Exosome freeze-drying protective dressing and preparation method thereof

Abstract

The invention provides an exosome freeze-drying protective dressing and a preparation method thereof, and belongs to the technical field of dressings. Mixing a protective agent and the exosome, and embedding the mixture in a chitosan-sodium tripolyphosphate microcapsule to obtain an embedded exosome; the preparation method comprises the following steps: adding an extracellular matrix modified by MXene and graphene, calcium peroxide microspheres and an embedded exosome into a poly (alpha, beta-aspartic hydrazide)-C16 copolymer-silver hydrogel, and carrying out freeze drying to obtain the exosome freeze-drying protective dressing, wherein the extracellular matrix modified by MXene and graphene, the calcium peroxide microspheres and the embedded exosome are added into the poly (alpha, beta-aspartic hydrazide)-C16 copolymer-silver hydrogel; the prepared exosome freeze-dried protective dressing has good biocompatibility, can rapidly promote wound healing, reduce inflammatory response of wounds, play antibacterial and bacteriostatic effects and avoid wound deterioration, is convenient to store and transport, still has good activity after long-term storage, and has wide application prospects in the field of rapid wound healing.

Description

Technical Field

[0001] The present invention relates to the technical field of dressings, and in particular to an exosome freeze-dried protective dressing and a preparation method thereof. Background Art

[0002] In daily life, people's skin is often damaged by burns, scalds and mechanical trauma, in addition to skin damage caused by diabetic foot, pressure sores, etc. The repair process after skin damage is generally divided into four stages: coagulation stage-inflammatory stage-proliferation stage-remodeling stage. During these repair stages, if infected with bacteria, it will cause wound inflammation and suppuration, which will not only inhibit wound repair, but also cause severe problems such as tissue necrosis. Therefore, promoting wound repair and effectively preventing wound infection is a challenging problem. During the treatment process, early detection and rapid, appropriate and effective sterilization and antibacterial are particularly important for improving wound repair effects. Especially in today's increasingly serious antibiotic resistance, it is also necessary to reduce the use of antibiotics as much as possible while ensuring the antibacterial effect.

[0003] Dressings are an essential component of modern wound care, protecting the wound surface from further damage and, to a certain extent, isolating it from microbial infection. Exosomes are small vesicles approximately 30-150 nanometers in diameter, membrane-enclosed structures that are released from the interior of cells via the vesicular transport pathway. Exosomes contain a variety of bioactive molecules, including proteins, nucleic acids, and lipids. Endothelial progenitor cells are a type of stem cell derived from the bone marrow or other tissues that have the potential to differentiate into endothelial cells. These cells secrete exosomes to regulate intercellular signaling and metabolic activity. Therefore, exosomes can be used to treat wounds by promoting angiogenesis in wound tissue.

[0004] With the introduction and application of moist healing theory, the use of moist dressings such as hydrogels can not only help maintain a moist environment on the wound surface and promote wound repair, but also improve the adhesion phenomenon produced during the wound repair process and prevent secondary damage. However, if moist dressings are used improperly, they can easily breed bacteria and cause infection. To solve the above problems, a variety of wound dressings suitable for different stages have appeared on the market, which contain antimicrobial drugs or antimicrobial agents. In order to avoid the problem of drug resistance caused by excessive use of antibiotics, antimicrobial agents that are not prone to drug resistance are mainly used, including organic antimicrobial agents, inorganic antimicrobial agents, natural antimicrobial agents and other major categories. Organic antimicrobial agents include imidazoles, thiazoles, isothiazolone derivatives, quaternary ammonium salts, biguanides, etc. Their biosafety is relatively poor and they can easily cause hemolysis, pain and other problems; inorganic antimicrobial agents are mainly metal nanoparticles, among which nanosilver occupies a dominant position due to its outstanding antibacterial properties, but during use, the deposition and adhesion of nanosilver may cause local skin darkening, affecting the appearance, and the cost is relatively high; natural antimicrobial agents include chitosan, honey, etc., which have relatively weak antibacterial properties and are usually combined with other types of antimicrobial agents or drugs to work together. Summary of the Invention

[0005] The purpose of the present invention is to provide a freeze-dried exosome protective dressing and its preparation method, which has good biocompatibility, can quickly promote wound healing, reduce wound inflammatory response, have antibacterial and antibacterial effects, avoid wound deterioration, and is easy to store and transport. It still has good activity after long-term storage and has broad application prospects in the field of rapid wound healing.

[0006] The technical solution of the present invention is achieved as follows:

[0007] The present invention provides a method for preparing an exosome freeze-dried protective dressing. The method comprises the following steps: mixing a protective agent with exosomes and embedding the mixed mixture into chitosan-sodium tripolyphosphate microcapsules to obtain embedded exosomes; adding MXene and graphene-modified extracellular matrix, calcium peroxide microspheres, and embedded exosomes into a poly(α,β-aspartyl hydrazide)-C16 copolymer-silver hydrogel, and freeze-drying the mixture to obtain the exosome freeze-dried protective dressing.

[0008] As a further improvement of the present invention, the following steps are included:

[0009] S1. Preparation of a protective agent: Sucrose, trehalose, mannitol, poloxamer 188, and taurine were mixed to prepare a protective agent;

[0010] S2. Extracellular Matrix Modification: A MXene nanosheet aqueous dispersion and a graphene oxide aqueous dispersion were uniformly mixed, added to the extracellular matrix, impregnated, freeze-dried, reduced with hydrazine hydrate vapor, and pulverized to produce a modified extracellular matrix.

[0011] S3. Embedding exosomes: adding exosomes and protective agents into water, adding sodium tripolyphosphate, stirring and mixing uniformly to prepare solution A; dissolving chitosan in acid solution to obtain solution B; loading solution A and solution B into syringes respectively, inserting into FNP device (multi-inlet vortex mixer), pressing the piston handle of the two syringes parallel downward to extrude the solution, freeze-drying the product to obtain embedded exosomes;

[0012] S4. Preparation of calcium peroxide microspheres: dissolving calcium salt in water, adding ammonia water dropwise, adding polyethylene glycol, stirring and mixing uniformly, then adding hydrogen peroxide, stirring and reacting, adjusting the pH value of the solution, standing and precipitating to obtain calcium peroxide particles; dissolving sodium alginate and emulsifier in water, adding calcium peroxide particles, stirring and mixing uniformly, adding fish oil, emulsifying, curing at room temperature, centrifuging, washing, and drying to obtain calcium peroxide microspheres.

[0013] S5. Preparation of exosome freeze-drying protective dressing: adding poly(α,β-aspartyl hydrazide)-C16 copolymer into phosphate buffer solution, adding modified extracellular matrix, embedding exosomes, calcium peroxide microspheres and sodium citrate, stirring and mixing uniformly, adding silver nitrate, ultrasonic dispersion uniformly, freeze-drying to obtain exosome freeze-drying protective dressing.

