A freeze-dried exosome protective dressing and its preparation method

Abstract

This invention proposes a lyophilized exosome protective dressing and its preparation method, belonging to the field of dressing technology. A protective agent is mixed with exosomes and embedded in chitosan-sodium tripolyphosphate microcapsules to obtain embedded exosomes. MXene and graphene-modified extracellular matrix, along with calcium peroxide microspheres and the embedded exosomes, are added to poly(α,β-aspartic acid hydrazine)-C16 copolymer-silver hydrogel and freeze-dried to obtain the lyophilized exosome protective dressing. The lyophilized exosome protective dressing prepared by this invention has good biocompatibility, can rapidly promote wound healing, reduce wound inflammation, exert antibacterial and bacteriostatic effects, prevent wound deterioration, and is convenient for storage and transportation. It retains good activity even after long-term storage, showing broad application prospects in the field of rapid wound healing.

Description

Technical Field

[0001] This invention relates to the field of dressing technology, specifically to an exosome freeze-dried protective dressing and its preparation method. Background Technology

[0002] In daily life, people's skin is often damaged by burns, scalds, and mechanical trauma, as well as skin injuries caused by diabetic foot ulcers and pressure sores. The skin repair process after injury generally consists of four stages: coagulation phase, inflammation phase, proliferation phase, and remodeling phase. During these repair stages, bacterial infection can cause wound inflammation and suppuration, not only inhibiting wound repair but also potentially leading to tissue necrosis. Therefore, promoting wound repair and effectively preventing wound infection is a challenging issue. In the treatment process, early detection and rapid, appropriate, and effective sterilization and inhibition are crucial for improving wound repair outcomes. Especially today, with increasing antibiotic resistance, it is necessary to minimize antibiotic use while ensuring antibacterial effectiveness.

[0003] Dressings are essential products in modern wound care, protecting the wound surface from further damage and providing some degree of protection against microbial infection. Exosomes are small vesicles, approximately 30-150 nanometers in diameter, membrane-bound structures released from inside cells via vesicle transport pathways. Exosomes contain various bioactive molecules, including proteins, nucleic acids, and lipids. Endothelial progenitor cells are stem cells derived from bone marrow or other tissues, possessing the potential to differentiate into endothelial cells. These cells regulate intercellular signaling and metabolic activities by secreting exosomes. Therefore, wound treatment can be achieved by using exosomes to promote angiogenesis in wound tissue.

[0004] With the development and application of the moist wound healing theory, the use of hydrogels and other moist dressings not only helps maintain a moist environment on the wound surface and promotes wound repair, but also improves adhesions that occur during the wound healing process and prevents secondary damage. However, if moist dressings are used improperly, they can easily breed bacteria and cause infection. To address these issues, various wound dressings suitable for different stages have emerged on the market, often containing added antibacterial drugs or agents. To avoid antibiotic resistance caused by overuse, antibacterial agents that are less likely to induce resistance are primarily used, including organic antibacterial agents, inorganic antibacterial agents, and natural antibacterial agents. Organic antibacterial agents include imidazoles, thiazoles, isothiazolone derivatives, quaternary ammonium salts, biguanides, etc., which have relatively poor biocompatibility and are prone to causing hemolysis, pain, and other problems. Inorganic antibacterial agents are mainly composed of metal nanoparticles, among which nano-silver dominates due to its outstanding antibacterial properties. However, during use, the deposition and adhesion of nano-silver may cause local skin darkening, affecting appearance, and the cost is relatively high. Natural antibacterial agents include chitosan, honey, etc., which have relatively weak antibacterial properties and are usually combined with other types of antibacterial agents or drugs for synergistic effects. Summary of the Invention

[0005] The purpose of this invention is to propose an exosome freeze-dried protective dressing and its preparation method. It has good biocompatibility, can rapidly promote wound healing, reduce wound inflammation, and has antibacterial and bacteriostatic effects, preventing wound deterioration. It is also convenient to store and transport, and retains good activity even after long-term storage. It has broad application prospects in the field of rapid wound healing.

[0006] The technical solution of this invention is implemented as follows:

[0007] This invention provides a method for preparing an exosome lyophilized protective dressing. The method involves mixing a protective agent with exosomes and embedding the mixture in chitosan-sodium tripolyphosphate microcapsules to obtain embedded exosomes. MXene and graphene-modified extracellular matrix, along with calcium peroxide microspheres and the embedded exosomes, are added to a poly(α,β-aspartic acid hydrazine)-C16 copolymer-silver hydrogel and freeze-dried to obtain the exosome lyophilized protective dressing.

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

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

[0010] S2. Modification of extracellular matrix: Mix MXene nanosheet aqueous dispersion and graphene oxide aqueous dispersion evenly, add extracellular matrix, impregnate, freeze dry, reduce with hydrazine hydrate vapor, pulverize, and obtain modified extracellular matrix;

[0011] S3. Encapsulating exosomes: Add exosomes and a protective agent to water, add sodium tripolyphosphate, stir and mix evenly to obtain solution A; dissolve chitosan in acid to obtain solution B; load solution A and solution B into syringes respectively, insert them into an FNP device (multi-inlet vortex mixer), press the piston handles of the two syringes downwards in parallel to squeeze out the solution, freeze-dry the product to obtain encapsulated exosomes;

[0012] S4. Preparation of calcium peroxide microspheres: Dissolve calcium salt in water, add ammonia, add polyethylene glycol, stir and mix evenly, add hydrogen peroxide, stir and react, adjust the pH of the solution, let it stand to precipitate, and obtain calcium peroxide particles; dissolve sodium alginate and emulsifier in water, add calcium peroxide particles, stir and mix evenly, add to fish oil, emulsify, solidify at room temperature, centrifuge, wash, and dry to obtain calcium peroxide microspheres;

[0013] S5. Preparation of exosome lyophilized protective dressing: Poly(α,β-aspartic acid hydrazide)-C16 copolymer was added to phosphate buffer, modified extracellular matrix, embedded exosomes, calcium peroxide microspheres and sodium citrate were added, stirred and mixed evenly, silver nitrate was added, ultrasonically dispersed evenly, and freeze-dried to obtain exosome lyophilized protective dressing.

