Mesenchymal stem cell exosome composition for repairing wounds and method of preparing the same

Abstract

The present application relates to the technical field of biological medicine, in particular to a mesenchymal stem cell exosome composition for repairing wound and a preparation method thereof.The preparation raw material of the exosome composition comprises the following components in parts by weight: mesenchymal stem cell exosome 0.15-0.2 parts, modified hyaluronic acid 3-5 parts, modified dandelion extract 5-8 parts, and carboxymethyl chitosan 2-3 parts.The exosome composition prepared by the present application can effectively promote wound repair.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a mesenchymal stem cell exosome composition for wound repair and its preparation method. Background Technology

[0002] The skin, the largest organ in the human body, serves as a natural barrier against external pathogens. In cases of acute and chronic skin injuries, burns, hypertension, and severe diabetes, the integrity of the skin is compromised, affecting its barrier function, increasing the risk of infection, and causing pain and decreased tactile sensation. Therefore, wound repair is crucial for the body. Mesenchymal stem cells (MSCs) can differentiate and replace damaged cells in injured tissues, regulate inflammatory responses, promote angiogenesis, form a healthy granulation matrix, and promote skin cell proliferation and migration, thereby accelerating skin repair and regeneration. However, direct use of MSCs for treatment may carry risks such as inducing tumors and immune responses. MSC-derived exosomes can carry cytokines and growth factors, signaling lipids, mRNA, and regulatory miRNAs from MSCs. After transporting these contents to recipient cells, exosomes can regulate cellular state and behavior. MSC-derived exosomes have also been shown to possess regenerative properties, promoting angiogenesis, skin cell proliferation, and epithelial regeneration in damaged tissues, and inhibiting scar formation. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention proposes a mesenchymal stem cell exosome composition for wound repair and its preparation method.

[0004] This invention is achieved through the following technical solution: A mesenchymal stem cell exosome composition for wound repair, comprising the following components in parts by weight: 0.15-0.2 parts mesenchymal stem cell exosomes, 3-5 parts modified hyaluronic acid, 5-8 parts modified dandelion extract, and 2-3 parts carboxymethyl chitosan.

[0005] Furthermore, the preparation method of the modified hyaluronic acid includes the following steps: L1. Dissolve hyaluronic acid in deionized water, add EDC·HCl and NHS, both at a concentration of 50 mmol / L, adjust the pH to 5.5 with 1 mol / L HCl, stir at room temperature in the dark for 30 min, add L-cysteine ​​hydrochloride, adjust the pH to 4.75, stir at room temperature in the dark for 5 h; L2. After the reaction in step V1 was completed, the solution was dialyzed for 3 days in the dark with HCl solution at pH 5, HCl solution at pH 5 containing 1% w / v NaCl, and HCl solution at pH 5. The solution was then freeze-dried to obtain SH-hyaluronic acid. L3. Mix DMSO and deionized water at a volume ratio of 1:1 to obtain a DMSO aqueous solution. Add the SH-hyaluronic acid obtained in step L2 to the DMSO aqueous solution and stir until homogeneous. Add DCC and DMAP, and stir in a water bath at 35°C for 2 h to obtain a mixed solution. Dissolve curcumin in DMSO to obtain a curcumin solution, and add it to the mixed solution. Continue the reaction in a water bath at 35°C in the dark for 12 h. After the reaction is complete, dialyze in DMSO for 48 h, dialyze in ultrapure water for 72 h, and freeze dry to obtain modified hyaluronic acid.

[0006] Furthermore, in step L1, the mass concentration of the hyaluronic acid in deionized water is 4 mg / mL.

[0007] Further, in step L1, the mass ratio of hyaluronic acid to L-cysteine ​​hydrochloride is 1:2.

[0008] Furthermore, in step L3, the mass ratio of SH-hyaluronic acid, DCC, DMAP, and curcumin is 10:5:0.75:1.

[0009] Furthermore, in step L3, the mass concentration of the SH-hyaluronic acid in the DMSO aqueous solution is 2-5 mg / mL.

[0010] Furthermore, in step L3, the mass concentration of curcumin in DMSO is 2-3 mg / mL.