[0014] As a further improvement of the present application, the mass ratio of sucrose, trehalose, mannitol, poloxamer 188 and taurine in step S1 is 2-3:3-4:5-7:1-2:1-2.

[0015] As a further improvement of the present application, the concentration of the MXene nanosheet aqueous dispersion in step S2 is 1-3 mg / mL, the concentration of the graphene oxide aqueous dispersion is 0.5-1.5 mg / mL, the mass ratio of the MXene nanosheet aqueous dispersion, graphene oxide aqueous dispersion and extracellular matrix is 100:100:12-17, the soaking time is 0.5-1.5 h, and the hydrazine hydrate vapor reduction time is 8-10 h.

[0016] As a further improvement of the present application, the preparation method of the MXene nanosheet aqueous dispersion is as follows: adding LiF into HCl solution, stirring and dissolving, adding Ti3AlC2, heating and etching, centrifuging, washing, adding water, ultrasonic dispersion uniformly, centrifuging, collecting supernatant, diluting with water to a specific concentration to obtain MXene nanosheet aqueous dispersion.

[0017] As a further improvement of the present application, the mass ratio of LiF and Ti3AlC2 is 1-3:1-3, the concentration of the HCl solution is 8-10 mol / L, and the heating and etching temperature is 35-45℃ for 45-53 h.

[0018] As a further improvement of the present invention, the mass ratio of the exosomes, protective agent, sodium tripolyphosphate and chitosan in step S3 is 2-3:0.1-0.2:2-3:6-9.

[0019] As a further improvement of the present invention, in step S4, the mass ratio of the calcium salt, ammonia water, polyethylene glycol, and hydrogen peroxide is 5-7:20-30:200-300:25-35, the calcium salt is calcium chloride or calcium nitrate, the concentration of the ammonia water is 1-2 mol / L, the concentration of the hydrogen peroxide is 25-35 wt%, the pH value of the adjustment solution is 10.5-11.5, the mass ratio of the sodium alginate, emulsifier, and calcium peroxide particles is 10-12:0.3-0.7:4-7, and the emulsifier is at least one of Tween-20, Tween-40, Tween-60, Tween-80, and Tween-85.

[0020] As a further improvement of the present invention, the pH value of the phosphate buffer in step S5 is 7.2-7.6, and the mass ratio of the poly(α,β-aspartyl hydrazide)-C16 copolymer, modified extracellular matrix, embedded exosomes, calcium peroxide microspheres, sodium citrate, and silver nitrate is 15-20:4-8:3-5:4-6:1-2:0.3-0.5.

[0021] The present invention further protects a freeze-dried exosome protective dressing prepared by the above-mentioned preparation method.

[0022] The present invention has the following beneficial effects:

[0023] The protective agent of this invention utilizes a combination of trehalose and mannitol, along with poloxamer 188, to enhance exosome activity through a triple mechanism: inhibiting ice crystal growth, maintaining membrane stability, and enhancing dispersibility. Trehalose forms a vitrified structure to protect membrane integrity, mannitol acts as a filler to prevent post-freeze collapse, and poloxamer 188 reduces surface tension to prevent aggregation. The synergistic effect of these three agents allows exosomes to maintain high activity even after freezing. Taurine also enhances the exosomes' antioxidant capacity, while sucrose partially replaces trehalose, maintaining stability while reducing raw material costs.

[0024] The structure of the extracellular matrix (ECM) is highly similar to natural collagen fibers, with high porosity, allowing it to load exosomes for sustained release and exhibiting excellent compatibility with skin tissue. Modification of the ECM by impregnation and doping with MXene and graphene significantly improves its electrical conductivity. The ECM's scaffolding structure significantly mitigates the self-accumulation of MXene. This enhanced conductivity can regulate cell signaling, while photothermal conversion enables on-demand release of exosomes, shortening wound healing time and inhibiting expression of the inflammatory factor IL-6.

[0025] After tissue injury, damaged blood vessels at the wound site hinder oxygen delivery, creating a hypoxic environment around the wound. This hypoxia exacerbates the recruitment of oxygen-consuming inflammatory cells. Chronic hypoxia in chronic wounds inhibits angiogenesis, slows the re-epithelialization healing process, and slows extracellular matrix synthesis. Therefore, increasing oxygen concentration in wound tissue is crucial for chronic wound healing. Calcium peroxide (CPO), under the action of water and catalase (Cat), generates oxygen, increasing dissolved oxygen in the tissue and improving the microenvironment at the site of injury. After being embedded in sodium alginate, the alginate microspheres' shell is destroyed in the wound environment, allowing the calcium peroxide to be slowly released, thereby increasing oxygen concentration near the wound.

[0026] The various proteins, RNA, and lipids carried by milk exosomes promote the repair of damaged tissues and have a significant proliferative effect on cells in damaged tissues. The synergistic effect of exosomes and oxygen is used to heal diabetic wounds and treat bone defects, respectively. Exosomes are encapsulated in chitosan-sodium tripolyphosphate nanocapsules, which dissolve and release in acidic wound environments. This pH-responsive formulation prevents premature release of exosomes under neutral storage conditions, while enabling precise delivery to the site of inflammation.

[0027] The present invention loads the amphiphilic poly (α, β-aspartyl hydrazide)-C16 copolymer hydrogel with modified extracellular matrix, embeds exosomes and calcium peroxide microspheres, and synthesizes nanosilver in situ, forming a cross-linked structure on the one hand and a "physical barrier + antibacterial release" dual protection on the other hand. It can play an antibacterial and antimicrobial effect while healing the wound, preventing wound deterioration, and accelerating re-epithelialization by promoting keratinocyte migration.

[0028] The exosome freeze-dried protective dressing prepared by the present invention has good biocompatibility, can quickly promote wound healing, reduce the inflammatory response of the wound, have antibacterial and antibacterial effects, avoid wound deterioration, and is easy to store and transport. It still has good activity after long-term storage, and has broad application prospects in the field of rapid wound healing. DETAILED DESCRIPTION

[0029] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0030] Extracellular matrix was purchased from ThermoFisher; poly(α,β-aspartic hydrazide)-C16 copolymer was purchased from Xi'an Qiyue Biotechnology Co., Ltd.; and milk exosomes were purchased from Beyotime Biotechnology Co., Ltd.

[0031] Preparation Example 1 MXene nanosheet aqueous dispersion

[0032] The preparation method is as follows: add 2g LiF to 40mL 9mol / L HCl solution, stir to dissolve, add 2g Ti3AlC2, etch at 40℃ for 48h, centrifuge at 3500r / min for 10min, wash to pH 6, add water, ultrasonically disperse at 1000W for 2h, centrifuge at 3500r / min for 30min, collect the supernatant, and dilute with water to a specific concentration to prepare a MXene nanosheet aqueous dispersion.