[0014] As a further improvement of the present invention, 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 invention, 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, the graphene oxide aqueous dispersion, and the extracellular matrix is ​​100:100:12-17, the impregnation 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 invention, the preparation method of the MXene nanosheet aqueous dispersion is as follows: LiF is added to HCl solution, stirred and dissolved, Ti3AlC2 is added, heated and etched, centrifuged, washed, added to water, ultrasonically dispersed evenly, centrifuged, the supernatant is collected, and diluted with water to a specific concentration to obtain the MXene nanosheet aqueous dispersion.

[0017] As a further improvement of the present invention, 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℃, and the time is 45-53h.

[0018] As a further improvement of the present invention, 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.

[0019] As a further improvement of the present invention, in step S4, the mass ratio of calcium salt, ammonia, 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 ammonia is 1-2 mol / L, the concentration of hydrogen peroxide is 25-35 wt%, the pH value of the adjusted solution is 10.5-11.5, the mass ratio of 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-aspartic acid 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 an exosome freeze-dried protective dressing prepared by the above-described preparation method.

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

[0023] This invention utilizes a protective agent composed of trehalose and mannitol, with the addition of poloxamer 188. This enhances exosome activity through a triple mechanism of inhibiting ice crystal growth, maintaining membrane stability, and improving dispersibility. Trehalose forms a glassy structure to protect membrane integrity, mannitol acts as a filler to prevent structural collapse after freeze-drying, 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. The added taurine enhances the antioxidant properties of exosomes, and the added sucrose partially replaces trehalose, reducing raw material costs while maintaining stability.

[0024] The extracellular matrix (ECM) has a structure highly similar to natural collagen fibers, with high porosity, allowing for the sustained release of exosomes and good biocompatibility with skin tissue. Modification of the ECM through MXene and graphene impregnation significantly improves its conductivity. Furthermore, the scaffold structure of the ECM effectively mitigates the self-accumulation problem of MXene. Increased conductivity modulates cell signal transduction, and photothermal conversion enables on-demand release of exosomes, shortening wound healing time and inhibiting the expression of the inflammatory factor IL-6.

[0025] Following 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. Prolonged hypoxia in chronic wounds inhibits angiogenesis, slows the re-epithelialization healing process, and reduces extracellular matrix synthesis. Therefore, enhancing oxygen concentration in wound tissue is crucial for the healing of chronic wounds. 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 injury site. After being encapsulated in sodium alginate, the sodium alginate microsphere shell is disrupted within the wound environment, allowing for the slow release of calcium peroxide, thereby increasing the oxygen concentration near the wound.

[0026] Milk exosomes, carrying various proteins, RNA, and lipids, promote tissue repair and exhibit significant cell proliferation at the site of injury. The synergistic effect of exosomes and oxygen has been used to heal diabetic wounds and treat bone defects. Exosomes are encapsulated in chitosan-sodium tripolyphosphate nanocapsules, which dissolve and release them in an acidic wound environment. This pH-responsive approach prevents premature release of exosomes under neutral storage conditions and ensures precise delivery to the site of inflammation.

[0027] This invention loads modified extracellular matrix, encapsulates exosomes and calcium peroxide microspheres onto amphiphilic poly(α,β-aspartic hydrazide)-C16 copolymer hydrogel, and synthesizes silver nanoparticles in situ. On the one hand, it forms a cross-linked structure, and on the other hand, it forms a dual protection of "physical barrier + antibacterial release". It can play an antibacterial and bacteriostatic effect while wound healing, prevent wound deterioration, and accelerate re-epithelialization by promoting the migration of keratinocytes.

[0028] The exosome freeze-dried protective dressing prepared by this invention has good biocompatibility, can rapidly promote wound healing, reduce wound inflammation, and has antibacterial and bacteriostatic effects, preventing wound deterioration. It is also convenient to store and transport, and retains good activity even after long-term storage, showing broad application prospects in the field of rapid wound healing. Detailed Implementation

[0029] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

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

[0031] Preparation Example 1: Aqueous Dispersion of MXene Nanosheets

[0032] The preparation method is as follows: 2g LiF was added to 40mL of 9mol / L HCl solution and stirred to dissolve. 2g Ti3AlC2 was added, etched at 40℃ for 48h, centrifuged at 3500r / min for 10min, washed until the pH value was 6, added to water, ultrasonically dispersed at 1000W for 2h, centrifuged at 3500r / min for 30min, the supernatant was collected, and diluted with water to a specific concentration to obtain an aqueous dispersion of MXene nanosheets.

[0033] Example 1

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

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

[0036] S2. Modification of extracellular matrix: 100g of 1mg / mL MXene nanosheet aqueous dispersion prepared in Preparation Example 1 and 100g of 0.5mg / mL graphene oxide aqueous dispersion were mixed evenly, 12g of extracellular matrix was added, impregnated for 0.5h, freeze-dried, reduced with hydrazine hydrate vapor for 8h, and pulverized to obtain modified extracellular matrix.

