Injectable temperature-sensitive hydrogel loaded with traditional Chinese medicine active exosome

Abstract

The invention relates to injectable temperature-sensitive hydrogel loaded with traditional Chinese medicine active exosomes, and belongs to the technical field of clinical treatment of diabetic foot refractory wounds, and the injectable temperature-sensitive hydrogel is obtained by poloxamer 407 hydrogel and a hyaluronic acid loaded composite carrier; the composite carrier body is formed by coating a traditional Chinese medicine active exosome with a coating solution; the traditional Chinese medicine active exosome is obtained by inducing adipose-derived mesenchymal stem cells with a modified astragalus extract. The injectable temperature-sensitive hydrogel is obtained by loading a composite carrier with poloxamer 407 hydrogel and hyaluronic acid, the poloxamer 407 and the hyaluronic acid quickly form a physical barrier on a wound surface after being injected, a moist healing environment is provided, and continuous and slow release of exosome and active ingredients is realized by means of the temperature-sensitive characteristic and the network structure of the poloxamer 407 and the hyaluronic acid, so that the wound healing effect is improved. And over-high local concentration or premature loss caused by sudden release of the medicine is avoided.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of clinical treatment of diabetic foot refractory wounds, and particularly relates to an injectable temperature-sensitive hydrogel loaded with traditional Chinese medicine active exosomes. BACKGROUND

[0002] Diabetic foot is a disease in which blood vessels and nerves in the foot of a diabetic patient distal to the ankle joint are diseased, leading to insufficient blood supply to the foot, and symptoms such as dry skin, muscle atrophy, and reduced sensation. In the clinical treatment of diabetic foot refractory wounds, injectable temperature-sensitive hydrogels, as an advanced local drug delivery carrier, are widely used. The core mechanism is to load therapeutic drugs such as growth factors, exosomes, or anti-inflammatory components, and to deliver them precisely to the wound site by injection. Under the action of body temperature, the hydrogel rapidly gels to form a local "drug reservoir".

[0003] However, when traditional temperature-sensitive hydrogels are loaded with drugs, the structure of the carrier material is often single, and there is a lack of stable binding between the drug and the matrix, leading to a burst release of the drug in the early stage of gel formation. This can cause a high local drug concentration in a short period of time, followed by a rapid decline, which cannot maintain a long-term effective therapeutic concentration on the wound site. Since the healing period of diabetic foot is long, the sustained anti-inflammatory, pro-angiogenic, and tissue regeneration-promoting abilities are severely limited, ultimately affecting the overall efficacy and possibly prolonging the healing time. SUMMARY

[0004] The present application provides an injectable temperature-sensitive hydrogel loaded with traditional Chinese medicine active exosomes.

[0005] The present application achieves the above-mentioned objectives through the following technical solutions: The present application provides an injectable temperature-sensitive hydrogel loaded with traditional Chinese medicine active exosomes. The injectable temperature-sensitive hydrogel is obtained from a poloxamer 407 hydrogel and a hyaluronic acid-loaded composite carrier. The composite carrier is formed by coating the traditional Chinese medicine active exosomes with a coating solution. The traditional Chinese medicine active exosomes are obtained by modifying the extract of Astragalus membranaceus after induction by the extract.

[0006] As a further optimization of the present application, the preparation process of the modified Astragalus membranaceus extract is as follows: (1) Add 40-80 mL of anhydrous pyridine to a dry three-necked flask, and stir at 300 rpm in a 0-4°C water bath. Add 13-27 mL of citric acid at a rate of 0.1-0.2 mL / min. After the addition is complete, continue stirring at 120 rpm in a 0-4°C water bath for 5 min. Add 10 g of Astragalus polysaccharide in batches and stir at 60 rpm until the addition is complete. After the end of the addition, perform constant temperature stirring at 30-40°C for 1-2 h to obtain a reaction solution. (ii) cooling the reaction solution to 3-6°C, adding 15-25 mL of 65% sodium hydroxide aqueous solution to the reaction solution until the pH is 7.5, then adding 150-250 mL of 75% ethanol aqueous solution to precipitate for 20-28 h, collecting the precipitate by suction filtration, and dialyzing against distilled water for 3-4 times to obtain the modified astragalus extract.

[0007] As a further optimization scheme of the present application, in the preparation process of the modified astragalus extract, the astragalus polysaccharide is added in five batches, 2 g per batch, and the interval between each batch is 2 min; and the volume of distilled water for dialysis is 1000 ml per round, and the molecular weight cut-off of the dialysis bag is 3500 Da.