[0033] Example 1

[0034] This embodiment provides a freeze-dried exosome protective dressing, comprising the following steps:

[0035] S1. Preparation of protective agent: 2 g sucrose, 3 g trehalose, 5 g mannitol, 1 g poloxamer 188, and 1 g taurine were mixed to prepare a protective agent;

[0036] S2. Extracellular matrix modification: 100 g of a 1 mg / mL aqueous dispersion of MXene nanosheets prepared in Preparation Example 1 and 100 g of a 0.5 mg / mL aqueous dispersion of graphene oxide were mixed uniformly, 12 g of extracellular matrix was added, the mixture was immersed for 0.5 h, freeze-dried, reduced with hydrazine hydrate vapor for 8 h, and pulverized to obtain a modified extracellular matrix.

[0037] S3. Exosome Encapsulation: 2g of milk exosomes and 0.1g of a protective agent were added to 200mL of water, followed by 2g of sodium tripolyphosphate, and stirred to obtain solution A. 6g of chitosan was dissolved in 200mL of a 2wt% acetic acid solution to obtain solution B. Equal volumes of solution A and solution B were loaded into syringes, inserted into the FNP device, and the pistons of the two syringes were pressed downward in parallel to uniformly extrude the solutions. Extrusion was completed within 2 minutes, and the solution was allowed to stand for 1 hour. The product was then freeze-dried to obtain the encapsulated exosomes.

[0038] S4. Preparation of calcium peroxide microspheres: 5g of calcium chloride was dissolved in 70mL of water, 20g of 1mol / L ammonia was added dropwise, 200g of PEG200 was added, and after stirring and mixing, 25g of 25wt% hydrogen peroxide was added dropwise, and the reaction was stirred for 4h. The pH of the solution was adjusted to 10.5, and the solution was allowed to settle for 1h. The mixture was centrifuged, washed, and dried to obtain calcium peroxide particles; 10g of sodium alginate and 0.3g of Tween-40 were dissolved in 300mL of water, 4g of calcium peroxide particles were added, stirred and mixed, and added to 500mL of fish oil. The mixture was emulsified at 5000r / min for 15min, cured at room temperature for 30min, centrifuged, washed, and dried to obtain calcium peroxide microspheres.

[0039] S5. Preparation of freeze-dried exosome protective dressing: 15 g of poly(α,β-aspartyl hydrazide)-C16 copolymer was added to 300 mL of pH 7.2 phosphate buffer, followed by 4 g of modified extracellular matrix, 3 g of embedded exosomes, 4 g of calcium peroxide microspheres, and 1 g of sodium citrate. The mixture was stirred until uniform, and 0.3 g of silver nitrate was added. Ultrasonic dispersion was performed at 1000 W for 15 min, and the mixture was freeze-dried to prepare the freeze-dried exosome protective dressing.

[0040] Example 2

[0041] This embodiment provides a freeze-dried exosome protective dressing, comprising the following steps:

[0042] S1. Preparation of protective agent: 3 g sucrose, 4 g trehalose, 7 g mannitol, 2 g poloxamer 188, and 2 g taurine were mixed to prepare a protective agent;

[0043] S2. Extracellular matrix modification: 100 g of a 3 mg / mL aqueous dispersion of MXene nanosheets prepared in Preparation Example 1 and 100 g of a 1.5 mg / mL aqueous dispersion of graphene oxide were mixed uniformly, 17 g of extracellular matrix was added, and the mixture was immersed for 1.5 h, freeze-dried, reduced with hydrazine hydrate vapor for 10 h, and pulverized to obtain a modified extracellular matrix.

[0044] S3. Exosome Encapsulation: 3g of milk exosomes and 0.2g of a protective agent were added to 200mL of water, followed by 3g of sodium tripolyphosphate, and stirred to obtain solution A. 9g of chitosan was dissolved in 200mL of a 2wt% acetic acid solution to obtain solution B. Equal volumes of solution A and solution B were loaded into syringes, inserted into the FNP device, and the pistons of the two syringes were pressed downward in parallel to uniformly extrude the solutions. Extrusion was completed within 2 minutes, and the solution was allowed to stand for 1 hour. The product was then freeze-dried to obtain the encapsulated exosomes.

[0045] S4. Preparation of calcium peroxide microspheres: 7g of calcium nitrate was dissolved in 70mL of water, 30g of 2mol / L ammonia was added dropwise, 300g of PEG200 was added, and after stirring and mixing, 35g of 35wt% hydrogen peroxide was added dropwise, and the reaction was stirred for 4h. The pH of the solution was adjusted to 11.5, and the solution was allowed to settle for 1h. The mixture was centrifuged, washed, and dried to obtain calcium peroxide particles; 12g of sodium alginate and 0.7g of Tween-60 were dissolved in 300mL of water, 7g of calcium peroxide particles were added, the mixture was stirred and mixed, and 500mL of fish oil was added. The mixture was emulsified at 5000r / min for 15min, cured at room temperature for 30min, centrifuged, washed, and dried to obtain calcium peroxide microspheres.

[0046] S5. Preparation of freeze-dried exosome protective dressing: 20 g of poly(α,β-aspartyl hydrazide)-C16 copolymer was added to 300 mL of pH 7.6 phosphate buffer, followed by 8 g of modified extracellular matrix, 5 g of embedded exosomes, 6 g of calcium peroxide microspheres, and 2 g of sodium citrate. The mixture was stirred until uniform, and 0.5 g of silver nitrate was added. Ultrasonic dispersion was performed at 1000 W for 15 min, and the mixture was freeze-dried to prepare the freeze-dried exosome protective dressing.

[0047] Example 3

[0048] This embodiment provides a freeze-dried exosome protective dressing, comprising the following steps:

[0049] S1. Preparation of protective agent: 2.5 g sucrose, 3.5 g trehalose, 6 g mannitol, 1.5 g poloxamer 188, 1.5 g taurine were mixed to prepare a protective agent;

[0050] S2. Extracellular matrix modification: 100 g of a 2 mg / mL aqueous dispersion of MXene nanosheets prepared in Preparation Example 1 and 100 g of a 1 mg / mL aqueous dispersion of graphene oxide were mixed uniformly, 15 g of extracellular matrix was added, the mixture was immersed for 1 h, freeze-dried, reduced with hydrazine hydrate vapor for 9 h, and pulverized to obtain a modified extracellular matrix.

[0051] S3. Exosome Encapsulation: 2.5 g of milk exosomes and 0.15 g of a protective agent were added to 200 mL of water, followed by 2.5 g of sodium tripolyphosphate, and stirred to obtain solution A. 7.5 g of chitosan was dissolved in 200 mL of a 2 wt% acetic acid solution to obtain solution B. Equal volumes of solution A and solution B were loaded into syringes, inserted into the FNP device, and the pistons of the two syringes were pressed downward in parallel to uniformly extrude the solutions. Extrusion was completed within 2 minutes, and the solution was allowed to stand for 1 hour. The product was then freeze-dried to obtain the encapsulated exosomes.