[0011] Furthermore, the preparation method of the modified dandelion polysaccharide includes the following steps: V1. Wash and dry dandelion leaves, pulverize them through an 80-100 mesh sieve to obtain dandelion powder. Add deionized water at a ratio of 1 g: 30 mL, heat at 90℃ for 2 h, filter and collect the filtrate. Add deionized water to the filter residue and repeat the above operation twice. Combine the filtrates from the three operations to obtain the extract. Concentrate the extract to 20% of its original volume by rotary evaporation. Add 4 times the volume of anhydrous ethanol and mix well. Place at 4℃ for 24 h, centrifuge at 8000 rpm for 10-15 min, reconstitute the precipitate with ultrapure water, and deproteinize it 5 times using the Sevage method. Remove the Sevage solution under reduced pressure, dialyze in ultrapure water for 3 days, and freeze-dry to obtain the dandelion extract. V2. Dissolve the dandelion extract obtained in step V1 in ultrapure water, add sodium periodate, stir at room temperature in the dark for 10-12 h, add ethylene glycol and stir for 30 min, dialyze in ultrapure water for 3 days, freeze dry to obtain oxidized dandelion extract; V3. Under a nitrogen atmosphere, caffeic acid and the oxidized dandelion extract obtained in step V2 were added to DMF and stirred at 30°C until homogeneous. EDC·HCl and DMAP were added, and the mixture was stirred at 30°C in the dark for 24 h. Four times the volume of ethanol was added, and the mixture was allowed to stand at 4°C for 12 h. The mixture was centrifuged at 8000 rpm for 10-15 min, and the precipitate was thoroughly washed with ethanol. The precipitate was dialyzed in distilled water for 3 days and then freeze-dried to obtain the modified dandelion extract.

[0012] Furthermore, in step V2, the mass concentration of the dandelion extract in ultrapure water is 20-30 mg / mL.

[0013] Furthermore, in step V2, the mass ratio of sodium periodate to dandelion extract is 1:3.

[0014] Furthermore, in step V2, the volume ratio of ethylene glycol to ultrapure water is 1:50.

[0015] Further, in step V3, the mass ratio of caffeic acid, oxidized dandelion extract, EDC·HCl and DMAP is 1:3:1.5:0.2.

[0016] Furthermore, in step V3, the caffeic acid concentration in DMF is 5 mg / mL.

[0017] Furthermore, the present invention also provides a method for preparing the aforementioned mesenchymal stem cell exosome composition for wound repair, comprising the following steps: S1. Umbilical cord mesenchymal stem cells were prepared by isolating umbilical cord tissue and cultured in DMEM medium containing 10% fetal bovine serum and 100 U / mL penicillin-streptomycin in a 5% CO2 incubator at 37°C, with the medium changed every 2-3 days. S2. When the cells reach 70% confluence, digest them with trypsin and passage them. When the confluence of the fourth generation cells reaches 80%, discard the culture medium, wash the cells with PBS, replace with serum-free culture medium and continue to culture for 48 h. Collect the supernatant, centrifuge at 300 g for 10 min at 4 °C, centrifuge the supernatant at 2000 g for 20 min at 4 °C, centrifuge the supernatant at 10000 g for 30 min at 4 °C, centrifuge the supernatant at 100000 g for 90 min at 4 °C, remove the supernatant and collect the precipitate. S3. Take the precipitate obtained in step S2, resuspend it in PBS buffer, centrifuge at 4℃ and 100000 g for 90 min, remove the supernatant, the precipitate is mesenchymal stem cell exosomes, resuspend it in PBS buffer to a concentration of 2 mg / mL, and obtain mesenchymal stem cell exosome solution. S4. Add the modified dandelion extract to PBS buffer at pH 8.5 and mix well. Add modified hyaluronic acid and stir for 1 h. Add carboxymethyl chitosan and adjust the pH to 7.4. Stir for 2 h. Add the mesenchymal stem cell exosome solution obtained in step S3 and continue stirring for 1 h. Let stand at 4℃ for 8-12 h to obtain the mesenchymal stem cell exosome composition for wound repair.

[0018] Further, in step S4, the mass concentration of the modified dandelion extract in PBS buffer is 20 mg / mL.