[0008] As a further optimization scheme of the present application, the preparation process of the traditional Chinese medicine active exosome is as follows: (i) taking 150-180 mL of α-MEM medium, adding 65-75 μg of recombinant human fibroblast growth factor and 25-30 μg of recombinant human epidermal growth factor, and 1.5-2.0 mL of penicillin-streptomycin double antibody, uniformly sterilizing with a 0.22 μm filter membrane, obtaining a stem cell culture medium, and storing at 4°C in the dark for standby use; (ii) the adipose mesenchymal stem cells are inoculated in T75 culture bottles at a concentration of 3×10 6 cells / vial, and 20 mL of the stem cell culture medium is added to the T75 culture bottle, which is cultured in a 37°C, 5% CO2 saturated humidity incubator, and the culture medium is replaced every 2 days until the cell confluence reaches 70%-80%, obtaining the cultured cells; (iii) the cultured cells are washed with 10 mL of sterile PBS buffer for 2-3 times, 15 mL of modified astragalus extract aqueous solution with a concentration of 2 mg / mL is added, and the cells are placed back in a 37°C, 5% CO2 incubator for 48-72 h of infiltration induction, after which the supernatant A is aspirated; the supernatant A is centrifuged at 300×g for 10 min, and the bottom cell precipitate is discarded, and the supernatant B is aspirated; the supernatant B is centrifuged at 10000×g for 30 min at 4°C, and the supernatant C is aspirated; (iv) an exosome enrichment agent is added to the supernatant C, which is gently inverted and mixed, and is placed at 3-5°C for 8-12 h, and is centrifuged at 12000×g for 25-35 min, and the supernatant is discarded to obtain a precipitate, 100-200 μL of sterile PBS buffer is added to the precipitate, and the mixture is blown and beaten for 10-15 times to obtain a crude exosome solution; the crude exosome solution is filtered through a 0.1 μm polycarbonate filter membrane to obtain the traditional Chinese medicine active exosome.

[0009] As a further optimization scheme of the present application, the volume ratio of the supernatant C to the exosome enrichment agent is 5:1.

[0010] As a further optimization scheme of the present application, the preparation raw materials of the coating liquid include, in percentage: 2-5% sodium alginate, 1-3% chitosan, 20-35% deproteinized calf blood extract, and 65-80% deionized water.

[0011] As a further optimization scheme of the present application, the preparation process of the coating liquid is as follows: (I) chitosan is dropped into sodium alginate under 0-4℃ water bath with a stirring speed of 300 rpm, deionized water is added, and stirring is carried out for 15-30 min to obtain a compound; (II) deproteinized calf blood extract is added into the compound, stirring is carried out in a 0-4℃ water bath for 10-20 min to obtain a coating liquid.

[0012] As a further optimization scheme of the present application, the preparation process of the injectable temperature-sensitive hydrogel is as follows: (I) active traditional Chinese medicine exosomes are taken and added into a sterile rolling coater, the rotating speed is set to 20-40 r / min, the temperature in the cavity is set to 25℃, a spray gun with a spray droplet diameter of 30-50 μm is used to spray the coating liquid into the coater, and the coating liquid is uniformly coated on the exosomes for 12-15 min to obtain a compound carrier; (II) the compound carrier is added into poloxamer 407 hydrogel and stirred and mixed, the stirring speed is set to 60-90 r / min, and stirring is carried out for 10-20 min, hyaluronic acid is added, the stirring speed is set to 120-150 r / min, and stirring is carried out for 10-20 min to obtain an injectable temperature-sensitive hydrogel.

[0013] As a further optimization scheme of the present application, the mass-volume ratio of the active traditional Chinese medicine exosomes to the coating liquid is 1 mg:5-8 mL; and the mass-volume ratio of the compound carrier, the poloxamer 407 hydrogel solution, and the hyaluronic acid is 1 mg:10-15 mL:8-12 mL.

[0014] As a further optimization scheme of the present application, the spray pressure of the spray gun is 0.2-0.3 MPa, and the spray rate is 0.5-1.0 mL / min.