[0052] S4. Preparation of calcium peroxide microspheres: 6g of calcium nitrate was dissolved in 70mL of water, 25g of 1.5mol / L ammonia was added dropwise, 250g of PEG200 was added, and after stirring and mixing, 30g of 30wt% hydrogen peroxide was added dropwise, and the reaction was stirred for 4h. The pH of the solution was adjusted to 11, and the solution was allowed to settle for 1h, centrifuged, washed, and dried to obtain calcium peroxide particles; 11g of sodium alginate and 0.5g of Tween-85 were dissolved in 300mL of water, 5.5g of calcium peroxide particles were added, stirred and mixed, and added to 500mL of fish oil. Emulsification was performed at 5000r / min for 15min, and the mixture was cured at room temperature for 30min, centrifuged, washed, and dried to obtain calcium peroxide microspheres.

[0053] S5. Preparation of freeze-dried exosome protective dressing: 17 g of poly(α,β-aspartyl hydrazide)-C16 copolymer was added to 300 mL of pH 7.4 phosphate buffer, followed by 6 g of modified extracellular matrix, 4 g of embedded exosomes, 5 g of calcium peroxide microspheres, and 1.5 g of sodium citrate. The mixture was stirred until uniform, and 0.4 g of silver nitrate was added. Ultrasonic dispersion was performed at 1000 W for 15 min, and the mixture was freeze-dried to prepare the freeze-dried exosome protective dressing.

[0054] Comparative Example 1

[0055] Compared with Example 3, the difference is that no MXene nanosheet aqueous dispersion is added in step S2.

[0056] The details are as follows:

[0057] S1. Preparation of protective agent: 2.5 g sucrose, 3.5 g trehalose, 6 g mannitol, 1.5 g poloxamer 188, 1.5 g taurine were mixed to prepare a protective agent;

[0058] S2. Extracellular matrix modification: 15 g of extracellular matrix was added to 200 g of a 1 mg / mL aqueous dispersion of graphene oxide, immersed for 1 h, freeze-dried, reduced with hydrazine hydrate vapor for 9 h, and pulverized to obtain a modified extracellular matrix;

[0059] S3. Exosome Encapsulation: 2.5 g of milk exosomes and 0.15 g of a protective agent were added to 200 mL of water, followed by 2.5 g of sodium tripolyphosphate, and stirred to obtain solution A. 7.5 g of chitosan was dissolved in 200 mL of a 2 wt% acetic acid solution to obtain solution B. Equal volumes of solution A and solution B were loaded into syringes, inserted into the FNP device, and the pistons of the two syringes were pressed downward in parallel to uniformly extrude the solutions. Extrusion was completed within 2 minutes, and the solution was allowed to stand for 1 hour. The product was then freeze-dried to obtain the encapsulated exosomes.

[0060] S4. Preparation of calcium peroxide microspheres: 6g of calcium nitrate was dissolved in 70mL of water, 25g of 1.5mol / L ammonia was added dropwise, 250g of PEG200 was added, and after stirring and mixing, 30g of 30wt% hydrogen peroxide was added dropwise, and the reaction was stirred for 4h. The pH of the solution was adjusted to 11, and the solution was allowed to settle for 1h, centrifuged, washed, and dried to obtain calcium peroxide particles; 11g of sodium alginate and 0.5g of Tween-85 were dissolved in 300mL of water, 5.5g of calcium peroxide particles were added, stirred and mixed, and added to 500mL of fish oil. Emulsification was performed at 5000r / min for 15min, and the mixture was cured at room temperature for 30min, centrifuged, washed, and dried to obtain calcium peroxide microspheres.

[0061] S5. Preparation of freeze-dried exosome protective dressing: 17 g of poly(α,β-aspartyl hydrazide)-C16 copolymer was added to 300 mL of pH 7.4 phosphate buffer, followed by 6 g of modified extracellular matrix, 4 g of embedded exosomes, 5 g of calcium peroxide microspheres, and 1.5 g of sodium citrate. The mixture was stirred until uniform, and 0.4 g of silver nitrate was added. Ultrasonic dispersion was performed at 1000 W for 15 min, and the mixture was freeze-dried to prepare the freeze-dried exosome protective dressing.

[0062] Comparative Example 2

[0063] Compared with Example 3, the difference is that no graphene oxide aqueous dispersion is added in step S2.

[0064] The details are as follows:

[0065] S1. Preparation of protective agent: 2.5 g sucrose, 3.5 g trehalose, 6 g mannitol, 1.5 g poloxamer 188, 1.5 g taurine were mixed to prepare a protective agent;

[0066] S2. Extracellular matrix modification: 15 g of extracellular matrix was added to 200 g of the 2 mg / mL aqueous dispersion of MXene nanosheets prepared in Preparation Example 1, immersed for 1 h, freeze-dried, reduced with hydrazine hydrate vapor for 9 h, and pulverized to obtain a modified extracellular matrix;

[0067] S3. Exosome Encapsulation: 2.5 g of milk exosomes and 0.15 g of a protective agent were added to 200 mL of water, followed by 2.5 g of sodium tripolyphosphate, and stirred to obtain solution A. 7.5 g of chitosan was dissolved in 200 mL of a 2 wt% acetic acid solution to obtain solution B. Equal volumes of solution A and solution B were loaded into syringes, inserted into the FNP device, and the pistons of the two syringes were pressed downward in parallel to uniformly extrude the solutions. Extrusion was completed within 2 minutes, and the solution was allowed to stand for 1 hour. The product was then freeze-dried to obtain the encapsulated exosomes.

[0068] S4. Preparation of calcium peroxide microspheres: 6g of calcium nitrate was dissolved in 70mL of water, 25g of 1.5mol / L ammonia was added dropwise, 250g of PEG200 was added, and after stirring and mixing, 30g of 30wt% hydrogen peroxide was added dropwise, and the reaction was stirred for 4h. The pH of the solution was adjusted to 11, and the solution was allowed to settle for 1h, centrifuged, washed, and dried to obtain calcium peroxide particles; 11g of sodium alginate and 0.5g of Tween-85 were dissolved in 300mL of water, 5.5g of calcium peroxide particles were added, stirred and mixed, and added to 500mL of fish oil. Emulsification was performed at 5000r / min for 15min, and the mixture was cured at room temperature for 30min, centrifuged, washed, and dried to obtain calcium peroxide microspheres.

[0069] S5. Preparation of freeze-dried exosome protective dressing: 17 g of poly(α,β-aspartyl hydrazide)-C16 copolymer was added to 300 mL of pH 7.4 phosphate buffer, followed by 6 g of modified extracellular matrix, 4 g of embedded exosomes, 5 g of calcium peroxide microspheres, and 1.5 g of sodium citrate. The mixture was stirred until uniform, and 0.4 g of silver nitrate was added. Ultrasonic dispersion was performed at 1000 W for 15 min, and the mixture was freeze-dried to prepare the freeze-dried exosome protective dressing.

[0070] Comparative Example 3

[0071] Compared with Example 3, the difference is that step S2 in Example 3 is not performed.