[0019] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a mesenchymal stem cell exosome composition for wound repair. Through chemical modification of hyaluronic acid and dandelion extract, the composition is endowed with excellent antibacterial and wound-healing activities. This invention prepares modified hyaluronic acid, which possesses basic moisturizing and cell migration-promoting effects. First, through an EDC / NHS-catalyzed amidation reaction, L-cysteine ​​hydrochloride is grafted onto the hyaluronic acid molecular chain, introducing thiol groups to prepare SH-hyaluronic acid, providing a basis for subsequent cross-linking. Then, through a DCC / DMAP-catalyzed esterification reaction, curcumin is covalently grafted onto the hyaluronic acid molecular chain, improving the water solubility of curcumin. Curcumin is a natural antibacterial agent, enhancing the anti-inflammatory, antibacterial, and antioxidant activities of hyaluronic acid. This invention also extracts polysaccharides from dandelion to prepare dandelion extract, which has antibacterial activity. Through a sodium periodate oxidation reaction, the ortho-dihydroxy groups in the dandelion polysaccharide molecules are oxidized to active aldehyde groups to prepare oxidized dandelion extract. The introduced aldehyde groups provide reaction sites for subsequent cross-linking. This invention utilizes EDC / DMAP catalysis to covalently graft caffeic acid onto the molecular chain of oxidized dandelion extract, yielding a modified dandelion extract. This modified extract synergistically enhances the natural activity of the dandelion extract, improving the anti-inflammatory, antibacterial, and antioxidant capabilities of the composition. The introduction of catechol groups from caffeic acid into the dandelion extract provides a foundation for subsequent cross-linking. The aldehyde groups introduced after oxidation of the modified dandelion extract can undergo a Schiff base reaction with the amino groups of carboxymethyl chitosan, forming a dynamic chemical cross-linking network. The thiol groups of modified hyaluronic acid react with the catechol groups on the modified dandelion extract, resulting in cross-linking. The cross-linking of modified hyaluronic acid, modified dandelion extract, and carboxymethyl chitosan in the composition of this invention forms a uniform, porous three-dimensional network structure, providing ample loading space for mesenchymal stem cell exosomes. The three-dimensional network structure encapsulates exosomes within the gel network, preventing them from being enzymatically degraded or rapidly cleared in the wound microenvironment, thus prolonging their local retention time. Simultaneously, the slow degradation of the network enables long-term sustained release of exosomes, enhancing their bioavailability. Dynamic Schiff base bonds endow the hydrogel with excellent self-healing properties and mechanical flexibility, adapting to the physiological deformation of the wound and preventing detachment. The network structure also possesses good air permeability and water retention, providing a physical barrier to the wound, isolating it from external pathogens, and maintaining a moist wound environment. Modified hyaluronic acid and carboxymethyl chitosan not only serve as carriers but also possess bioactivity that promotes cell adhesion, proliferation, and migration. Attached Figure Description

[0020] Figure 1 This demonstrates the antibacterial activity of the compositions described in Examples 1-3 and Comparative Examples 1-4 of the present invention. Figure 2 This demonstrates the wound repair effects of the compositions described in Examples 1-3 and Comparative Examples 1-4 of the present invention. Figure 3This is a scanning electron microscope image of the product described in Experimental Example 3 of the present invention. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. However, this invention is not limited to the following embodiments. It should be noted that, unless otherwise specified, all chemical reagents involved in this invention are purchased through commercial channels.

[0022] The main materials and reagents used in the embodiments of this invention were sourced from the following sources: Hyaluronic acid: Shandong Focus Freda Biotechnology Co., Ltd.; Carboxymethyl chitosan and caffeic acid: Shanghai Maclean Biotechnology Co., Ltd. L-cysteine ​​hydrochloride: Sigma-Aldrich; EDC·HCl, NHS, DMAP, DCC, Curcumin, Sodium Periodate: Shanghai Aladdin Biochemical Technology Co., Ltd.

[0023] Example 1: A mesenchymal stem cell exosome composition for wound repair, the raw materials of which include the following components in parts by weight: 0.2 parts mesenchymal stem cell exosomes, 5 parts modified hyaluronic acid, 8 parts modified dandelion extract, and 3 parts carboxymethyl chitosan.

[0024] The preparation method of modified hyaluronic acid includes the following steps: L1. Dissolve 2 g of hyaluronic acid in 500 mL of deionized water, add EDC·HCl and NHS, both at a concentration of 50 mmol / L, adjust the pH to 5.5 with 1 mol / L HCl, stir at room temperature in the dark for 30 min, add 4 g of L-cysteine ​​hydrochloride, adjust the pH to 4.75, stir at room temperature in the dark for 5 h. L2. After the reaction in step V1 was completed, the solution was dialyzed for 3 days in the dark with HCl solution at pH 5, HCl solution at pH 5 containing 1% w / v NaCl, and HCl solution at pH 5. The solution was then freeze-dried to obtain SH-hyaluronic acid. L3. Mix DMSO and deionized water at a volume ratio of 1:1 to obtain a DMSO aqueous solution. Add 1.2 g of SH-hyaluronic acid obtained in step L2 to 240 mL of DMSO aqueous solution and stir well. Add 0.6 g of DCC and 0.09 g of DMAP, and stir in a water bath at 35°C for 2 h to obtain a mixed solution. Dissolve 0.12 g of curcumin in 40 mL of DMSO to obtain a curcumin solution, add it to the mixed solution, and continue the reaction in a water bath at 35°C in the dark for 12 h. After the reaction is completed, dialyze in DMSO for 48 h, dialyze in ultrapure water for 72 h, and freeze dry to obtain modified hyaluronic acid.