[0015] The present application has the following beneficial effects: the injectable temperature-sensitive hydrogel in the present application is obtained by loading the compound carrier into poloxamer 407 hydrogel and hyaluronic acid, poloxamer 407 and hyaluronic acid can rapidly form a physical barrier on the wound surface after injection, and provide a moist healing environment, and relying on the temperature-sensitive characteristics and network structure, the sustained and slow release of exosomes and active ingredients is realized, and the local high concentration or premature loss caused by drug burst release is avoided; The compound carrier is formed by coating the active traditional Chinese medicine exosomes with the coating liquid, and the active traditional Chinese medicine exosomes are protected again, and the stability of the active traditional Chinese medicine exosomes in a complex wound environment is further enhanced; The traditional Chinese medicine active exosome is obtained by inducing adipose mesenchymal stem cells by modified astragalus extract, and is induced by co-culturing adipose mesenchymal stem cells, so that the obtained traditional Chinese medicine active exosome not only has the basic repair function of stem cell exosome, but also is rich in immunomodulatory components of astragalus polysaccharide and proliferation-promoting signals of growth factors, thereby inhibiting inflammation and promoting angiogenesis, and is suitable for local treatment of diabetic foot ulcer refractory wounds, and prolonging the action time of drugs and enhancing the tissue repair efficiency. DETAILED DESCRIPTION

[0016] The following further describes the present application, and it is necessary to point out here that the following detailed description is only used to further illustrate the present application, and cannot be understood as limiting the protection scope of the present application, and the skilled in the art can make some non-essential improvements and adjustments to the present application according to the above application content.

[0017] Materials The method used in the present application is a conventional method known to those skilled in the art, and the reagents and other materials used, such as commercially available products, are commercially available products.

[0018] In the present application: a-MEM culture medium is produced by Vincent Biotechnology (Nanjing) Co., Ltd., with the model number CHO-200 Penicillin-streptomycin double antibody is produced by Shanghai Blue Sky Biotechnology Co., Ltd., with the model number C0222; Exosome enrichment agent can be produced by Shanghai Blue Sky Biotechnology Co., Ltd., with the model number C3622S; Recombinant human fibroblast growth factor can be produced by SelleRx (Beijing) Life Science and Technology Co., Ltd., with the model number PF0301; Recombinant human epidermal growth factor can be produced by Beijing Equation Biotechnology Co., Ltd., with the model number 100-15; Calf blood deproteinized extract injection is produced by Heilongjiang Zhenbaodao Pharmaceutical Co., Ltd.; Sterile PBS buffer is Thermo 10010001; Poloxamer 407 hydrogel is BASF Kolliphor P407.

[0019] In the present application, the modified astragalus extract aqueous solution is obtained by mixing modified astragalus extract and distilled water.

[0020] Example 1 The preparation process of the modified astragalus extract is as follows: (a) Add 40 mL of anhydrous pyridine to a dry three-necked flask, place it in a 0°C water bath, stir at 300 rpm, and add 13 mL of citric acid dropwise at 0.1 mL / min. After the addition is complete, continue stirring at 120 rpm for 5 min in a 0°C water bath. Add astragalus polysaccharide in five batches, 2 g per batch, with a 2 min interval between each batch, and stir at 60 rpm until the addition is complete. After the addition is complete, stir at a constant temperature of 30°C water bath for 1 h to obtain the reaction solution. (ii) Cool the reaction solution to 3°C, add 15 mL of 65% sodium hydroxide aqueous solution to the reaction solution until the pH is 7.5, then add 150 mL of 75% ethanol aqueous solution to precipitate for 20 h, filter and collect the precipitate, and dialyze it three times with distilled water (1000 mL of distilled water per round of dialysis, and the molecular weight cutoff of the dialysis bag is 3500 Da) to obtain the modified Astragalus extract; The preparation process of active exosomes of traditional Chinese medicine is as follows: (a) Take 150 mL of α-MEM medium, add 65 μg of recombinant human fibroblast growth factor, 25 μg of recombinant human epidermal growth factor and 1.5 mL of penicillin-streptomycin antibiotic, mix well and sterilize with a 0.22 μm filter membrane to obtain stem cell culture medium, and store at 4°C in the dark for later use. (ii) Adipose-derived mesenchymal stem cells are prepared at a ratio of 3 × 10⁻⁶. 6 Each cell / flask was seeded into a T75 culture flask, and 20 mL of stem cell culture medium was added to the T75 culture flask. The flask was then placed in a 37°C, 5% CO2 saturated humidity incubator and cultured. The culture medium was changed every 2 days until the cell confluence reached 70% to obtain cultured cells. (III) Wash the cultured cells twice with 10 mL of sterile PBS buffer, add 15 mL of 2 mg / mL modified Astragalus extract aqueous solution, return to 37℃, 5% CO2 incubator, and incubate for 48 h. After incubation, aspirate supernatant A; centrifuge supernatant A at 300×g for 10 min, discard the bottom cell pellet, and aspirate supernatant B; centrifuge supernatant B at 10000×g at 4℃ for 30 min, and aspirate supernatant C. (iv) Add exosome enrichment agent to supernatant C (volume ratio of supernatant C to exosome enrichment agent is 5:1), gently invert and mix, let stand at 3℃ for 8 h, centrifuge at 12000×g for 25 min, discard the supernatant, and obtain the precipitate. Add 100 μL of sterile PBS buffer to the precipitate and repeat the pipetting and refluxing 10 times to obtain crude exosome extract solution. Filter the crude exosome extract solution through a 0.1 μm polycarbonate filter membrane to obtain active exosomes of traditional Chinese medicine. The preparation process of the coating solution is as follows: (a) Add 1% chitosan to 2% sodium alginate in a 0℃ water bath at 300 rpm, add 77% deionized water, and stir for 20 min to obtain the complex. (ii) 20% calf blood deproteinized extract was added to the complex and stirred in a 0°C water bath for 10 min to obtain the coating solution; The preparation process of injectable thermosensitive hydrogel is as follows: (i) Take active exosomes of traditional Chinese medicine and add them into a sterile rolling coating machine. Set the rotation speed to 20 r / min and the temperature inside the chamber to 25℃. Use a spray gun with a droplet diameter of 30 μm (spray pressure of 0.2 MPa and spray rate of 0.5 mL / min) to spray the coating liquid into the coating machine. Continue for 12 min to allow the coating liquid to evenly coat the exosomes (mass-volume ratio of active exosomes of traditional Chinese medicine to coating liquid is 1 mg: 5 mL) to obtain a composite carrier. (ii) The composite carrier is added to the poloxamer 407 hydrogel and stirred for 10 min at 60 r / min. Then, hyaluronic acid is added (the mass-volume ratio of the composite carrier, the poloxamer 407 hydrogel aqueous solution and the hyaluronic acid is 1 mg: 10 mL: 8 mL). The mixture is stirred for 10 min at 120 r / min to obtain an injectable thermosensitive hydrogel.

