animal experiment operation of Experimental Example 5.

FIG37 is a diagram of wounds at different time points in the animal experiment of Experimental Example 5;

FIG38 is a diagram of the wound healing pattern of Experimental Example 5;

FIG39 is a statistical chart of wound healing percentages in Experimental Example 5;

FIG40 is an H&E and Masson staining image of tissue sections of the infected wound area of Experimental Example 5 on the 8th and 16th days;

41 is a graph showing the results of detecting the lipid-associated protein Perilipin A and the angiogenesis-associated protein Platelet-endothelial cell adhesion molecule (CD31) in the skin tissues of each group collected on the 16th day of Experimental Example 5.

FIG42 is a graph showing the results of the investigation of new skin thickness on the 16th day of Experimental Example 5;

FIG43 is a graph showing the results of investigating the expression of CD31 in skin tissues of each group collected on the 16th day of Experimental Example 5;

FIG44 is a graph showing the results of investigating the expression of Perilipin A in skin tissues of each group collected on the 16th day of Experimental Example 5;

FIG45 is an H&E and Masson staining image of tissue sections of the infected wound area of Experimental Example 5 on the 8th and 16th days;

FIG46 is a graph showing the expression levels of collagen type I (Col 1), α-smooth muscle actin (α-SMA) and collagen type III (Col 3) in skin tissues of each group collected on the 16th day of Experimental Example 5;

FIG47 is a statistical diagram of the hair follicle results of rats on the 16th day of Experimental Example 5;

FIG48 is a statistical graph of the expression level of α-SMA on the 16th day of Experimental Example 5;

FIG. 49 is a graph showing the Col3/Col1 investigation results on the 16th day of Experimental Example 5.

DETAILED DESCRIPTION

The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. The reagents and other instruments used without specifying the manufacturer are all conventional reagent products that can be purchased on the market.

The adipose tissue apoptotic vesicles described in the embodiments of the present invention are prepared according to the method of Example 1 of the patent publication number CN116350659 A.

Examples 1-3 disclose the preparation of a soluble antibacterial microneedle patch capable of delivering apoptotic vesicles from adipose tissue.

Example 1

This embodiment discloses the preparation of the soluble antibacterial microneedle patch (ApoEVs-AT@MN) capable of delivering apoptotic vesicles in adipose tissue of the present invention; the schematic diagram of the process is shown in FIG1. The specific steps are:

S1. Extract apoptotic vesicles from adipose tissue using echelon centrifugation;

S2. Prepare bio-ink: mix adipose tissue apoptotic vesicles with methacrylated hyaluronic acid hydrogel solution to form bio-ink; the content of adipose tissue apoptotic vesicles in the bio-ink is 500 μg/ml, and the concentration of methacrylated hyaluronic acid is 5% w/v.

S3. Prepare a mixed antibacterial hydrogel solution: take methacrylated hyaluronic acid hydrogel and methacrylated polylysine, add water to mix and dissolve, and prepare a mixed antibacterial hydrogel solution; wherein the concentration of methacrylated hyaluronic acid hydrogel is 5% w/v, and the concentration of methacrylated polylysine hydrogel is 4% w/v.

S4. Construct a resin microneedle positive mold by 3D printing and prepare a polydimethylsiloxane mold by molding method.

S5. Take 400 μl of the bio-ink prepared in step S2, drop it into the mold, remove bubbles in vacuum, heat in an oven at 35°C and concentrate for 5 hours; add 400 μl of the bio-ink a second time, heat in an oven at 35°C and concentrate for 5 hours.

S6. Add 400 μl of the mixed antibacterial hydrogel solution prepared in step S3, heat and concentrate in an oven at 35° C. for 12 hours to prepare an antibacterial substrate.

S7. UV light curing for 15 seconds, demolding, and finally obtaining a soluble antibacterial microneedle patch that can deliver apoptotic vesicles in adipose tissue.

The microneedle base diameter of the microneedle patch prepared in this embodiment is 300 μm, the needle height is 800 μm, and the needle tip distance is 700 μm.

Example 2

This embodiment discloses the preparation of the soluble antibacterial microneedle patch capable of delivering apoptotic vesicles of adipose tissue of the present invention; the schematic flow chart thereof is shown in FIG1. The specific steps are:

S1. Extract apoptotic vesicles from adipose tissue using echelon centrifugation;

S2. Prepare bio-ink: mix adipose tissue apoptotic vesicles with methacrylated hyaluronic acid hydrogel solution to form bio-ink; the content of adipose tissue apoptotic vesicles in the bio-ink is 400 μg/ml, and the concentration of methacrylated hyaluronic acid is 10% w/v.