[0025] The preparation method of modified dandelion polysaccharide includes the following steps: V1. After washing and drying dandelion leaves, pulverize them through a 100-mesh sieve to obtain dandelion powder. Add deionized water at a ratio of 1 g: 30 mL, heat at 90℃ for 2 h, filter and collect the filtrate. Add deionized water to the filter residue and repeat the above operation twice. Combine the filtrates from the three operations to obtain the extract. Concentrate the extract to 20% of its original volume by rotary evaporation. Add 4 times the volume of anhydrous ethanol and mix well. Place at 4℃ for 24 h, centrifuge at 8000 rpm for 15 min, reconstitute the precipitate with ultrapure water, and deproteinize it 5 times using the Sevage method. Remove the Sevage solution under reduced pressure, dialyze in ultrapure water for 3 days, and freeze-dry to obtain the dandelion extract. V2. Dissolve 3 g of dandelion extract obtained in step V1 in 100 mL of ultrapure water, add 1 g of sodium periodate, stir at room temperature in the dark for 12 h, add 2 mL of ethylene glycol and stir for 30 min, dialyze in ultrapure water for 3 days, freeze dry to obtain oxidized dandelion extract. V3. Under a nitrogen atmosphere, 1 g of caffeic acid and 3 g of oxidized dandelion extract obtained in step V2 were added to 200 mL of DMF and stirred evenly at 30 °C. 1.5 g of EDC·HCl and 0.2 g of DMAP were added, and the mixture was stirred at 30 °C in the dark for 24 h. Four times the volume of ethanol was added, and the mixture was allowed to stand at 4 °C for 12 h. After centrifugation at 8000 rpm for 15 min, the precipitate was thoroughly washed with ethanol, dialyzed in distilled water for 3 days, and freeze-dried to obtain the modified dandelion extract.

[0026] This embodiment also provides a method for preparing the mesenchymal stem cell exosome composition for wound repair, including the following steps: S1. Umbilical cord mesenchymal stem cells were prepared by isolating umbilical cord tissue and cultured in DMEM medium containing 10% fetal bovine serum and 100 U / mL penicillin-streptomycin in a 5% CO2 incubator at 37°C, with the medium changed every 3 days. S2. When the cells reach 70% confluence, digest them with trypsin and passage them. When the confluence of the fourth generation cells reaches 80%, discard the culture medium, wash the cells with PBS, replace with serum-free culture medium and continue to culture for 48 h. Collect the supernatant, centrifuge at 300 g for 10 min at 4 °C, centrifuge the supernatant at 2000 g for 20 min at 4 °C, centrifuge the supernatant at 10000 g for 30 min at 4 °C, centrifuge the supernatant at 100000 g for 90 min at 4 °C, remove the supernatant and collect the precipitate. S3. Take the precipitate obtained in step S2, resuspend it in PBS buffer, centrifuge at 4℃ and 100000 g for 90 min, remove the supernatant, the precipitate is mesenchymal stem cell exosomes, resuspend it in PBS buffer to a concentration of 2 mg / mL, and obtain mesenchymal stem cell exosome solution. S4. Add 8 g of modified dandelion extract to 400 mL of PBS buffer at pH 8.5 and mix well. Add 5 g of modified hyaluronic acid and stir for 1 h. Add 3 g of carboxymethyl chitosan and adjust the pH to 7.4. Stir for 2 h. Add 100 mL of the mesenchymal stem cell exosome solution obtained in step S3 and continue stirring for 1 h. Let stand at 4℃ for 8-12 h to obtain the mesenchymal stem cell exosome composition for wound repair.

[0027] Example 2: A mesenchymal stem cell exosome composition for wound repair, the raw materials of which include the following components in parts by weight: 0.15 parts mesenchymal stem cell exosomes, 3 parts modified hyaluronic acid, 5 parts modified dandelion extract, and 2 parts carboxymethyl chitosan.