[0037] S3. Encapsulation of exosomes: Add 2g of milk exosomes and 0.1g of protective agent to 200mL of water, add 2g of sodium tripolyphosphate, stir and mix evenly to obtain solution A; dissolve 6g of chitosan in 200mL of 2wt% acetic acid solution to obtain solution B; put equal volumes of solution A and solution B into syringes, insert them into the FNP device, press the piston handles of the two syringes downwards in parallel to squeeze out the solution at a uniform speed, and squeeze out within 2min. Let stand for 1h, freeze dry the product to obtain encapsulated exosomes;

[0038] S4. Preparation of calcium peroxide microspheres: Dissolve 5g of calcium chloride in 70mL of water, add 20g of 1mol / L ammonia water, add 200g of PEG200, stir and mix evenly, then add 25g of 25wt% hydrogen peroxide, stir and react for 4h, adjust the pH of the solution to 10.5, let it stand to precipitate for 1h, centrifuge, wash, and dry to obtain calcium peroxide particles; Dissolve 10g of sodium alginate and 0.3g of Tween-40 in 300mL of water, add 4g of calcium peroxide particles, stir and mix evenly, add to 500mL of fish oil, emulsify at 5000r / min for 15min, solidify at room temperature for 30min, centrifuge, wash, and dry to obtain calcium peroxide microspheres;

[0039] S5. Preparation of exosome lyophilized protective dressing: 15g of poly(α,β-aspartic acid hydrazide)-C16 copolymer was added to 300mL of phosphate buffer with a pH of 7.2, along with 4g of modified extracellular matrix, 3g of embedded exosomes, 4g of calcium peroxide microspheres and 1g of sodium citrate. The mixture was stirred and mixed evenly, and 0.3g of silver nitrate was added. The mixture was ultrasonically dispersed at 1000W for 15min and then freeze-dried to obtain the exosome lyophilized protective dressing.

[0040] Example 2

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

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

[0043] S2. Modification of extracellular matrix: 100g of MXene nanosheet aqueous dispersion with a concentration of 3mg / mL prepared in Preparation Example 1 and 100g of graphene oxide aqueous dispersion with a concentration of 1.5mg / mL were mixed evenly, 17g of extracellular matrix was added, impregnated for 1.5h, freeze-dried, reduced with hydrazine hydrate vapor for 10h, and pulverized to obtain modified extracellular matrix;

[0044] S3. Encapsulating exosomes: Add 3g of milk exosomes and 0.2g of preservative to 200mL of water, add 3g of sodium tripolyphosphate, stir and mix evenly to obtain solution A; dissolve 9g of chitosan in 200mL of 2wt% acetic acid solution to obtain solution B; put equal volumes of solution A and solution B into syringes, insert them into the FNP device, press the piston handles of the two syringes downwards in parallel to squeeze out the solution at a uniform speed, and squeeze out within 2min. Let stand for 1h, freeze dry the product to obtain encapsulated exosomes;

[0045] S4. Preparation of calcium peroxide microspheres: Dissolve 7g of calcium nitrate in 70mL of water, add 30g of 2mol / L ammonia water, add 300g of PEG200, stir and mix evenly, then add 35g of 35wt% hydrogen peroxide, stir and react for 4h, adjust the pH of the solution to 11.5, let it stand to precipitate for 1h, centrifuge, wash, and dry to obtain calcium peroxide particles; Dissolve 12g of sodium alginate and 0.7g of Tween-60 in 300mL of water, add 7g of calcium peroxide particles, stir and mix evenly, add to 500mL of fish oil, emulsify at 5000r / min for 15min, solidify at room temperature for 30min, centrifuge, wash, and dry to obtain calcium peroxide microspheres;

[0046] S5. Preparation of exosome lyophilized protective dressing: 20g of poly(α,β-aspartic acid hydrazide)-C16 copolymer was added to 300mL of phosphate buffer with a pH of 7.6, along with 8g of modified extracellular matrix, 5g of embedded exosomes, 6g of calcium peroxide microspheres and 2g of sodium citrate. The mixture was stirred and mixed evenly, and 0.5g of silver nitrate was added. The mixture was ultrasonically dispersed at 1000W for 15min and then freeze-dried to obtain the exosome lyophilized protective dressing.

[0047] Example 3

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

[0049] S1. Preparation of the protective agent: Mix 2.5g sucrose, 3.5g trehalose, 6g mannitol, 1.5g poloxamer 188 and 1.5g taurine evenly to prepare the protective agent;

[0050] S2. Modification of extracellular matrix: 100g of 2mg / mL MXene nanosheet aqueous dispersion prepared in Preparation Example 1 and 100g of 1mg / mL graphene oxide aqueous dispersion were mixed evenly, 15g of extracellular matrix was added, impregnated for 1h, freeze-dried, reduced with hydrazine hydrate vapor for 9h, and pulverized to obtain modified extracellular matrix.

[0051] S3. Encapsulation of exosomes: 2.5g of milk exosomes and 0.15g of preservative were added to 200mL of water, and 2.5g of sodium tripolyphosphate was added. The mixture was stirred and stirred until homogeneous to obtain solution A. 7.5g of chitosan was dissolved in 200mL of 2wt% acetic acid solution to obtain solution B. Equal volumes of solutions A and B were loaded into syringes and inserted into the FNP device. The piston handles of the two syringes were pressed down parallel to each other to squeeze out the solution at a uniform speed. The extrusion was completed within 2 minutes. The mixture was allowed to stand for 1 hour, and the product was freeze-dried to obtain encapsulated exosomes.

[0052] S4. Preparation of calcium peroxide microspheres: Dissolve 6g of calcium nitrate in 70mL of water, add 25g of 1.5mol / L ammonia water, add 250g of PEG200, stir and mix evenly, then add 30g of 30wt% hydrogen peroxide, stir and react for 4h, adjust the pH of the solution to 11, let it stand to precipitate for 1h, centrifuge, wash, and dry to obtain calcium peroxide particles; Dissolve 11g of sodium alginate and 0.5g of Tween-85 in 300mL of water, add 5.5g of calcium peroxide particles, stir and mix evenly, add to 500mL of fish oil, emulsify at 5000r / min for 15min, solidify at room temperature for 30min, centrifuge, wash, and dry to obtain calcium peroxide microspheres;

[0053] S5. Preparation of exosome lyophilized protective dressing: 17g of poly(α,β-aspartic acid hydrazide)-C16 copolymer was added to 300mL of phosphate buffer with a pH of 7.4, along with 6g of modified extracellular matrix, 4g of embedded exosomes, 5g of calcium peroxide microspheres and 1.5g of sodium citrate. The mixture was stirred and mixed evenly, and 0.4g of silver nitrate was added. The mixture was ultrasonically dispersed at 1000W for 15min and then freeze-dried to obtain the exosome lyophilized protective dressing.