S3. Prepare a mixed antibacterial hydrogel solution: take methacrylated hyaluronic acid hydrogel and methacrylated polylysine, add water to mix and dissolve, and prepare a mixed antibacterial hydrogel solution; wherein the concentration of methacrylated hyaluronic acid hydrogel is 10% w/v, and the concentration of methacrylated polylysine hydrogel is 3% w/v.

S4. Construct a resin microneedle positive mold by 3D printing and prepare a polydimethylsiloxane mold by molding method.

S5. Take 500 μl of the bio-ink prepared in step S2, drop it into the mold, remove bubbles in vacuum, heat in an oven at 37°C and concentrate for 6 hours; add 500 μl of bio-ink a second time, heat in an oven at 37°C and concentrate for 6 hours.

S6. Add 300 μl of the mixed antibacterial hydrogel solution prepared in step S3, heat and concentrate in an oven at 35° C. for 12 hours to prepare an antibacterial substrate.

S7. UV light curing for 15 seconds, demolding, and finally obtaining a soluble antibacterial microneedle patch that can deliver apoptotic vesicles in adipose tissue.

The microneedle base diameter of the microneedle patch prepared in this embodiment is 250 μm, the needle height is 600 μm, and the needle tip distance is 600 μm.

Example 3

This embodiment discloses the preparation of the soluble antibacterial microneedle patch capable of delivering apoptotic vesicles of adipose tissue of the present invention; the schematic flow chart thereof is shown in FIG1. The specific steps are:

S1. Extract apoptotic vesicles from adipose tissue using echelon centrifugation;

S2. Prepare bio-ink: mix adipose tissue apoptotic vesicles with methacrylated hyaluronic acid hydrogel solution to form bio-ink; the content of adipose tissue apoptotic vesicles in the bio-ink is 600 μg/ml, and the concentration of methacrylated hyaluronic acid is 8% w/v.

S3. Prepare a mixed antibacterial hydrogel solution: take methacrylated hyaluronic acid hydrogel and methacrylated polylysine, add water to mix and dissolve, and prepare a mixed antibacterial hydrogel solution; wherein the concentration of methacrylated hyaluronic acid hydrogel is 8% w/v, and the concentration of methacrylated polylysine hydrogel is 5% w/v.

S4. Construct a resin microneedle positive mold by 3D printing and prepare a polydimethylsiloxane mold by molding method.

S5. Take 300 μl of the bio-ink prepared in step S2, drop it into the mold, remove bubbles in vacuum, heat in an oven at 37°C and concentrate for 6 hours; add 300 μl of bio-ink a second time, heat in an oven at 37°C and concentrate for 6 hours.

S6. Add 400 μl of the mixed antibacterial hydrogel solution prepared in step S3, heat and concentrate in an oven at 35° C. for 12 hours to prepare an antibacterial substrate.

S7. UV light curing for 15 seconds, demolding, and finally obtaining a soluble antibacterial microneedle patch that can deliver apoptotic vesicles in adipose tissue.

The microneedle base diameter of the microneedle patch prepared in this embodiment is 280 μm, the needle height is 700 μm, and the needle tip distance is 550 μm.

Test Example 1

In this test example, the adipose tissue apoptotic vesicles obtained in Example 1 were identified.

Apoptosis of adipose tissue was assessed by H&E and TUNEL staining.

Compared with native adipose tissue, the structure of apoptotic adipose tissue is loose and irregular, as shown in Figure 3.

The TUNEL staining images show that apoptosis occurs in many cells of apoptotic adipose tissue; in contrast, there are no TUNEL-labeled nuclei in native adipose tissue, as shown in Figure 4. Green indicates TUNEL-stained apoptotic cells; blue indicates DAPI-stained nuclei.

Test Example 2

In this test example, the mechanical properties and antibacterial properties of HAMA/PLMA mixed antibacterial hydrogels with different concentration ratios were evaluated.

1. Mechanical properties inspection

The mechanical properties of HAMA/PLMA hybrid antibacterial hydrogels were evaluated to obtain information about the rheological behavior of HAMA/PLMA hybrid antibacterial hydrogel solutions.

1.1 Preparation of hydrogels with different concentrations.

HAMA hydrogel: Take methacrylated hyaluronic acid and add water to prepare a 5% w/v HAMA hydrogel solution.

HAMA+4%/PLMA hydrogel: Methacrylated hyaluronic acid and methacrylated polylysine were added with water to prepare a mixed hydrogel solution with a HAMA concentration of 5% w/v and a PLMA concentration of 4% w/v, thereby preparing a HAMA+4%/PLMA hydrogel.