[0028] The preparation method of modified hyaluronic acid includes the following steps: L1. Dissolve 2 g of hyaluronic acid in 500 mL of deionized water, add EDC·HCl and NHS, both at a concentration of 50 mmol / L, adjust the pH to 5.5 with 1 mol / L HCl, stir at room temperature in the dark for 30 min, add 4 g of L-cysteine ​​hydrochloride, adjust the pH to 4.75, stir at room temperature in the dark for 5 h. L2. After the reaction in step V1 was completed, the solution was dialyzed for 3 days in the dark with HCl solution at pH 5, HCl solution at pH 5 containing 1% w / v NaCl, and HCl solution at pH 5. The solution was then freeze-dried to obtain SH-hyaluronic acid. L3. Mix DMSO and deionized water at a volume ratio of 1:1 to obtain a DMSO aqueous solution. Take 1 g of SH-hyaluronic acid obtained in step L2 and add it to 500 mL of DMSO aqueous solution and stir well. Add 0.5 g of DCC and 0.075 g of DMAP and stir in a water bath at 35℃ for 2 h to obtain a mixed solution. Dissolve 0.1 g of curcumin in 50 mL of DMSO to obtain a curcumin solution and add it to the mixed solution. Continue to react in a water bath at 35℃ in the dark for 12 h. After the reaction is completed, dialyze in DMSO for 48 h and dialyze in ultrapure water for 72 h. Freeze dry to obtain modified hyaluronic acid.

[0029] The preparation method of modified dandelion polysaccharide includes the following steps: V1. After washing and drying dandelion leaves, pulverize them through an 80-mesh sieve to obtain dandelion powder. Add deionized water at a ratio of 1 g: 30 mL, heat at 90℃ for 2 h, filter and collect the filtrate. Add deionized water to the filter residue and repeat the above operation twice. Combine the filtrates from the three operations to obtain the extract. Concentrate the extract to 20% of its original volume by rotary evaporation. Add 4 times the volume of anhydrous ethanol and mix well. Place at 4℃ for 24 h, centrifuge at 8000 rpm for 10 min, reconstitute the precipitate with ultrapure water, and deproteinize it 5 times using the Sevage method. Remove the Sevage solution under reduced pressure, dialyze in ultrapure water for 3 days, and freeze-dry to obtain the dandelion extract. V2. Dissolve 3 g of dandelion extract obtained in step V1 in 150 mL of ultrapure water, add 1 g of sodium periodate, stir at room temperature in the dark for 10 h, add 3 mL of ethylene glycol and stir for 30 min, dialyze in ultrapure water for 3 days, freeze dry to obtain oxidized dandelion extract. V3. Under a nitrogen atmosphere, 1 g of caffeic acid and 3 g of oxidized dandelion extract obtained in step V2 were added to 200 mL of DMF and stirred evenly at 30 °C. 1.5 g of EDC·HCl and 0.2 g of DMAP were added, and the mixture was stirred at 30 °C in the dark for 24 h. Four times the volume of ethanol was added, and the mixture was allowed to stand at 4 °C for 12 h. The mixture was centrifuged at 8000 rpm for 10 min, and the precipitate was thoroughly washed with ethanol. The precipitate was dialyzed in distilled water for 3 days and then freeze-dried to obtain the modified dandelion extract.

[0030] This embodiment also provides a method for preparing the mesenchymal stem cell exosome composition for wound repair, including the following steps: S1. Umbilical cord mesenchymal stem cells were prepared by isolating umbilical cord tissue and cultured in DMEM medium containing 10% fetal bovine serum and 100 U / mL penicillin-streptomycin in a 5% CO2 incubator at 37°C, with the medium changed every 2 days. S2. When the cells reach 70% confluence, digest them with trypsin and passage them. When the confluence of the fourth generation cells reaches 80%, discard the culture medium, wash the cells with PBS, replace with serum-free culture medium and continue to culture for 48 h. Collect the supernatant, centrifuge at 300 g for 10 min at 4 °C, centrifuge the supernatant at 2000 g for 20 min at 4 °C, centrifuge the supernatant at 10000 g for 30 min at 4 °C, centrifuge the supernatant at 100000 g for 90 min at 4 °C, remove the supernatant and collect the precipitate. S3. Take the precipitate obtained in step S2, resuspend it in PBS buffer, centrifuge at 4℃ and 100000 g for 90 min, remove the supernatant, the precipitate is mesenchymal stem cell exosomes, resuspend it in PBS buffer to a concentration of 2 mg / mL, and obtain mesenchymal stem cell exosome solution. S4. Add 5 g of modified dandelion extract to 250 mL of PBS buffer at pH 8.5 and mix well. Add 3 g of modified hyaluronic acid and stir for 1 h. Add 2 g of carboxymethyl chitosan and adjust the pH to 7.4. Stir for 2 h. Add 75 mL of the mesenchymal stem cell exosome solution obtained in step S3 and continue stirring for 1 h. Let stand at 4℃ for 8 h to obtain the mesenchymal stem cell exosome composition for wound repair.