[0072] The details are as follows:

[0073] S1. Preparation of protective agent: 2.5 g sucrose, 3.5 g trehalose, 6 g mannitol, 1.5 g poloxamer 188, 1.5 g taurine were mixed to prepare a protective agent;

[0074] S2. Exosome Encapsulation: 2.5 g of milk exosomes and 0.15 g of a protective agent were added to 200 mL of water, followed by 2.5 g of sodium tripolyphosphate, and stirred to obtain solution A. 7.5 g of chitosan was dissolved in 200 mL of a 2 wt% acetic acid solution to obtain solution B. Equal volumes of solution A and solution B were loaded into syringes, inserted into the FNP device, and the pistons of the two syringes were pressed downward in parallel to uniformly extrude the solutions. Extrusion was completed within 2 minutes. The solution was allowed to stand for 1 hour, and the product was freeze-dried to obtain the encapsulated exosomes.

[0075] S3. Preparation of calcium peroxide microspheres: 6g of calcium nitrate was dissolved in 70mL of water, 25g of 1.5mol / L ammonia was added dropwise, 250g of PEG200 was added, and after stirring and mixing, 30g of 30wt% hydrogen peroxide was added dropwise, and the reaction was stirred for 4h. The pH of the solution was adjusted to 11, and the solution was allowed to settle for 1h, centrifuged, washed, and dried to obtain calcium peroxide particles; 11g of sodium alginate and 0.5g of Tween-85 were dissolved in 300mL of water, 5.5g of calcium peroxide particles were added, stirred and mixed, and added to 500mL of fish oil, emulsified at 5000r / min for 15min, cured at room temperature for 30min, centrifuged, washed, and dried to obtain calcium peroxide microspheres;

[0076] S4. Preparation of freeze-dried exosome protective dressing: 17 g of poly(α,β-aspartyl hydrazide)-C16 copolymer was added to 300 mL of pH 7.4 phosphate buffer, followed by 6 g of extracellular matrix, 4 g of embedded exosomes, 5 g of calcium peroxide microspheres, and 1.5 g of sodium citrate. The mixture was stirred until uniform, and 0.4 g of silver nitrate was added. Ultrasonic dispersion was performed at 1000 W for 15 min, and the mixture was freeze-dried to prepare the freeze-dried exosome protective dressing.

[0077] Comparative Example 4

[0078] The difference compared with Example 3 is that no protective agent is added in step S2.

[0079] The specific process is as follows:

[0080] S1. Extracellular matrix modification: 100 g of 2 mg / mL MXene nanosheet water dispersion prepared in Preparation Example 1 and 100 g of 1 mg / mL graphene oxide water dispersion are mixed uniformly, 15 g of extracellular matrix is added, soaked for 1 h, freeze-dried, reduced by hydrazine hydrate steam for 9 h, and crushed to obtain a modified extracellular matrix;

[0081] S2. Exosome embedding: 2.65 g of milk exosome is added to 200 mL of water, 2.5 g of sodium tripolyphosphate is added, and stirred and mixed uniformly to obtain solution A; 7.5 g of chitosan is dissolved in 200 mL of 2 wt% acetic acid solution to obtain solution B; equal volumes of solutions A and B are respectively loaded into syringes, and the plungers of the two syringes are inserted into the FNP device and pressed downward in parallel to uniformly extrude the solutions, which is completed within 2 min, and the product is freeze-dried after standing for 1 h to obtain embedded exosomes;

[0082] S3. Preparation of calcium peroxide microspheres: 6 g of calcium nitrate is dissolved in 70 mL of water, 25 g of 1.5 mol / L ammonia water is added dropwise, 250 g of PEG200 is added, and stirred and mixed uniformly, then 30 g of 30 wt% hydrogen peroxide is added dropwise, and stirred and reacted for 4 h, the pH value of the solution is adjusted to 11, and the solution is left to stand for 1 h, centrifuged, washed, and dried to obtain calcium peroxide particles; 11 g of sodium alginate and 0.5 g of Tween-85 are dissolved in 300 mL of water, 5.5 g of calcium peroxide particles are added, stirred and mixed uniformly, and then added into 500 mL of fish oil, emulsified at 5000 r / min for 15 min, and cured at room temperature for 30 min, then centrifuged, washed, and dried to obtain calcium peroxide microspheres;

[0083] S4. Preparation of exosome freeze-drying protection dressing: 17 g of poly(α,β-aspartyl hydrazide)-C16 copolymer is added to 300 mL of phosphate buffer solution with a pH value of 7.4, 6 g of modified extracellular matrix, 4 g of embedded exosomes, 5 g of calcium peroxide microspheres, and 1.5 g of sodium citrate are added, stirred and mixed uniformly, 0.4 g of silver nitrate is added, and ultrasonically dispersed at 1000 W for 15 min, and freeze-dried to obtain an exosome freeze-drying protection dressing.

[0084] Comparative Example 5

[0085] The difference compared with Example 3 is that no embedding is performed in step S3.

[0086] The specific process is as follows:

[0087] S1. Preparation of protective agent: 2.5 g sucrose, 3.5 g trehalose, 6 g mannitol, 1.5 g poloxamer 188, 1.5 g taurine were mixed to prepare a protective agent;

[0088] S2. Extracellular matrix modification: 100 g of a 2 mg / mL aqueous dispersion of MXene nanosheets prepared in Preparation Example 1 and 100 g of a 1 mg / mL aqueous dispersion of graphene oxide were mixed uniformly, 15 g of extracellular matrix was added, the mixture was immersed for 1 h, freeze-dried, reduced with hydrazine hydrate vapor for 9 h, and pulverized to obtain a modified extracellular matrix.

[0089] S3. Preparation of a mixture: 2.5 g of milk exosomes and 0.15 g of a protective agent were mixed to obtain a mixture;

[0090] S4. Preparation of calcium peroxide microspheres: 6g of calcium nitrate was dissolved in 70mL of water, 25g of 1.5mol / L ammonia was added dropwise, 250g of PEG200 was added, and after stirring and mixing, 30g of 30wt% hydrogen peroxide was added dropwise, and the reaction was stirred for 4h. The pH of the solution was adjusted to 11, and the solution was allowed to settle for 1h, centrifuged, washed, and dried to obtain calcium peroxide particles; 11g of sodium alginate and 0.5g of Tween-85 were dissolved in 300mL of water, 5.5g of calcium peroxide particles were added, stirred and mixed, and added to 500mL of fish oil. Emulsification was performed at 5000r / min for 15min, and the mixture was cured at room temperature for 30min, centrifuged, washed, and dried to obtain calcium peroxide microspheres.

[0091] S5. Preparation of freeze-dried exosome protective dressing: 17 g of poly(α,β-aspartyl hydrazide)-C16 copolymer was added to 300 mL of pH 7.4 phosphate buffer, followed by 6 g of modified extracellular matrix, 4 g of the mixture, 5 g of calcium peroxide microspheres, and 1.5 g of sodium citrate. The mixture was stirred until uniformly mixed, and 0.4 g of silver nitrate was added. Ultrasonic dispersion was performed at 1000 W for 15 min, and the mixture was freeze-dried to prepare the freeze-dried exosome protective dressing.