HAMA+2%/PLMA hydrogel: Compared with HAMA+4%/PLMA hydrogel, the difference is that the concentration of PLMA in the mixed hydrogel solution is 2% w/v, and the rest are the same.

HAMA+6%/PLMA hydrogel: Compared with HAMA+4%/PLMA hydrogel, the difference is that the concentration of PLMA in the mixed hydrogel solution is 6% w/v, and the rest are the same.

The appearance of hydrogels with different concentrations is shown in Figure 9.

1.2 Investigation of rheological behavior of hydrogel.

Viscoelastic analysis was performed using a HAAKE viscometer iQ Air, and experiments were performed using a C35 1°/Ti conical rotator with a gap distance of 1 mm. Shear rate sweeps were performed at 25°C from 0.1 to 100 s ^-1 to determine the shear viscosity of each group of HAMA/PLMA mixed antibacterial hydrogel solutions. The dynamic modulus of the bio-ink was characterized by frequency sweeps: at 25°C, in the frequency range of 0.1-10 Hz, the applied strain constant was 0.01.

The results showed that within the set concentration range, the viscosity and dynamic modulus of the mixed antibacterial hydrogel were the highest when the PLMA concentration was 4% w/v, as shown in Figures 10 and 11. Rheology is an effective means to determine whether the HAMA/PLMA mixed antibacterial hydrogel solution can be used as a microneedle substrate and skin wound dressing. The results showed that the HAMA/PLMA bio-ink can resist deformation and exhibit the elasticity of a robust hydrogel network.

2. Investigation of antibacterial properties

To further evaluate the antibacterial properties of the HAMA/PLMA mixed antibacterial hydrogel solution, the embodiment of the present invention incubated a high concentration of Staphylococcus aureus bacterial suspension (about 10 ⁶ CFU/ml) with HAMA/PLMA photocured mixed antibacterial hydrogel containing different concentrations of PLMA at 37°C for 24 hours, and incubated 400μl of photocured hydrogel for every 1ml of Staphylococcus aureus bacterial suspension. After the incubation, the bacterial suspension was taken and counted by OD value; and the bacterial suspension was diluted 1:10000 and inoculated on the surface of the agar culture medium. After culturing at 37°C for 24 hours, the culture dish was taken out to take pictures and count.

The preparation method of each photocured mixed antibacterial hydrogel is as follows: prepare each hydrogel according to the method of "preparing hydrogels of different concentrations" under 1.1 of this embodiment, and then cure with UV light for 15 seconds.

The results showed that the addition of PLMA could effectively inhibit the growth and proliferation of high concentration Staphylococcus aureus, as shown in Figures 12-14.

Test Example 3

This test example investigated the material properties of soluble microneedles.

1. Investigation of the integrity of the microneedle as a whole and the needle tip

The soluble antibacterial microneedle patch capable of delivering apoptotic vesicles in adipose tissue prepared in Example 1 was taken, and the macroscopic digital photo showed that the ApoEVs-AT@MN as a whole and the needle tip were intact, as shown in FIG. 15 .

2. Investigation of microneedle tip angle

The needle tip angles were observed and counted under a microscope, and the results showed that the needle tip angles of the microneedle matrix were between 30-38°, which was convenient for insertion into the skin, as shown in FIG16 .

3. Fluorescence inspection of microneedle tip integrity

Fluorescently labeled ApoEVs-AT@MN was prepared using fluorescently labeled methacrylated hyaluronic acid hydrogel according to the method of Example 1, and three-dimensional reconstruction was performed under a fluorescence confocal microscope. The results further demonstrated the integrity of the needle tip, as shown in FIG17 .

4. Scanning electron microscopy observation

Scanning electron microscopy showed that there were pores on the surface of the ApoEVs-AT@MN that could be used to release apoptotic vesicles of adipose tissue (ApoEVs-AT), as shown in FIG18 .

5. Axial compression test

Place different weights and sizes on ApoEVs-AT@MN, remove them, observe the deformation of the needle tip, and take pictures. In addition, place ApoEVs-AT@MN on an electronic universal testing machine and perform an axial compression test. Apply force from 0g until the needle tip is deformed, refer to 2.5* ^10-3 MPa, and measure the microneedle stress-strain curve based on the deformation of the needle tip. The axial compression test shows that the needle tip morphology of ApoEVs-AT@MN is relatively stable within the range of 0-1000g resistance weight, as shown in Figure 19, and the maximum mechanical strength that each needle tip can withstand is greater than the insertion force required for the needle to enter the skin barrier, which is about 0.125N/needle, as shown in Figure 20.