[0031] Example 3: A mesenchymal stem cell exosome composition for wound repair, the raw materials of which include the following components in parts by weight: 0.18 parts mesenchymal stem cell exosomes, 4 parts modified hyaluronic acid, 6 parts modified dandelion extract, and 2.5 parts carboxymethyl chitosan.

[0032] The preparation method of modified hyaluronic acid includes the following steps: L1. Dissolve 2 g of hyaluronic acid in 500 mL of deionized water, add EDC·HCl and NHS, both at a concentration of 50 mmol / L, adjust the pH to 5.5 with 1 mol / L HCl, stir at room temperature in the dark for 30 min, add 4 g of L-cysteine ​​hydrochloride, adjust the pH to 4.75, stir at room temperature in the dark for 5 h. L2. After the reaction in step V1 was completed, the solution was dialyzed for 3 days in the dark with HCl solution at pH 5, HCl solution at pH 5 containing 1% w / v NaCl, and HCl solution at pH 5. The solution was then freeze-dried to obtain SH-hyaluronic acid. L3. Mix DMSO and deionized water at a volume ratio of 1:1 to obtain a DMSO aqueous solution. Take 1 g of SH-hyaluronic acid obtained in step L2 and add it to 250 mL of DMSO aqueous solution. Stir well. Add 0.5 g of DCC and 0.075 g of DMAP. Stir in a water bath at 35℃ for 2 h to obtain a mixed solution. Dissolve 0.1 g of curcumin in 40 mL of DMSO to obtain a curcumin solution. Add it to the mixed solution and continue the reaction in a water bath at 35℃ in the dark for 12 h. After the reaction is completed, dialyze in DMSO for 48 h and dialyze in ultrapure water for 72 h. Freeze dry to obtain modified hyaluronic acid.

[0033] The preparation method of modified dandelion polysaccharide includes the following steps: V1. After washing and drying dandelion leaves, pulverize them through a 90-mesh sieve to obtain dandelion powder. Add deionized water at a ratio of 1 g: 30 mL, heat at 90℃ for 2 h, filter and collect the filtrate. Add deionized water to the filter residue and repeat the above operation twice. Combine the filtrates from the three operations to obtain the extract. Concentrate the extract to 20% of its original volume by rotary evaporation. Add 4 times the volume of anhydrous ethanol and mix well. Place at 4℃ for 24 h, centrifuge at 8000 rpm for 12 min, reconstitute the precipitate with ultrapure water, and deproteinize it 5 times using the Sevage method. Remove the Sevage solution under reduced pressure, dialyze in ultrapure water for 3 days, and freeze-dry to obtain the dandelion extract. V2. Dissolve 3 g of dandelion extract obtained in step V1 in 120 mL of ultrapure water, add 1 g of sodium periodate, stir at room temperature in the dark for 11 h, add 2.4 mL of ethylene glycol and stir for 30 min, dialyze in ultrapure water for 3 days, freeze dry to obtain oxidized dandelion extract. V3. Under a nitrogen atmosphere, 1 g of caffeic acid and 3 g of oxidized dandelion extract obtained in step V2 were added to 200 mL of DMF and stirred evenly at 30 °C. 1.5 g of EDC·HCl and 0.2 g of DMAP were added, and the mixture was stirred at 30 °C in the dark for 24 h. Four times the volume of ethanol was added, and the mixture was allowed to stand at 4 °C for 12 h. After centrifugation at 8000 rpm for 12 min, the precipitate was thoroughly washed with ethanol, dialyzed in distilled water for 3 days, and then freeze-dried to obtain the modified dandelion extract.