[0054] Comparative Example 1

[0055] The difference from Example 3 is that MXene nanosheet aqueous dispersion was not added in step S2.

[0056] Specifically as follows:

[0057] S1. Preparation of the protective agent: Mix 2.5g sucrose, 3.5g trehalose, 6g mannitol, 1.5g poloxamer 188 and 1.5g taurine evenly to prepare the protective agent;

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

[0059] S3. Encapsulation of exosomes: 2.5g of milk exosomes and 0.15g of preservative were added to 200mL of water, and 2.5g of sodium tripolyphosphate was added. The mixture was stirred and stirred until homogeneous to obtain solution A. 7.5g of chitosan was dissolved in 200mL of 2wt% acetic acid solution to obtain solution B. Equal volumes of solutions A and B were loaded into syringes and inserted into the FNP device. The piston handles of the two syringes were pressed down parallel to each other to squeeze out the solution at a uniform speed. The extrusion was completed within 2 minutes. The mixture was allowed to stand for 1 hour, and the product was freeze-dried to obtain encapsulated exosomes.

[0060] S4. Preparation of calcium peroxide microspheres: Dissolve 6g of calcium nitrate in 70mL of water, add 25g of 1.5mol / L ammonia water, add 250g of PEG200, stir and mix evenly, then add 30g of 30wt% hydrogen peroxide, stir and react for 4h, adjust the pH of the solution to 11, let it stand to precipitate for 1h, centrifuge, wash, and dry to obtain calcium peroxide particles; Dissolve 11g of sodium alginate and 0.5g of Tween-85 in 300mL of water, add 5.5g of calcium peroxide particles, stir and mix evenly, add to 500mL of fish oil, emulsify at 5000r / min for 15min, solidify at room temperature for 30min, centrifuge, wash, and dry to obtain calcium peroxide microspheres;

[0061] S5. Preparation of exosome lyophilized protective dressing: 17g of poly(α,β-aspartic acid hydrazide)-C16 copolymer was added to 300mL of phosphate buffer with a pH of 7.4, along with 6g of modified extracellular matrix, 4g of embedded exosomes, 5g of calcium peroxide microspheres and 1.5g of sodium citrate. The mixture was stirred and mixed evenly, and 0.4g of silver nitrate was added. The mixture was ultrasonically dispersed at 1000W for 15min and then freeze-dried to obtain the exosome lyophilized protective dressing.

[0062] Comparative Example 2

[0063] The difference from Example 3 is that no graphene oxide aqueous dispersion was added in step S2.

[0064] Specifically as follows:

[0065] S1. Preparation of the protective agent: Mix 2.5g sucrose, 3.5g trehalose, 6g mannitol, 1.5g poloxamer 188 and 1.5g taurine evenly to prepare the protective agent;

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

[0067] S3. Encapsulation of exosomes: 2.5g of milk exosomes and 0.15g of preservative were added to 200mL of water, and 2.5g of sodium tripolyphosphate was added. The mixture was stirred and stirred until homogeneous to obtain solution A. 7.5g of chitosan was dissolved in 200mL of 2wt% acetic acid solution to obtain solution B. Equal volumes of solutions A and B were loaded into syringes and inserted into the FNP device. The piston handles of the two syringes were pressed down parallel to each other to squeeze out the solution at a uniform speed. The extrusion was completed within 2 minutes. The mixture was allowed to stand for 1 hour, and the product was freeze-dried to obtain encapsulated exosomes.

[0068] S4. Preparation of calcium peroxide microspheres: Dissolve 6g of calcium nitrate in 70mL of water, add 25g of 1.5mol / L ammonia water, add 250g of PEG200, stir and mix evenly, then add 30g of 30wt% hydrogen peroxide, stir and react for 4h, adjust the pH of the solution to 11, let it stand to precipitate for 1h, centrifuge, wash, and dry to obtain calcium peroxide particles; Dissolve 11g of sodium alginate and 0.5g of Tween-85 in 300mL of water, add 5.5g of calcium peroxide particles, stir and mix evenly, add to 500mL of fish oil, emulsify at 5000r / min for 15min, solidify at room temperature for 30min, centrifuge, wash, and dry to obtain calcium peroxide microspheres;

[0069] S5. Preparation of exosome lyophilized protective dressing: 17g of poly(α,β-aspartic acid hydrazide)-C16 copolymer was added to 300mL of phosphate buffer with a pH of 7.4, along with 6g of modified extracellular matrix, 4g of embedded exosomes, 5g of calcium peroxide microspheres and 1.5g of sodium citrate. The mixture was stirred and mixed evenly, and 0.4g of silver nitrate was added. The mixture was ultrasonically dispersed at 1000W for 15min and then freeze-dried to obtain the exosome lyophilized protective dressing.

[0070] Comparative Example 3

[0071] The difference from Example 3 is that step S2 in Example 3 was not performed.

[0072] Specifically as follows:

[0073] S1. Preparation of the protective agent: Mix 2.5g sucrose, 3.5g trehalose, 6g mannitol, 1.5g poloxamer 188 and 1.5g taurine evenly to prepare the protective agent;

[0074] S2. Encapsulation of exosomes: 2.5g of milk exosomes and 0.15g of preservative were added to 200mL of water, and 2.5g of sodium tripolyphosphate was added. The mixture was stirred and stirred until homogeneous to obtain solution A. 7.5g of chitosan was dissolved in 200mL of 2wt% acetic acid solution to obtain solution B. Equal volumes of solutions A and B were loaded into syringes and inserted into the FNP device. The piston handles of the two syringes were pressed down parallel to each other to extrude the solution at a uniform speed. The extrusion was completed within 2 minutes. The mixture was allowed to stand for 1 hour, and the product was freeze-dried to obtain encapsulated exosomes.