6. 3D reconstruction of DiO-labeled ApoEVs-AT@MN

Take 100 μg ApoEVs-AT, add 1 ml α-MEM culture medium, and add 1 μl cell membrane green fluorescent probe (DiO dye), mix well and incubate in a 37°C, 5% CO ₂ incubator for 20 minutes. Centrifuge at 10,000g, 4°C for 1 hour to obtain DiO-labeled ApoEVs-AT. Then use DiO-labeled ApoEVs-AT to prepare ApoEVs-AT@MN according to Example 1.S2-7 to obtain DiO-labeled ApoEVs-AT@MN. Afterwards, the DiO-labeled ApoEVs-AT@MN was placed under a fluorescence confocal microscope for scanning and three-dimensional reconstruction. The results showed that the microneedle matrix in the ApoEVs-AT@MN had a large drug loading capacity, as shown in Figure 21.

7. BCA test

The BCA test steps are as follows:

(1) ApoEVs-AT@MN was placed in 4 ml PBS, and 20 μl was drawn into EP tubes on days 0, 1, 2, 3, 4, 5, 6, 7, and 8 as test samples;

(2) Set up standard wells in a 96-well plate and add the reagents listed in the following table:

(3) Set up sample wells, take 10 μl of the sample to be tested and dilute it to 100 μl, and add 20 μl of the diluted sample to each well;

(4) Prepare the color developing solution according to the ratio of solution A: solution B = 50:1 in the instruction manual and shake to mix;

(5) Add 200 μl of color development solution to each well;

(6) The 96-well plate was shaken and mixed in an ELISA reader and incubated at 37°C for 30 min;

(7) Measure the absorbance (OD) value of each well under 562 nm excitation light;

(8) Draw a standard curve based on the OD value of each well.

The BCA test results are shown in Figure 22; the results show that ApoEVs-AT@MN can gradually release the ApoEV-AT inside it in PBS until the 8th day.

Experimental Example 4 Cell Biology Experiment

In order to explore the effect of ApoEVs-AT@MN on important cells in the skin regeneration process, this experimental example selected two important cells in the skin regeneration process: fibroblasts and endothelial cells, and conducted a co-culture in vitro experiment. The DiO-labeled ApoEVs-AT@MN in this experimental example was prepared according to the method of Experimental Example 3.

1. Investigation of the effect on fibroblasts

1. DiO-labeled ApoEVs-AT@MN and fibroblast co-culture experiment

DiO-labeled ApoEVs-AT@MN was placed in a confocal dish with fibroblasts attached to the wall, and α-MEM supplemented with 10% fetal bovine serum was added to cover the cells and ApoEVs-AT@MN for co-culture. After 24 hours, ApoEVs-AT@MN was taken out, and the fibroblasts were stained with fluorescent proteins for the cytoskeleton. DiO-labeled ApoEVs-AT can be effectively detected in the fibroblasts, as shown in Figure 23, indicating that fibroblasts can take up ApoEVs-AT released by ApoEVs-AT@MN. Among them, red represents fibroblasts stained with phalloidin, green represents DiO-labeled ApoEVs-AT, and blue represents DAPI-stained cell nuclei.

The experimental steps for cytoskeleton (phalloidin) fluorescent protein staining are as follows:

(1) Take the glass-bottomed dish out of the incubator, remove the culture medium, and wash with PBS three times, 5 min each time;

(2) Fix with 4% paraformaldehyde at room temperature for 20 min and wash with PBS three times, 5 min each time;

(3) Add 0.1% Triton X-100 for 5 min and wash with PBS three times, 5 min each time;

(4) Block with 1% BSA at room temperature for 20 min to prevent nonspecific staining, and wash with PBS three times, 5 min each time;

(5) Add 5 μg/mL FITC-phalloidin to stain 200 μL, place in a 37°C incubator for staining for 1 h, protect from light, and wash with PBS three times, 5 min each time;

(6) DAPI staining of nuclei for 5 min, washing with PBS three times, 5 min each time, in the dark;

(7) Observe and take photos using a laser confocal microscope.

2. Fibroblast Proliferation Assay

ApoEVs-AT@MN was placed in a 15 ml centrifuge tube, and 10 ml of α-MEM supplemented with 10% fetal bovine serum was added. The tube was placed in a cell culture incubator at 37°C for 48 hours to obtain the conditioned medium.