[0092] Comparative Example 6

[0093] Compared with Example 3, the difference is that no embedding is performed in step S4.

[0094] The details are as follows:

[0095] S1. Preparation of protective agent: 2.5 g sucrose, 3.5 g trehalose, 6 g mannitol, 1.5 g poloxamer 188, 1.5 g taurine were mixed to prepare a protective agent;

[0096] S2. Extracellular matrix modification: 100 g of a 2 mg / mL aqueous dispersion of MXene nanosheets prepared in Preparation Example 1 and 100 g of a 1 mg / mL aqueous dispersion of graphene oxide were mixed uniformly, 15 g of extracellular matrix was added, the mixture was immersed for 1 h, freeze-dried, reduced with hydrazine hydrate vapor for 9 h, and pulverized to obtain a modified extracellular matrix.

[0097] S3. Exosome Encapsulation: 2.5 g of milk exosomes and 0.15 g of a protective agent were added to 200 mL of water, followed by 2.5 g of sodium tripolyphosphate, and stirred to obtain solution A. 7.5 g of chitosan was dissolved in 200 mL of a 2 wt% acetic acid solution to obtain solution B. Equal volumes of solution A and solution B were loaded into syringes, inserted into the FNP device, and the pistons of the two syringes were pressed downward in parallel to uniformly extrude the solutions. Extrusion was completed within 2 minutes, and the solution was allowed to stand for 1 hour. The product was then freeze-dried to obtain the encapsulated exosomes.

[0098] S4. Preparation of calcium peroxide particles: 6 g of calcium nitrate was dissolved in 70 mL of water, 25 g of 1.5 mol / L ammonia solution was added dropwise, 250 g of PEG200 was added, and after stirring and mixing, 30 g of 30 wt% hydrogen peroxide was added dropwise, and the reaction was stirred for 4 h. The pH of the solution was adjusted to 11, and the solution was allowed to settle for 1 h, centrifuged, washed, and dried to obtain calcium peroxide particles;

[0099] S5. Preparation of freeze-dried exosome protective dressing: 17 g of poly(α,β-aspartyl hydrazide)-C16 copolymer was added to 300 mL of pH 7.4 phosphate buffer, followed by 6 g of modified extracellular matrix, 4 g of embedded exosomes, 5 g of calcium peroxide particles, and 1.5 g of sodium citrate. The mixture was stirred and evenly mixed. 0.4 g of silver nitrate was added, and ultrasonic dispersion was performed at 1000 W for 15 min. The mixture was freeze-dried to prepare the freeze-dried exosome protective dressing.

[0100] Comparative Example 7

[0101] Compared with Example 3, the difference is that silver nitrate is not added in step S5.

[0102] The details are as follows:

[0103] S1. Preparation of protective agent: 2.5 g sucrose, 3.5 g trehalose, 6 g mannitol, 1.5 g poloxamer 188, 1.5 g taurine were mixed to prepare a protective agent;

[0104] S2. Extracellular matrix modification: 100 g of a 2 mg / mL aqueous dispersion of MXene nanosheets prepared in Preparation Example 1 and 100 g of a 1 mg / mL aqueous dispersion of graphene oxide were mixed uniformly, 15 g of extracellular matrix was added, the mixture was immersed for 1 h, freeze-dried, reduced with hydrazine hydrate vapor for 9 h, and pulverized to obtain a modified extracellular matrix.

[0105] S3. Exosome Encapsulation: 2.5 g of milk exosomes and 0.15 g of a protective agent were added to 200 mL of water, followed by 2.5 g of sodium tripolyphosphate, and stirred to obtain solution A. 7.5 g of chitosan was dissolved in 200 mL of a 2 wt% acetic acid solution to obtain solution B. Equal volumes of solution A and solution B were loaded into syringes, inserted into the FNP device, and the pistons of the two syringes were pressed downward in parallel to uniformly extrude the solutions. Extrusion was completed within 2 minutes, and the solution was allowed to stand for 1 hour. The product was then freeze-dried to obtain the encapsulated exosomes.

[0106] S4. Preparation of calcium peroxide microspheres: 6g of calcium nitrate was dissolved in 70mL of water, 25g of 1.5mol / L ammonia was added dropwise, 250g of PEG200 was added, and after stirring and mixing, 30g of 30wt% hydrogen peroxide was added dropwise, and the reaction was stirred for 4h. The pH of the solution was adjusted to 11, and the solution was allowed to settle for 1h, centrifuged, washed, and dried to obtain calcium peroxide particles; 11g of sodium alginate and 0.5g of Tween-85 were dissolved in 300mL of water, 5.5g of calcium peroxide particles were added, stirred and mixed, and added to 500mL of fish oil. Emulsification was performed at 5000r / min for 15min, and the mixture was cured at room temperature for 30min, centrifuged, washed, and dried to obtain calcium peroxide microspheres.

[0107] S5. Preparation of freeze-dried exosome protective dressing: 17 g of poly(α,β-aspartyl hydrazide)-C16 copolymer was added to 300 mL of pH 7.4 phosphate buffer, followed by 6 g of modified extracellular matrix, 4 g of embedded exosomes, and 5 g of calcium peroxide microspheres. The mixture was stirred and thoroughly dispersed under ultrasonication at 1000 W for 15 min. The dressing was freeze-dried to prepare the freeze-dried exosome protective dressing.

[0108] Comparative Example 8

[0109] Compared with Example 3, the difference is that calcium peroxide microspheres are not added in step S5, and step S4 in Example 3 is not performed.

[0110] The details are as follows:

[0111] S1. Preparation of protective agent: 2.5 g sucrose, 3.5 g trehalose, 6 g mannitol, 1.5 g poloxamer 188, 1.5 g taurine were mixed to prepare a protective agent;

[0112] S2. Extracellular matrix modification: 100 g of a 2 mg / mL aqueous dispersion of MXene nanosheets prepared in Preparation Example 1 and 100 g of a 1 mg / mL aqueous dispersion of graphene oxide were mixed uniformly, 15 g of extracellular matrix was added, the mixture was immersed for 1 h, freeze-dried, reduced with hydrazine hydrate vapor for 9 h, and pulverized to obtain a modified extracellular matrix.

[0113] S3. Exosome Encapsulation: 2.5 g of milk exosomes and 0.15 g of a protective agent were added to 200 mL of water, followed by 2.5 g of sodium tripolyphosphate, and stirred to obtain solution A. 7.5 g of chitosan was dissolved in 200 mL of a 2 wt% acetic acid solution to obtain solution B. Equal volumes of solution A and solution B were loaded into syringes, inserted into the FNP device, and the pistons of the two syringes were pressed downward in parallel to uniformly extrude the solutions. Extrusion was completed within 2 minutes, and the solution was allowed to stand for 1 hour. The product was then freeze-dried to obtain the encapsulated exosomes.