[0075] S3. Preparation of calcium peroxide microspheres: Dissolve 6g of calcium nitrate in 70mL of water, add 25g of 1.5mol / L ammonia water, add 250g of PEG200, stir and mix evenly, then add 30g of 30wt% hydrogen peroxide, stir and react for 4h, adjust the pH of the solution to 11, let it stand to precipitate for 1h, centrifuge, wash, and dry to obtain calcium peroxide particles; Dissolve 11g of sodium alginate and 0.5g of Tween-85 in 300mL of water, add 5.5g of calcium peroxide particles, stir and mix evenly, add to 500mL of fish oil, emulsify at 5000r / min for 15min, solidify at room temperature for 30min, centrifuge, wash, and dry to obtain calcium peroxide microspheres;

[0076] S4. Preparation of exosome lyophilized protective dressing: 17g of poly(α,β-aspartic acid hydrazide)-C16 copolymer was added to 300mL of phosphate buffer with a pH of 7.4, along with 6g of extracellular matrix, 4g of embedded exosomes, 5g of calcium peroxide microspheres and 1.5g of sodium citrate. The mixture was stirred and mixed evenly, and 0.4g of silver nitrate was added. The mixture was ultrasonically dispersed at 1000W for 15min and then freeze-dried to obtain the exosome lyophilized protective dressing.

[0077] Comparative Example 4

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

[0079] Specifically as follows:

[0080] S1. Modification of extracellular matrix: 100g of 2mg / mL MXene nanosheet aqueous dispersion prepared in Preparation Example 1 and 100g of 1mg / mL graphene oxide aqueous dispersion were mixed evenly, 15g of extracellular matrix was added, impregnated for 1h, freeze-dried, reduced with hydrazine hydrate vapor for 9h, and pulverized to obtain modified extracellular matrix.

[0081] S2. Encapsulation of exosomes: 2.65g of milk exosomes were added to 200mL of water, and 2.5g of sodium tripolyphosphate was added. The mixture was stirred and stirred until homogeneous to obtain solution A. 7.5g of chitosan was dissolved in 200mL of 2wt% acetic acid solution to obtain solution B. Equal volumes of solutions A and B were loaded into syringes and inserted into the FNP device. The piston handles of the two syringes were pressed down parallel to each other to squeeze out the solution at a uniform speed. The extrusion was completed within 2 minutes. The mixture was allowed to stand for 1 hour, and the product was freeze-dried to obtain encapsulated exosomes.

[0082] S3. Preparation of calcium peroxide microspheres: Dissolve 6g of calcium nitrate in 70mL of water, add 25g of 1.5mol / L ammonia water, add 250g of PEG200, stir and mix evenly, then add 30g of 30wt% hydrogen peroxide, stir and react for 4h, adjust the pH of the solution to 11, let it stand to precipitate for 1h, centrifuge, wash, and dry to obtain calcium peroxide particles; Dissolve 11g of sodium alginate and 0.5g of Tween-85 in 300mL of water, add 5.5g of calcium peroxide particles, stir and mix evenly, add to 500mL of fish oil, emulsify at 5000r / min for 15min, solidify at room temperature for 30min, centrifuge, wash, and dry to obtain calcium peroxide microspheres;

[0083] S4. Preparation of exosome lyophilized protective dressing: 17g of poly(α,β-aspartic acid hydrazide)-C16 copolymer was added to 300mL of phosphate buffer with a pH of 7.4, along with 6g of modified extracellular matrix, 4g of embedded exosomes, 5g of calcium peroxide microspheres and 1.5g of sodium citrate. The mixture was stirred and mixed evenly, and 0.4g of silver nitrate was added. The mixture was ultrasonically dispersed at 1000W for 15min and then freeze-dried to obtain the exosome lyophilized protective dressing.

[0084] Comparative Example 5

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

[0086] Specifically as follows:

[0087] S1. Preparation of the protective agent: Mix 2.5g sucrose, 3.5g trehalose, 6g mannitol, 1.5g poloxamer 188 and 1.5g taurine evenly to prepare the protective agent;

[0088] S2. Modification of extracellular matrix: 100g of 2mg / mL MXene nanosheet aqueous dispersion prepared in Preparation Example 1 and 100g of 1mg / mL graphene oxide aqueous dispersion were mixed evenly, 15g of extracellular matrix was added, impregnated for 1h, freeze-dried, reduced with hydrazine hydrate vapor for 9h, and pulverized to obtain modified extracellular matrix.

[0089] S3. Preparation of the mixture: Mix 2.5g of milk exosomes and 0.15g of preservative evenly to obtain the mixture;

[0090] S4. Preparation of calcium peroxide microspheres: Dissolve 6g of calcium nitrate in 70mL of water, add 25g of 1.5mol / L ammonia water, add 250g of PEG200, stir and mix evenly, then add 30g of 30wt% hydrogen peroxide, stir and react for 4h, adjust the pH of the solution to 11, let it stand to precipitate for 1h, centrifuge, wash, and dry to obtain calcium peroxide particles; Dissolve 11g of sodium alginate and 0.5g of Tween-85 in 300mL of water, add 5.5g of calcium peroxide particles, stir and mix evenly, add to 500mL of fish oil, emulsify at 5000r / min for 15min, solidify at room temperature for 30min, centrifuge, wash, and dry to obtain calcium peroxide microspheres;

[0091] S5. Preparation of exosome lyophilized protective dressing: 17g of poly(α,β-aspartic acid hydrazide)-C16 copolymer was added to 300mL of phosphate buffer with a pH of 7.4, along with 6g of modified extracellular matrix, 4g of the mixture, 5g of calcium peroxide microspheres and 1.5g of sodium citrate. The mixture was stirred and mixed evenly, and 0.4g of silver nitrate was added. The mixture was ultrasonically dispersed at 1000W for 15min and then freeze-dried to obtain the exosome lyophilized protective dressing.