Fibroblasts were seeded into 96-well plates at 2×10 ³ cells per well and incubated overnight at 37°C with 5% CO _2. After the cells adhered to the wall, the original culture medium was removed. 200 μl of conditioned medium was added to each well of the experimental group, and full culture medium was added to each well of the blank group. The culture medium was changed every 2 days according to the corresponding required culture medium. After 1, 2, 3, 4, 5, 6, and 7 days of culture, the original culture medium was removed and 110 μl of CCK-8 working solution was added to each well (10 μl of CCK-8 detection solution was added to every 100 μl of α-MEM). Incubate at 37°C for 1 hour, measure the OD value with an enzyme marker, and perform CCK-8 detection. The results show that compared with the blank group, the conditioned medium can significantly increase the proliferation of fibroblasts, as shown in Figure 24.

3. Transwell assay

Fibroblasts were seeded in the upper chamber of 24-well Transwell at a density of 1.5×10 ⁴ /well; experimental group (conditioned medium) and blank group (α-MEM) culture medium were added to the lower layer of the Transwell chemotaxis chamber respectively; incubated in a 37°C, 5% CO ₂ incubator for 12 hours; the Transwell chamber was removed and fixed with 4% paraformaldehyde at room temperature for 2 hours; the cells on the upper layer of the membrane were carefully wiped off with a cotton swab and washed 3 times with PBS; Giemsa staining was performed and observed and photographed under a light microscope; 5 fields of view were taken for each sample, and the number of cells that migrated through the membrane was counted and analyzed. The results of the Transwell test showed that the conditioned medium significantly promoted the migration of fibroblasts after 12 hours of co-culture, as shown in Figures 25 and 26.

4. Experiment to promote the expression of adipogenesis-related genes

After the fibroblasts were treated with conditioned medium for 20 days, total RNA was extracted and reverse transcribed according to routine procedures. Then, samples were added according to the following primer table and the expression of adipogenesis-related genes PPARγ, C/EBPα, Adiponectin and FABP4 was evaluated by qRT-PCR. The results showed that ApoEVs-AT can promote the expression of adipogenesis-related genes, as shown in Figure 27.

Table 1 Primer sequence list

SEQ ID NO sequence PPARγ2-F 1 GACCACTCCCACTCCTTTGA PPARγ2-R 2 CAGGCTCCACTTTGATTGC C/EBPα-F 3 AGGTTTCCTGCCTCCTTCC C/EBPα-R 4 CCCAAGTCCCTATGTTTCCA Adiponectin-F 5 CCCATTCGCTTTACCAAGAT Adiponectin-R 6 GGCTGACCTTCACATCCTTC FABP4-F 7 CAGGAAAGTCAAGAGCACCA FABP4-R 8 TCCACCACCAGTTTATCATCC

2. Investigation of the effect on endothelial cells

1. Co-culture experiment of DiO-labeled ApoEVs-AT@MN and endothelial cells

DiO-labeled ApoEVs-AT@MN was placed in a confocal dish with endothelial cells attached to the wall, and α-MEM supplemented with 10% fetal bovine serum was added to cover the cells and ApoEVs-AT@MN for co-culture. After 24 hours, ApoEVs-AT@MN was taken out, and the endothelial cells were stained with fluorescent proteins for the cytoskeleton. DiO-labeled ApoEVs-AT can be effectively detected in the endothelial cells, as shown in Figure 28, indicating that endothelial cells can take up ApoEVs-AT released by ApoEVs-AT@MN. Among them, red represents endothelial cells stained with phalloidin, green represents DiO-labeled ApoEVs-AT, and blue represents DAPI-stained cell nuclei.

2. Endothelial Cell Proliferation Assay

ApoEVs-AT@MN was placed in a 15 ml centrifuge tube, and 10 ml of α-MEM supplemented with 10% fetal bovine serum was added. The tube was placed in a cell culture incubator at 37°C for 48 hours to obtain the conditioned medium.

Endothelial cells were seeded into 96-well plates at 2×10 ³ cells per well and incubated overnight at 37°C with 5% CO _2. After the cells adhered to the wall, the original culture medium was removed. 200 μl of conditioned medium was added to each well of the experimental group, and full culture medium was added to each well of the blank group. The culture medium was changed every 2 days according to the corresponding required culture medium. After 1, 2, 3, 4, and 5 days of culture, the original culture medium was removed and 110 μl of CCK-8 working solution was added to each well (10 μl of CCK-8 detection solution was added to every 100 μl of α-MEM). Incubate at 37°C for 1 hour, measure the OD value with an enzyme marker, and perform CCK-8 detection. The results show that compared with the blank group, the conditioned medium can significantly increase the proliferation of endothelial cells, as shown in Figure 29.