[0114] S4. Preparation of freeze-dried exosome protective dressing: 17 g of poly(α,β-aspartyl hydrazide)-C16 copolymer was added to 300 mL of pH 7.4 phosphate buffer, followed by 6 g of modified extracellular matrix, 4 g of embedded exosomes, and 1.5 g of sodium citrate. The mixture was stirred until uniform, and 0.4 g of silver nitrate was added. Ultrasonic dispersion was performed at 1000 W for 15 min, and the mixture was freeze-dried to prepare the freeze-dried exosome protective dressing.

[0115] Comparative Example 9

[0116] Compared with Example 3, the difference is that no modified extracellular matrix is ​​added in step S5.

[0117] The details are as follows:

[0118] S1. Preparation of protective agent: 2.5 g sucrose, 3.5 g trehalose, 6 g mannitol, 1.5 g poloxamer 188, 1.5 g taurine were mixed to prepare a protective agent;

[0119] S2. Exosome Encapsulation: 2.5 g of milk exosomes and 0.15 g of a protective agent were added to 200 mL of water, followed by 2.5 g of sodium tripolyphosphate, and stirred to obtain solution A. 7.5 g of chitosan was dissolved in 200 mL of a 2 wt% acetic acid solution to obtain solution B. Equal volumes of solution A and solution B were loaded into syringes, inserted into the FNP device, and the pistons of the two syringes were pressed downward in parallel to uniformly extrude the solutions. Extrusion was completed within 2 minutes. The solution was allowed to stand for 1 hour, and the product was freeze-dried to obtain the encapsulated exosomes.

[0120] S3. Preparation of calcium peroxide microspheres: 6g of calcium nitrate was dissolved in 70mL of water, 25g of 1.5mol / L ammonia was added dropwise, 250g of PEG200 was added, and after stirring and mixing, 30g of 30wt% hydrogen peroxide was added dropwise, and the reaction was stirred for 4h. The pH of the solution was adjusted to 11, and the solution was allowed to settle for 1h, centrifuged, washed, and dried to obtain calcium peroxide particles; 11g of sodium alginate and 0.5g of Tween-85 were dissolved in 300mL of water, 5.5g of calcium peroxide particles were added, stirred and mixed, and added to 500mL of fish oil, emulsified at 5000r / min for 15min, cured at room temperature for 30min, centrifuged, washed, and dried to obtain calcium peroxide microspheres;

[0121] S4. Preparation of freeze-dried exosome protective dressing: 17 g of poly(α,β-aspartyl hydrazide)-C16 copolymer was added to 300 mL of pH 7.4 phosphate buffer, followed by 4 g of embedded exosomes, 5 g of calcium peroxide microspheres, and 1.5 g of sodium citrate. The mixture was stirred and evenly mixed. 0.4 g of silver nitrate was added, and ultrasonic dispersion was performed at 1000 W for 15 min. The mixture was freeze-dried to prepare the freeze-dried exosome protective dressing.

[0122] Comparative Example 10

[0123] Compared with Example 3, the difference is that no embedded exosomes are added in step S5, and step S3 in Example 3 is not performed.

[0124] The details are as follows:

[0125] S1. Extracellular matrix modification: 100 g of a 2 mg / mL aqueous dispersion of MXene nanosheets prepared in Preparation Example 1 and 100 g of a 1 mg / mL aqueous dispersion of graphene oxide were mixed uniformly, 15 g of extracellular matrix was added, the mixture was immersed for 1 h, freeze-dried, reduced with hydrazine hydrate vapor for 9 h, and pulverized to obtain a modified extracellular matrix.

[0126] S2. Preparation of calcium peroxide microspheres: 6g of calcium nitrate was dissolved in 70mL of water, 25g of 1.5mol / L ammonia was added dropwise, 250g of PEG200 was added, and after stirring and mixing, 30g of 30wt% hydrogen peroxide was added dropwise, and the reaction was stirred for 4h. The pH of the solution was adjusted to 11, and the solution was allowed to settle for 1h. The mixture was centrifuged, washed, and dried to obtain calcium peroxide particles; 11g of sodium alginate and 0.5g of Tween-85 were dissolved in 300mL of water, 5.5g of calcium peroxide particles were added, stirred and mixed, and added to 500mL of fish oil. The mixture was emulsified at 5000r / min for 15min, cured at room temperature for 30min, centrifuged, washed, and dried to obtain calcium peroxide microspheres.

[0127] S3. Preparation of freeze-dried exosome protective dressing: 17 g of poly(α,β-aspartyl hydrazide)-C16 copolymer was added to 300 mL of pH 7.4 phosphate buffer, followed by 6 g of modified extracellular matrix, 5 g of calcium peroxide microspheres, and 1.5 g of sodium citrate. The mixture was stirred and thoroughly mixed. 0.4 g of silver nitrate was added, and the mixture was ultrasonically dispersed at 1000 W for 15 min. The dressing was freeze-dried to prepare the dressing.

[0128] Test Example 1

[0129] The products obtained in Examples 1-3 and Comparative Examples 1-10 were added to water to a concentration of 60 g / L, and stirred to mix evenly to form a hydrogel.

[0130] (1) Adhesion strength. Using a syringe, squeeze the hydrogel into the middle of a 4 cm long and 2.5 cm wide pigskin strip, with the overlapping area of ​​the two strips being 1 cm × 2.5 cm, to obtain a test product. The test was conducted in accordance with YY / T0729.1 Test Method for Adhesion Properties of Tissue Adhesives Part 1: Lap-Shear Tensile Strength.

[0131] (2) In vitro degradation performance. In this experiment, no hydrogel sample was prepared. First, a dry gel dressing with a weight of m0 was placed in an aqueous solution. The degradation test was carried out in an air bath shaker at a temperature of 25°C and an oscillation speed of 100 rpm. At regular intervals, the sample was taken out, freeze-dried, and weighed as m1. Then, a new degradation solution was replaced. When (m0-m1 / m0) was greater than 0.99, it was considered to be completely degraded. The complete degradation time was recorded.

[0132] (3) In vitro cytotoxicity: The hydrogel was tested according to the ISO 10993-5 standard test method.

[0133] (4) Tensile strength. The tensile properties of the hydrogels were tested at room temperature using a CMT4102 universal testing machine. The specimens used in the tensile test had a diameter of 5 mm, a length of 30 mm, and a tensile rate of 50 mm / min.

[0134] The results are shown in Table 1.

[0135] Table 1

[0136]

[0137] As can be seen from the above table, the products prepared in Examples 1-3 of the present invention have good bonding strength and tensile strength, low cytotoxicity and fast degradation rate.