[0034] This embodiment also provides a method for preparing the mesenchymal stem cell exosome composition for wound repair, including the following steps: S1. Umbilical cord mesenchymal stem cells were prepared by isolating umbilical cord tissue and cultured in DMEM medium containing 10% fetal bovine serum and 100 U / mL penicillin-streptomycin in a 5% CO2 incubator at 37°C, with the medium changed every 2-3 days. S2. When the cells reach 70% confluence, digest them with trypsin and passage them. When the confluence of the fourth generation cells reaches 80%, discard the culture medium, wash the cells with PBS, replace with serum-free culture medium and continue to culture for 48 h. Collect the supernatant, centrifuge at 300 g for 10 min at 4 °C, centrifuge the supernatant at 2000 g for 20 min at 4 °C, centrifuge the supernatant at 10000 g for 30 min at 4 °C, centrifuge the supernatant at 100000 g for 90 min at 4 °C, remove the supernatant and collect the precipitate. S3. Take the precipitate obtained in step S2, resuspend it in PBS buffer, centrifuge at 4℃ and 100000 g for 90 min, remove the supernatant, the precipitate is mesenchymal stem cell exosomes, resuspend it in PBS buffer to a concentration of 2 mg / mL, and obtain mesenchymal stem cell exosome solution. S4. Add 6 g of modified dandelion extract to 300 mL of PBS buffer at pH 8.5 and mix well. Add 4 g of modified hyaluronic acid and stir for 1 h. Add 2.5 g of carboxymethyl chitosan and adjust the pH to 7.4. Stir for 2 h. Add 90 mL of the mesenchymal stem cell exosome solution obtained in step S3 and continue stirring for 1 h. Let stand at 4℃ for 10 h to obtain the mesenchymal stem cell exosome composition for wound repair.

[0035] The only difference between Comparative Example 1 and Example 1 is that hyaluronic acid is used instead of modified hyaluronic acid.

[0036] The only difference between Comparative Example 2 and Example 1 is that dandelion extract was used instead of modified dandelion extract.

[0037] The only difference between Comparative Example 3 and Example 1 is that SH-hyaluronic acid is used instead of modified hyaluronic acid.

[0038] The only difference between Comparative Example 4 and Example 1 is that oxidized dandelion extract was used instead of modified dandelion extract.

[0039] Experimental Example 1: Resuscitated Staphylococcus aureus was diluted with LB medium to a concentration of 5 × 10⁻⁶. 6 CFU / mL was used to obtain a bacterial suspension. The compositions prepared in Examples 1-3 and Comparative Examples 1-4 were sterilized under UV light and added to the bacterial suspension at a ratio of 50 mg / mL as the experimental group. A pure bacterial suspension was used as the control group. The samples were incubated at 37°C for 12 h. The bacterial suspension was diluted and spread onto agar medium, incubated overnight at 37°C, and the colonies were counted. The inhibition rate was calculated as follows: Inhibition rate (%) = [(Control group colony count - Experimental group colony count) / Control group colony count] × 100%. Results are as follows... Figure 1 As shown.

[0040] Figure 1 The results showed that the antibacterial rate of Examples 1-3 was significantly better than that of Comparative Examples 1-4. In Comparative Example 1, the use of hyaluronic acid instead of modified hyaluronic acid resulted in a decrease in antibacterial activity; in Comparative Example 2, the lack of modification of dandelion polysaccharide led to a decrease in antibacterial activity; in Comparative Example 3, SH-hyaluronic acid was used instead of modified hyaluronic acid without curcumin grafting; and in Comparative Example 4, oxidized dandelion extract was used instead of modified dandelion extract without caffeic acid grafting, resulting in a decrease in antibacterial activity. These results indicate that the exosome composition prepared according to the present invention has a good antibacterial effect and can inhibit wound infection.

[0041] Experimental Example 2: SPF-grade female mice were randomly divided into 5 groups of 12 mice each. Mice were anesthetized by intraperitoneal injection of 4% chloral hydrate. The fur on the backs of the mice was shaved, and after disinfection with alcohol, a circular wound with a diameter of 1 cm was created on the back. The exosome composition prepared in Example 1 and Comparative Examples 1-4 of this invention was applied to the wound surface to a thickness of 1 mm and secured with medical tape, which was changed every 3 days. Wound healing was observed on postoperative days 3, 7, 10, and 15, and the wound area was measured to calculate the wound healing rate. Results are as follows: Figure 2 As shown.

[0042] Figure 2 The results showed that the wound healing rate of Example 1 was significantly better than that of Comparative Examples 1-4. In Comparative Example 1, the wound healing rate decreased because hyaluronic acid was used instead of modified hyaluronic acid; in Comparative Example 2, the wound healing rate decreased because dandelion polysaccharide was not modified; in Comparative Example 3, the wound healing rate decreased because SH-hyaluronic acid was used instead of modified hyaluronic acid and curcumin was not grafted; and in Comparative Example 4, the wound healing rate decreased because oxidized dandelion extract was used instead of modified dandelion extract and caffeic acid was not grafted. These results indicate that the exosome composition prepared by this invention can effectively inhibit bacteria, has good moisturizing effects, and promotes wound healing.