3. Scratch assay and Transwell assay

Endothelial cells were seeded in a 12-well plate at a density of 1×10 ⁵ /well; incubated in a 37°C, 5% CO ₂ incubator until the cells grew to a density of more than 90%; a horizontal line was drawn with the help of a ruler using a gun tip; the drawn cells were gently washed off with PBS; the experimental group (conditioned medium) and the blank group (α-MEM) medium were added; incubated in a 37°C, 5% CO ₂ incubator, and photographed, counted, and statistically analyzed after 24 hours. The results of the scratch test showed that the conditioned medium significantly promoted the migration of endothelial cells after 24 hours, as shown in Figures 30 and 31.

Endothelial cells were seeded in the upper chamber of 24-well Transwell at a density of 1.5×10 ⁴ /well; experimental group (conditioned medium) and blank group (α-MEM) culture medium were added to the lower layer of the Transwell chemotaxis chamber respectively; incubated in a 37°C, 5% CO ₂ incubator for 12 hours; the Transwell chamber was removed and fixed with 4% paraformaldehyde at room temperature for 2 hours; the cells on the upper layer of the membrane were carefully wiped off with a cotton swab and washed 3 times with PBS; Giemsa staining was performed and observed and photographed under a light microscope; 5 fields of view were taken for each sample, and the number of cells that migrated through the membrane was counted and analyzed. The results of the Transwell test showed that the conditioned medium significantly promoted the migration of endothelial cells after 12 hours of action, as shown in Figures 32 and 33.

4. Tube Formation Experiment

Endothelial cells were seeded in a 96-well plate coated with Matrigel at a cell density of 2×10 ⁴ , and epithelial cell culture medium was used as the positive control group. Cultured for 6 hours at 37°C and 5% CO ₂ , observed and photographed under an inverted phase contrast microscope. Image Pro Plus software was used to count the number of nodes, crosslinks, and blood vessels in each group, and plotted. The results of the tube formation experiment showed that the conditioned medium can significantly induce angiogenesis of endothelial cells, and the results of the increase in the number of nodes, the number of grids, and the total length are shown in Figures 34 and 35.

It can be seen from the results of this experiment that ApoEVs-AT@MN can act on fibroblasts and endothelial cells by releasing ApoEVs-AT and regulate the cellular behaviors of both.

Experimental Example 5: Rat Infection Wound Model Experiment

In this experiment, in order to evaluate the role of ApoEVs-AT@MN in full-thickness skin wound healing, two full-thickness skin wounds (2 cm in diameter) were established on the back of each SD rat and coated with a high-concentration Staphylococcus aureus suspension, which was recorded as day -2. After 48 hours, the suppuration of the skin wound was observed, which was recorded as day 0.

The experiment was divided into ApoEVs-AT@MN group and blank group. In the ApoEVs-AT@MN group, ApoEVs-AT@MN were implanted once on day 0 and day 8. In the blank group, 100 μl PBS was used as a control.

A schematic diagram of ApoEVs-AT@MN acting on the wound surface is shown in Figure 2, and the animal experiment operation timeline is shown in Figure 36.

1. Wound healing study

Digital macrophotographs were taken on days 0, 4, 8, 12, and 16 to measure the healing rate, as shown in Figure 37. A pattern diagram was drawn to more clearly indicate the speed of wound healing, as shown in Figure 38, in which the dotted circle represents the range of the initial wound area on day 0, and the colored part represents the area of the unhealed wound at different time points.

In the embodiment of the present invention, the wound area (%) is defined as: the unhealed wound area on day X/initial wound area×100%.

In this test example, through statistical analysis of the wound areas of rats in the blank group and the ApoEVs-AT@MN group, it was found that after implantation on day 0, ApoEVs-AT@MN showed the ability to accelerate wound healing. ApoEVs-AT@MN reduced the average wound area on day 8 from 16.04% of the blank group to 4.07% of the ApoEVs-AT group, as shown in Figure 39. Similarly, after the second ApoEVs-AT@MN treatment, ApoEVs-AT@MN reduced the average wound area on day 12 from 6.74% of the blank group to 0.45%, as shown in Figure 39. In addition, according to the digital macrophotographs, on day 16, the full-thickness skin wounds in the ApoEVs-AT@MN group healed, with only linear scars. In contrast, the wounds in the blank group were not completely healed, with an average wound area of 1.55%, and the presence of scabs was still visible. Moreover, the scar area of each group was significantly larger than that of the ApoEVs-AT@MN group, as shown in Figure 37.