[0092] Comparative Example 6

[0093] The difference from Example 3 is that no embedding was performed in step S4.

[0094] Specifically as follows:

[0095] S1. Preparation of the protective agent: Mix 2.5g sucrose, 3.5g trehalose, 6g mannitol, 1.5g poloxamer 188 and 1.5g taurine evenly to prepare the protective agent;

[0096] S2. Modification of extracellular matrix: 100g of 2mg / mL MXene nanosheet aqueous dispersion prepared in Preparation Example 1 and 100g of 1mg / mL graphene oxide aqueous dispersion were mixed evenly, 15g of extracellular matrix was added, impregnated for 1h, freeze-dried, reduced with hydrazine hydrate vapor for 9h, and pulverized to obtain modified extracellular matrix.

[0097] S3. Encapsulation of exosomes: 2.5g of milk exosomes and 0.15g of preservative were added to 200mL of water, and 2.5g of sodium tripolyphosphate was added. The mixture was stirred and stirred until homogeneous to obtain solution A. 7.5g of chitosan was dissolved in 200mL of 2wt% acetic acid solution to obtain solution B. Equal volumes of solutions A and B were loaded into syringes and inserted into the FNP device. The piston handles of the two syringes were pressed down parallel to each other to squeeze out the solution at a uniform speed. The extrusion was completed within 2 minutes. The mixture was allowed to stand for 1 hour, and the product was freeze-dried to obtain encapsulated exosomes.

[0098] S4. Preparation of calcium peroxide particles: Dissolve 6g of calcium nitrate in 70mL of water, add 25g of 1.5mol / L ammonia water dropwise, add 250g of PEG200, stir and mix evenly, then add 30g of 30wt% hydrogen peroxide dropwise, stir and react for 4h, adjust the pH of the solution to 11, let it stand to precipitate for 1h, centrifuge, wash, and dry to obtain calcium peroxide particles;

[0099] S5. Preparation of exosome lyophilized protective dressing: 17g of poly(α,β-aspartic acid hydrazide)-C16 copolymer was added to 300mL of phosphate buffer with a pH of 7.4, along with 6g of modified extracellular matrix, 4g of embedded exosomes, 5g of calcium peroxide particles and 1.5g of sodium citrate. The mixture was stirred and mixed evenly, and 0.4g of silver nitrate was added. The mixture was ultrasonically dispersed at 1000W for 15min and then freeze-dried to obtain the exosome lyophilized protective dressing.

[0100] Comparative Example 7

[0101] The difference from Example 3 is that silver nitrate was not added in step S5.

[0102] Specifically as follows:

[0103] S1. Preparation of the protective agent: Mix 2.5g sucrose, 3.5g trehalose, 6g mannitol, 1.5g poloxamer 188 and 1.5g taurine evenly to prepare the protective agent;

[0104] S2. Modification of extracellular matrix: 100g of 2mg / mL MXene nanosheet aqueous dispersion prepared in Preparation Example 1 and 100g of 1mg / mL graphene oxide aqueous dispersion were mixed evenly, 15g of extracellular matrix was added, impregnated for 1h, freeze-dried, reduced with hydrazine hydrate vapor for 9h, and pulverized to obtain modified extracellular matrix.

[0105] S3. Encapsulation of exosomes: 2.5g of milk exosomes and 0.15g of preservative were added to 200mL of water, and 2.5g of sodium tripolyphosphate was added. The mixture was stirred and stirred until homogeneous to obtain solution A. 7.5g of chitosan was dissolved in 200mL of 2wt% acetic acid solution to obtain solution B. Equal volumes of solutions A and B were loaded into syringes and inserted into the FNP device. The piston handles of the two syringes were pressed down parallel to each other to squeeze out the solution at a uniform speed. The extrusion was completed within 2 minutes. The mixture was allowed to stand for 1 hour, and the product was freeze-dried to obtain encapsulated exosomes.

[0106] S4. Preparation of calcium peroxide microspheres: Dissolve 6g of calcium nitrate in 70mL of water, add 25g of 1.5mol / L ammonia water, add 250g of PEG200, stir and mix evenly, then add 30g of 30wt% hydrogen peroxide, stir and react for 4h, adjust the pH of the solution to 11, let it stand to precipitate for 1h, centrifuge, wash, and dry to obtain calcium peroxide particles; Dissolve 11g of sodium alginate and 0.5g of Tween-85 in 300mL of water, add 5.5g of calcium peroxide particles, stir and mix evenly, add to 500mL of fish oil, emulsify at 5000r / min for 15min, solidify at room temperature for 30min, centrifuge, wash, and dry to obtain calcium peroxide microspheres;

[0107] S5. Preparation of exosome lyophilized protective dressing: 17g of poly(α,β-aspartic acid hydrazide)-C16 copolymer was added to 300mL of phosphate buffer with a pH of 7.4, along with 6g of modified extracellular matrix, 4g of embedded exosomes, and 5g of calcium peroxide microspheres. The mixture was stirred and mixed evenly, ultrasonically dispersed at 1000W for 15min, and then freeze-dried to obtain the exosome lyophilized protective dressing.

[0108] Comparative Example 8

[0109] The difference from Example 3 is that calcium peroxide microspheres were not added in step S5, and step S4 in Example 3 was not performed.