[0138] Test Example 2

[0139] Male C57BL / 6J mice were selected for modeling after a week of acclimatization. The day before modeling, after fasting for 16 hours and changing bedding, diabetic mice were intraperitoneally injected with 1 wt% streptozotocin solution at a dose of 50 mg / kg once daily for five consecutive days. Three days after the final streptozotocin injection, blood samples were collected via the tail vein for random blood glucose measurement. A successful type 1 diabetes model was confirmed if the random blood glucose value was >16.7 mmol / L and the mice exhibited symptoms of polydipsia, polyphagia, polyuria, and weight loss. The mice were monitored continuously for one week until their blood glucose levels stabilized and were then included in the formal experiments. Mice were anesthetized with an intraperitoneal injection of 1.25% avertin (0.2 mL / 10 g). The dorsal skin of each group was depilated and disinfected with medical alcohol. A 1 cm diameter section of skin and fascia was removed along the edge of the incision using sterile ophthalmic scissors and forceps. After wound modeling, the rats were randomly divided into 14 groups, each with 10 rats, namely the model group, the Example 1-3 group, and the Comparative Example 1-10 group. The model group was not treated, while the Example 1-3 and Comparative Example 1-10 groups were treated with the corresponding products on the wounds at 0.2 g / cm 2 The wound was secured with 3M film dressing to prevent skin retraction. Day 0 was designated the day of wound initiation. Wound healing was recorded by photographing the mice. Before each measurement, 1.25% avertin was injected intraperitoneally to anesthetize and position the mice. Wounds were photographed on days 0, 6, and 10 after injury. Wound healing rates (%) were calculated.

[0140] Wound healing rate (%) = (wound area on day 0 - residual wound area on the day of observation) / wound area on day 0 × 100%

[0141] The results are shown in Table 2.

[0142] Table 2

[0143]

[0144] It can be seen from the above table that the products prepared in Examples 1-3 of the present invention have a good effect of promoting wound healing.

[0145] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a freeze-dried exosome protective dressing, characterized in that: The protective agent and exosomes were mixed and embedded in chitosan-sodium tripolyphosphate microcapsules to obtain embedded exosomes; MXene and graphene-modified extracellular matrix, calcium peroxide microspheres, and embedded exosomes were added to poly(α,β-aspartic hydrazide)-C16 copolymer-silver hydrogel and freeze-dried to prepare exosome freeze-dried protective dressing.

2. The preparation method according to claim 1, characterized in that The following steps are involved: S1. Preparation of a protective agent: Sucrose, trehalose, mannitol, poloxamer 188, and taurine were mixed to prepare a protective agent; S2. Extracellular Matrix Modification: A MXene nanosheet aqueous dispersion and a graphene oxide aqueous dispersion were uniformly mixed, added to the extracellular matrix, impregnated, freeze-dried, reduced with hydrazine hydrate vapor, and pulverized to produce a modified extracellular matrix. S3. Exosome Encapsulation: Exosomes and a protective agent were added to water, followed by sodium tripolyphosphate, and stirred to obtain solution A. Chitosan was dissolved in acid to obtain solution B. Solutions A and B were separately loaded into syringes, inserted into the FNP device, and the pistons of the two syringes were pressed downward in parallel to squeeze out the solutions. The product was freeze-dried to obtain the encapsulated exosomes. S4. Preparation of calcium peroxide microspheres: Calcium salt is dissolved in water, ammonia is added dropwise, polyethylene glycol is added, and the mixture is stirred until uniformly mixed. Hydrogen peroxide is then added dropwise, the mixture is stirred, the pH of the solution is adjusted, and the mixture is allowed to settle to obtain calcium peroxide particles. Sodium alginate and an emulsifier are dissolved in water, calcium peroxide particles are added, the mixture is stirred until uniformly mixed, and the mixture is added to fish oil, emulsified, cured at room temperature, centrifuged, washed, and dried to obtain calcium peroxide microspheres. S5. Preparation of freeze-dried exosome protective dressing: Poly(α,β-aspartyl hydrazide)-C16 copolymer was added to phosphate buffer, followed by the addition of modified extracellular matrix, embedded exosomes, calcium peroxide microspheres, and sodium citrate. The mixture was stirred and evenly mixed. Silver nitrate was added, and the mixture was evenly dispersed by ultrasonication. The mixture was freeze-dried to prepare a freeze-dried exosome protective dressing.

3. The preparation method according to claim 2, characterized in that The mass ratio of sucrose, trehalose, mannitol, poloxamer 188 and taurine in step S1 is 2-3:3-4:5-7:1-2:1-2.

4. The preparation method according to claim 2, characterized in that In step S2, the concentration of the MXene nanosheet aqueous dispersion is 1-3 mg / mL, the concentration of the graphene oxide aqueous dispersion is 0.5-1.5 mg / mL, the mass ratio of the MXene nanosheet aqueous dispersion to the graphene oxide aqueous dispersion and the extracellular matrix is ​​100:100:12-17, the immersion time is 0.5-1.5 h, and the hydrazine hydrate vapor reduction time is 8-10 h.

5. The preparation method according to claim 2, characterized in that The preparation method of the MXene nanosheet aqueous dispersion is as follows: LiF is added to an HCl solution, stirred to dissolve, Ti3AlC2 is added, heated and etched, centrifuged, washed, added to water, ultrasonically dispersed evenly, centrifuged, collected the supernatant, and diluted with water to a specific concentration to prepare the MXene nanosheet aqueous dispersion.

6. The preparation method according to claim 5, characterized in that The mass ratio of LiF to Ti3AlC2 is 1-3:1-3, the concentration of the HCl solution is 8-10 mol / L, the temperature of the heating etching is 35-45°C, and the time is 45-53h.

7. The preparation method according to claim 2, characterized in that The mass ratio of exosomes, protective agent, sodium tripolyphosphate and chitosan in step S3 is 2-3:0.1-0.2:2-3:6-9.

8. The preparation method according to claim 2, characterized in that In step S4, the mass ratio of the calcium salt, ammonia water, polyethylene glycol, and hydrogen peroxide is 5-7:20-30:200-300:25-35, the calcium salt is calcium chloride or calcium nitrate, the concentration of the ammonia water is 1-2 mol / L, the concentration of the hydrogen peroxide is 25-35 wt%, the pH value of the adjustment solution is 10.5-11.5, the mass ratio of the sodium alginate, emulsifier, and calcium peroxide particles is 10-12:0.3-0.7:4-7, and the emulsifier is at least one of Tween-20, Tween-40, Tween-60, Tween-80, and Tween-85.

9. The preparation method according to claim 2, characterized in that The pH value of the phosphate buffer in step S5 is 7.2-7.6, and the mass ratio of the poly(α,β-aspartyl hydrazide)-C16 copolymer, modified extracellular matrix, embedded exosomes, calcium peroxide microspheres, sodium citrate, and silver nitrate is 15-20:4-8:3-5:4-6:1-2:0.3-0.

5.

10. A freeze-dried exosome protective dressing prepared by the preparation method according to any one of claims 1 to 9.

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