2. Evaluate the quality of granulation tissue on day 8 and new skin tissue on day 16 in the infected wound area.

2.1 Collect wound area tissue on the 8th day and make sections for subsequent staining. H&E staining of longitudinal sections revealed that the new tissue in the ApoEVs-AT@MN group had a clear new epidermis, and its dermis had abundant regularly arranged new skin appendages, including new hair follicles, sweat glands, sebaceous glands, etc. The unhealed area in the center of the wound was connected by granulation tissue, and the granulation tissue was covered by scab, as shown in Figure 40. In the blank group, only a very small amount of irregular skin-like appendage tissue appeared at the edge of the wound, while most of the wound area was connected by granulation tissue, and the surface was covered by a large number of scabs, as shown in Figure 40, which is consistent with the results of the gross photograph. It shows that ApoEVs-AT@MN accelerates the speed of re-epithelialization and regeneration of skin appendages. Among them, the black inverted triangle indicates the edge of the wound area/the edge of re-epithelialization, the black solid box indicates the enlarged area, and in the enlarged area on the right, the black dotted line indicates the area of regenerated epidermis.

2.2 In this test example, the healed skin of the wound site on the 16th day was collected, and sections were made for subsequent staining. The H&E stained images showed that the wound of the ApoEVs-AT@MN group was completely healed on the 16th day, the scar area was extremely narrow, and there were a large number of regularly arranged new hair follicles, hair, sebaceous glands, etc. in the non-scar area, similar to natural skin, and there were a large number of large-diameter, extensive new blood vessels infiltration, as shown in Figure 40. In contrast, the blank group was not completely healed on the 16th day, and there was still a small amount of scab on the wound surface. The scar area was significantly wider than that of the ApoEVs-AT@MN group. The wound surface was occupied by fibrous tissue over a large area, and only a very small amount of irregular skin attachments appeared at the edge of the wound surface. No obvious new blood vessel infiltration was observed on the surface of the new skin, and a small amount of new microvascular infiltration was observed in the basal layer of the skin, as shown in Figure 40. The above results are consistent with the digital macrophotographs.

2.3 In this test example, immunofluorescence staining analysis was performed on the skin tissues of each group collected on the 16th day to evaluate the expression of different proteins in the new tissue. The results showed that in the non-scar area, compared with the blank group, the ApoEVs-AT@MN group could promote the expression of fibroblast-related proteins type I collagen (Col 1) and type III collagen (Col 3), adipogenic-related protein perilipin (Perilipin A), and angiogenic-related protein platelet-endothelial cell adhesion molecule (CD31), as shown in Figures 41 and 46. Among them, the positive signal of Col 3 in the ApoEVs-AT@MN group was more than that in the blank group. According to statistics, ApoEVs-AT@MN increased the relative fluorescence density of Col 3/Col 1 in each field of view from 0.099 (blank group) to 0.43 (ApoEVs-AT@MN group), as shown in Figures 46 and 49. It is reported that an increase in the ratio of Col 3/Col 1 is closely associated with a reduction in scars and an increase in skin softness. In addition, α-smooth muscle actin (α-SMA), which can be expressed in mature fibroblasts, perivascular cells, and hair follicle root sheaths, is also expressed in large quantities in the new skin of the ApoEVs-AT@MN group, as shown in Figures 46 to 48.

The steps for immunofluorescence staining of sections are as follows:

(1) Place the paraffin sections in a 60°C oven overnight;

(2) Dewaxing with xylene I and xylene II, 10 min each;

(3) 100% I, 100% II, 95% I, 95% II, 85%, 75% gradient alcohol hydration, 5 min each, and then rinse with running water;

(4) Incubate with peroxidase inhibitor at room temperature for 20 min and wash with PBS three times (5 min/time);

(5) Bring the citric acid solution to a boil in a microwave oven. Remove the bubbles from the empty slide rack and gently

Put the slices in to prevent them from falling off, adjust the microwave to the lowest setting, keep the solution temperature but not boiling, and cook for 15 minutes.

Remove, cool the slides at room temperature, and wash 3 times with PBS (5 min/time);

(6) Block with goat serum at room temperature for 30 min, and remove the goat serum (without washing);

(7) Add primary antibodies (Col 1, α-SMA, Col 3, Perilipin A, CD31), incubate overnight at 4°C, and wash three times with PBS (5 min/time);

(8) Add secondary antibody, incubate at 37°C in the dark for 60 min, and wash three times with PBS (5 min/time);

(9) DAPI staining for 5 min, and washing with PBS three times (5 min/time);

(10) Observation under a confocal microscope.

These results indicate that ApoEVs-AT@MN exhibit the ability to promote high-quality full-thickness skin wound healing while reducing scar formation and promoting hair follicle regeneration.