[0110] Specifically as follows:

[0111] S1. Preparation of the protective agent: Mix 2.5g sucrose, 3.5g trehalose, 6g mannitol, 1.5g poloxamer 188 and 1.5g taurine evenly to prepare the protective agent;

[0112] S2. Modification of extracellular matrix: 100g of 2mg / mL MXene nanosheet aqueous dispersion prepared in Preparation Example 1 and 100g of 1mg / mL graphene oxide aqueous dispersion were mixed evenly, 15g of extracellular matrix was added, impregnated for 1h, freeze-dried, reduced with hydrazine hydrate vapor for 9h, and pulverized to obtain modified extracellular matrix.

[0113] S3. Encapsulation of exosomes: 2.5g of milk exosomes and 0.15g of preservative were added to 200mL of water, and 2.5g of sodium tripolyphosphate was added. The mixture was stirred and stirred until homogeneous to obtain solution A. 7.5g of chitosan was dissolved in 200mL of 2wt% acetic acid solution to obtain solution B. Equal volumes of solutions A and B were loaded into syringes and inserted into the FNP device. The piston handles of the two syringes were pressed down parallel to each other to squeeze out the solution at a uniform speed. The extrusion was completed within 2 minutes. The mixture was allowed to stand for 1 hour, and the product was freeze-dried to obtain encapsulated exosomes.

[0114] S4. Preparation of exosome lyophilized protective dressing: 17g of poly(α,β-aspartic acid hydrazide)-C16 copolymer was added to 300mL of phosphate buffer with a pH of 7.4, along with 6g of modified extracellular matrix, 4g of embedded exosomes and 1.5g of sodium citrate. The mixture was stirred and mixed evenly, and 0.4g of silver nitrate was added. The mixture was ultrasonically dispersed at 1000W for 15min and then freeze-dried to obtain the exosome lyophilized protective dressing.

[0115] Comparative Example 9

[0116] The difference from Example 3 is that no modified extracellular matrix was added in step S5.

[0117] Specifically as follows:

[0118] S1. Preparation of the protective agent: Mix 2.5g sucrose, 3.5g trehalose, 6g mannitol, 1.5g poloxamer 188 and 1.5g taurine evenly to prepare the protective agent;

[0119] S2. Encapsulation of exosomes: 2.5g of milk exosomes and 0.15g of preservative were added to 200mL of water, and 2.5g of sodium tripolyphosphate was added. The mixture was stirred and stirred until homogeneous to obtain solution A. 7.5g of chitosan was dissolved in 200mL of 2wt% acetic acid solution to obtain solution B. Equal volumes of solutions A and B were loaded into syringes and inserted into the FNP device. The piston handles of the two syringes were pressed down parallel to each other to extrude the solution at a uniform speed. The extrusion was completed within 2 minutes. The mixture was allowed to stand for 1 hour, and the product was freeze-dried to obtain encapsulated exosomes.

[0120] S3. Preparation of calcium peroxide microspheres: Dissolve 6g of calcium nitrate in 70mL of water, add 25g of 1.5mol / L ammonia water, add 250g of PEG200, stir and mix evenly, then add 30g of 30wt% hydrogen peroxide, stir and react for 4h, adjust the pH of the solution to 11, let it stand to precipitate for 1h, centrifuge, wash, and dry to obtain calcium peroxide particles; Dissolve 11g of sodium alginate and 0.5g of Tween-85 in 300mL of water, add 5.5g of calcium peroxide particles, stir and mix evenly, add to 500mL of fish oil, emulsify at 5000r / min for 15min, solidify at room temperature for 30min, centrifuge, wash, and dry to obtain calcium peroxide microspheres;

[0121] S4. Preparation of exosome lyophilized protective dressing: 17g of poly(α,β-aspartic acid hydrazide)-C16 copolymer was added to 300mL of phosphate buffer with a pH of 7.4, along with 4g of embedded exosomes, 5g of calcium peroxide microspheres and 1.5g of sodium citrate. The mixture was stirred and mixed evenly, and 0.4g of silver nitrate was added. The mixture was ultrasonically dispersed at 1000W for 15min and then freeze-dried to obtain the exosome lyophilized protective dressing.

[0122] Comparative Example 10

[0123] The difference from Example 3 is that no embedded exosomes were added in step S5, and step S3 in Example 3 was not performed.

[0124] Specifically as follows:

[0125] S1. Modification of extracellular matrix: 100g of 2mg / mL MXene nanosheet aqueous dispersion prepared in Preparation Example 1 and 100g of 1mg / mL graphene oxide aqueous dispersion were mixed evenly, 15g of extracellular matrix was added, impregnated for 1h, freeze-dried, reduced with hydrazine hydrate vapor for 9h, and pulverized to obtain 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 solution was added dropwise, 250g of PEG200 was added, and the mixture was stirred until homogeneous. Then, 30g of 30wt% hydrogen peroxide was added dropwise, and the mixture was stirred for 4h. The pH of the solution was adjusted to 11, and the solution was allowed to stand for 1h to precipitate. After centrifugation, washing, and drying, calcium peroxide particles were obtained. 11g of sodium alginate and 0.5g of Tween-85 were dissolved in 300mL of water, 5.5g of calcium peroxide particles were added, and the mixture was stirred until homogeneous. The mixture was then added to 500mL of fish oil, emulsified at 5000r / min for 15min, solidified at room temperature for 30min, centrifuged, washed, and dried to obtain calcium peroxide microspheres.

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

[0128] Test Example 1

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

[0130] (1) Bond strength. Using a syringe, the hydrogel was extruded into the middle of a pigskin strip 4 cm long and 2.5 cm wide, with the overlapping area of ​​the two strips being 1 cm × 2.5 cm, to obtain the test specimen. The test was conducted according to YY / T0729.1 Test method for bonding properties of tissue adhesives, Part 1: Overlap-shear tensile bearing strength.