[0043] Experimental Example 3: Following the method in Example 1, modified dandelion extract was added to PBS buffer at pH 8.5 and mixed well. Modified hyaluronic acid was added, and the mixture was stirred for 1 h. 3 g of carboxymethyl chitosan was added, the pH was adjusted to 7.4, and the mixture was stirred for 2 h. PBS buffer without mesenchymal stem cell exosomes was added, and the mixture was stirred for another 1 h. The mixture was then incubated at 4°C for 12 h. The resulting product was freeze-dried, observed and photographed using a scanning electron microscope. The results are as follows: Figure 3 As shown.

[0044] Figure 3 The results showed that the modified dandelion extract, modified hyaluronic acid, and carboxymethyl chitosan of the present invention cross-linked to form a three-dimensional network structure with uniform structure and rich porosity, which can provide sufficient space for the loading of exosomes and improve the wound repair effect.

[0045] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of the invention is limited to these examples; within the framework of the invention, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity.

Claims

1. A mesenchymal stem cell exosome composition for wound repair, characterized in that, The raw materials for preparation include the following components in parts by weight: 0.15-0.2 parts of mesenchymal stem cell exosomes, 3-5 parts of modified hyaluronic acid, 5-8 parts of modified dandelion extract, and 2-3 parts of carboxymethyl chitosan; The preparation method of modified hyaluronic acid includes the following steps: L1. Dissolve hyaluronic acid in deionized water, add EDC·HCl and NHS, adjust the pH, stir, add L-cysteine ​​hydrochloride, adjust the pH, and stir. L2. After the reaction in step V1 is completed, dialyze and freeze-dry to obtain SH-hyaluronic acid; L3. Take the SH-hyaluronic acid obtained in step L2 and add it to DMSO aqueous solution and stir until homogeneous. Add DCC and DMAP, stir, and obtain a mixture. Dissolve curcumin in DMSO to obtain curcumin solution, add it to the mixture, react, dialyze, freeze dry, and obtain modified hyaluronic acid. The preparation method of modified dandelion polysaccharide includes the following steps: V1. After washing and drying the dandelion leaves, pulverize them to obtain dandelion powder. Add deionized water, heat to extract, concentrate by rotary evaporation, add anhydrous ethanol, precipitate and deproteinize using the Sevage method, dialyze, freeze dry to obtain dandelion extract. V2. Dissolve the dandelion extract obtained in step V1 in ultrapure water, add sodium periodate, stir, add ethylene glycol, dialyze, freeze dry, and obtain oxidized dandelion extract; V3. Add caffeic acid and the oxidized dandelion extract obtained in step V2 to DMF, stir, add EDC·HCl and DMAP, stir, add ethanol, precipitate and wash, dialyze, freeze dry to obtain modified dandelion extract.

2. The mesenchymal stem cell exosome composition for wound repair according to claim 1, characterized in that, In step L1, the mass ratio of hyaluronic acid to L-cysteine ​​hydrochloride is 1:

2.

3. The mesenchymal stem cell exosome composition for wound repair according to claim 2, characterized in that, In step L3, the mass ratio of SH-hyaluronic acid, DCC, DMAP and curcumin is 10:5:0.75:

1.

4. The mesenchymal stem cell exosome composition for wound repair according to claim 3, characterized in that, In step V2, the mass ratio of sodium periodate to dandelion extract is 1:

3.

5. The mesenchymal stem cell exosome composition for wound repair according to claim 4, characterized in that, In step V3, the mass ratio of caffeic acid, oxidized dandelion extract, EDC·HCl and DMAP is 1:3:1.5:0.

2.

6. The method for preparing the mesenchymal stem cell exosome composition for wound repair according to any one of claims 1-5, characterized in that, Includes the following steps: S1. Umbilical cord mesenchymal stem cells were prepared by isolating umbilical cord tissue and cultured. S2. Cell conveyor belt: After the confluence of the 4th generation cells reaches 80%, the culture medium is discarded, replaced with serum-free culture medium and cultured for a longer period. The supernatant is collected and centrifuged multiple times, and finally the precipitate is collected. S3. Resuspend the precipitate obtained in step S2, centrifuge, and resuspend the precipitate in PBS buffer to obtain a mesenchymal stem cell exosome solution; S4. Add the modified dandelion extract to PBS buffer at pH 8.5 and mix well. Add the modified hyaluronic acid and stir. Add carboxymethyl chitosan and adjust the pH to 7.

4. Stir. Add the mesenchymal stem cell exosome solution obtained in step S3. Stir and let stand to obtain a mesenchymal stem cell exosome composition for wound repair.

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