Finally, it should be noted that the above embodiments are only preferred embodiments of the present invention to illustrate the technical solution of the present invention, but not to limit it, and certainly not to limit the patent scope of the present invention. Any changes or modifications that are made to the main design concept and spirit of the present invention without any substantial meaning, and the technical problems they solve are still consistent with the present invention, should be included in the protection scope of the present invention; in addition, the direct or indirect application of the technical solution of the present invention in other related technical fields is also included in the patent protection scope of the present invention.

Claims (10)

1. A soluble antibacterial microneedle patch capable of delivering apoptotic vesicles of adipose tissue, characterized in that it comprises a microneedle matrix and an antibacterial substrate; the microneedle matrix comprises apoptotic vesicles of adipose tissue and methacryloyl hyaluronic acid hydrogel; the substrate comprises methacryloyl hyaluronic acid hydrogel and methacryloyl polylysine hydrogel. 2. A soluble antibacterial microneedle patch capable of delivering apoptotic vesicles of adipose tissue according to claim 1, characterized in that the mass ratio of the methacryloyl hyaluronic acid hydrogel and the methacryloyl polylysine hydrogel in the substrate is 3 to 10:3 to 5; preferably 5:4. 3. A soluble antibacterial microneedle patch capable of delivering apoptotic vesicles from adipose tissue according to claim 1 or 2, characterized in that the content of apoptotic vesicles from adipose tissue in each microneedle patch is 320-480 μg/piece; preferably 400 μg/piece. 4. A soluble antibacterial microneedle patch capable of delivering apoptotic vesicles of adipose tissue according to claim 3, characterized in that the diameter of the microneedle base is 250-300 μm, the needle height is 600-800 μm, and the needle tip distance is 550-700 μm; preferably, the patch size is adapted to the number of microneedles and the size of the wound. 5. A method for preparing a soluble antibacterial microneedle patch capable of delivering apoptotic vesicles of adipose tissue according to any one of claims 1 to 4, characterized in that it comprises the following steps: Step 1. Prepare bio-ink: mix adipose tissue apoptotic vesicles with methacrylated hyaluronic acid hydrogel solution to form bio-ink; Step 2. preparing a mixed antibacterial hydrogel solution of methacrylated hyaluronic acid and methacrylated polylysine water; Step 3. dripping the bio-ink into a mold, removing bubbles, heating, and concentrating; Preferably, after concentration, the bio-ink is added dropwise again, heated, and concentrated; Step 4. adding the mixed antibacterial hydrogel solution dropwise into the mold, heating and concentrating to prepare an antibacterial substrate; Step 5. UV light curing and demolding to obtain a soluble antibacterial microneedle patch capable of delivering apoptotic vesicles in adipose tissue. 6. The preparation method according to claim 5, characterized in that the content of apoptotic vesicles of adipose tissue in the bio-ink is 400-600 μg/ml; preferably 500 μg/ml; The concentration of the methacrylated hyaluronic acid is 3 to 10% w/v, preferably 5 to 10% w/v, and more preferably 5% w/v. 7. The preparation method according to claim 5, characterized in that the concentration of methacrylated hyaluronic acid hydrogel in the mixed antibacterial hydrogel solution is 3 to 10% w/v; preferably 5 to 10% w/v, more preferably 5% w/v; The concentration of or/and methacryloyl polylysine hydrogel is 3-5% (w/v), preferably 4%. 8. The preparation method according to claim 5, characterized in that in step 3, the bio-ink is dripped into the mold and bubbles are removed under vacuum conditions; Preferably, after vacuum degassing, heating and concentrating in an oven at 35-37° C. for 5-6 hours; Preferably, after the second drop of the bio-ink, the mixture is heated and concentrated in an oven at 35 to 37° C. for 5 to 6 hours; Preferably, 300 to 600 μl of the bio-ink is added dropwise each time, more preferably 400 μl. 9. The preparation method according to claim 5, characterized in that in step 4, 300-400 μl of the mixed antibacterial hydrogel solution is added dropwise; Preferably, after the antibacterial hydrogel solution is added dropwise, the mixture is heated and concentrated in an oven at 35 to 37° C. for 10 to 18 hours, more preferably for 12 hours, to prepare the antibacterial substrate. 10. The use of a soluble antibacterial microneedle patch capable of delivering apoptotic vesicles of adipose tissue according to any one of claims 1 to 4, characterized in that it is used in the preparation of an external preparation for wound treatment; preferably, the wound includes an acute or/and chronic infected suppurative wound; Preferably, the wound surface includes difficult-to-heal infected wound surfaces accompanied by various underlying diseases.