[0131] (2) In vitro degradation performance. In this experiment, no hydrogel sample was prepared initially. A dry gel dressing weighing m0 was placed in an aqueous solution. The degradation test was conducted in an air bath shaker at 25℃ and a shaking speed of 100 rpm. Every so often, the sample was removed, freeze-dried, and weighed as m1. Then, a new degradation solution was applied. Complete degradation was considered achieved when (m0-m1 / m0) was greater than 0.99. The time to complete degradation 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 hydrogel were tested at room temperature using a CMT4102 universal testing machine. The specimen diameter was 5 mm, the length was 30 mm, and the tensile rate was 50 mm / min.

[0134] The results are shown in Table 1.

[0135] Table 1

[0136]

[0137] As shown in the table above, the products obtained in Examples 1-3 of this invention have good bonding strength and tensile strength, low cytotoxicity, and fast degradation rate.

[0138] Test Example 2

[0139] Male C57BL / 6J mice, after a week of acclimatization, were selected for modeling. The mice were fasted for 16 hours and their bedding was changed the day before modeling. The diabetic mice were then intraperitoneally injected with 1 wt% streptozotocin solution at a dose of 50 mg / kg, once daily for 5 consecutive days. On the third day after the streptozotocin injection, blood samples were collected via the tail vein to measure random blood glucose. A random blood glucose level >16.7 mmol / L, along with symptoms of polydipsia, polyphagia, polyuria, and weight loss, confirmed the successful establishment of the type 1 diabetic mouse model. Monitoring continued for one week until blood glucose levels stabilized, at which point the mice were included in the formal experiment. Mice were anesthetized by intraperitoneal injection of 1.25% aphthylazine (0.2 mL / 10 g). The dorsal skin of each group of mice was shaved, and the dorsal skin was disinfected with medical alcohol. Using sterilized ophthalmic surgical scissors and forceps, a 1 cm diameter section of skin and fascia was removed along the edge of the incision. After wound modeling, the animals were randomly divided into 14 groups of 10 each: model group, Examples 1-3 groups, and Comparative Examples 1-10 groups. The model group received no treatment. Examples 1-3 and Comparative Examples 1-10 groups had their corresponding prepared products applied to the wounds at a concentration of 0.2 g / cm³. 2 The wound was secured with 3M dressing film to prevent skin retraction. Day 0 was designated as the day the wound was created. Wound healing was recorded by photographs. Before each measurement, mice were anesthetized and secured with an intraperitoneal injection of 1.25% aphthylamine. Wounds were photographed on days 0, 6, and 10 post-injury. The wound healing rate (%) was 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] As can be seen from the table above, the products obtained in Examples 1-3 of the present invention have a good effect on 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 within the protection scope of the present invention.

Claims

1. A method for preparing an exosome freeze-dried protective dressing, characterized in that, The protective agent was mixed with exosomes and embedded in chitosan-sodium tripolyphosphate microcapsules to obtain embedded exosomes. The extracellular matrix was modified by reducing hydrazine hydrate with MXene nanosheets and graphene oxide to obtain modified extracellular matrix. The modified extracellular matrix was added to poly(α,β-aspartic acid hydrazine)-C16 copolymer-silver hydrogel with sodium alginate-embedded calcium peroxide microspheres and embedded exosomes, and then freeze-dried to obtain exosome freeze-dried protective dressing. The protective agent was composed of sucrose, trehalose, mannitol, poloxamer 188 and taurine.

2. The preparation method according to claim 1, characterized in that, Includes the following steps: S1. Preparation of the protective agent: Sucrose, trehalose, mannitol, poloxamer 188 and taurine are mixed evenly to prepare the protective agent; S2. Modification of extracellular matrix: Mix MXene nanosheet aqueous dispersion and graphene oxide aqueous dispersion evenly, add extracellular matrix, impregnate, freeze dry, reduce with hydrazine hydrate vapor, pulverize, and obtain modified extracellular matrix; S3. Encapsulating exosomes: Add exosomes and a protective agent to water, add sodium tripolyphosphate, stir and mix evenly to obtain solution A; dissolve chitosan in acid to obtain solution B; load solution A and solution B into syringes respectively, insert them into the FNP device, press the piston handles of the two syringes downwards in parallel to squeeze out the solution, freeze-dry the product to obtain encapsulated exosomes; S4. Preparation of calcium peroxide microspheres: Dissolve calcium salt in water, add ammonia, add polyethylene glycol, stir and mix evenly, add hydrogen peroxide, stir to react, adjust the pH of the solution, let it stand to precipitate, and obtain calcium peroxide particles; dissolve sodium alginate and emulsifier in water, add calcium peroxide particles, stir and mix evenly, add to fish oil, emulsify, solidify at room temperature, centrifuge, wash, and dry to obtain calcium peroxide microspheres; S5. Preparation of exosome lyophilized protective dressing: Poly(α,β-aspartic acid hydrazide)-C16 copolymer was added to phosphate buffer, modified extracellular matrix, embedded exosomes, calcium peroxide microspheres and sodium citrate were added, stirred and mixed evenly, silver nitrate was added, ultrasonically dispersed evenly, and freeze-dried to obtain exosome lyophilized 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, the graphene oxide aqueous dispersion, and the extracellular matrix is ​​100:100:12-17, the impregnation 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 HCl solution, stirred and dissolved, Ti3AlC2 is added, heated and etched, centrifuged, washed, added to water, ultrasonically dispersed evenly, centrifuged, the supernatant is collected, and diluted with water to a specific concentration to obtain 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, and the temperature for the heating etching is 35-45℃ for 45-53 h.

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 calcium salt, ammonia, 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 ammonia is 1-2 mol / L. The concentration of hydrogen peroxide is 25-35 wt%. The pH value of the solution is adjusted to 10.5-11.

5. The mass ratio of sodium alginate, emulsifier, and calcium peroxide particles is 10-12:0.3-0.7:4-7. 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 solution in step S5 is 7.2-7.6, and the mass ratio of the poly(α,β-aspartic acid 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 lyophilized exosome protective dressing prepared by the preparation method according to any one of claims 1-9.

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