[0021] Example 2 The preparation process of modified Astragalus extract is as follows: (a) Add 60 mL of anhydrous pyridine to a dry three-necked flask, place it in a 2°C water bath, stir at 300 rpm, and add 20 mL of citric acid dropwise at 0.15 mL / min. After the addition is complete, continue stirring at 120 rpm for 5 min in a 2°C water bath. Add astragalus polysaccharide in five batches, 2 g per batch, with a 2 min interval between each batch, and stir at 60 rpm until the addition is complete. After the addition is complete, stir at a constant temperature of 35°C water bath for 1.5 h to obtain the reaction solution. (ii) Cool the reaction solution to 5°C, add 20 mL of 65% sodium hydroxide aqueous solution to the reaction solution until the pH is 7.5, then add 200 mL of 75% ethanol aqueous solution to precipitate for 24 h, filter and collect the precipitate, and dialyze it three times with distilled water (1000 mL of distilled water per round of dialysis, and the molecular weight cutoff of the dialysis bag is 3500 Da) to obtain the modified Astragalus extract; The preparation process of active exosomes of traditional Chinese medicine is as follows: (a) Take 160 mL of α-MEM medium, add 70 μg of recombinant human fibroblast growth factor, 28 μg of recombinant human epidermal growth factor and 1.8 mL of penicillin-streptomycin antibiotics, mix well and sterilize with a 0.22 μm filter membrane to obtain stem cell culture medium, and store at 4°C in the dark for later use. (ii) Adipose-derived mesenchymal stem cells are prepared at a ratio of 3 × 10⁻⁶. 6Each cell / flask was seeded into a T75 culture flask, and 20 mL of stem cell culture medium was added to the T75 culture flask. The flask was then placed in a 37°C, 5% CO2 saturated humidity incubator and cultured. The culture medium was changed every 2 days until the cell confluence reached 75% to obtain cultured cells. (III) Wash the cultured cells three times with 10 mL of sterile PBS buffer, add 15 mL of modified Astragalus extract aqueous solution with a concentration of 2 mg / mL, and return to the 37℃, 5% CO2 incubator for incubation for 60 h. After incubation, aspirate the supernatant A; centrifuge the supernatant A at 300×g for 10 min, discard the bottom cell pellet, and aspirate the supernatant B; centrifuge the supernatant B at 10000×g at 4℃ for 30 min, and aspirate the supernatant C. (iv) Add exosome enrichment agent to supernatant C (volume ratio of supernatant C to exosome enrichment agent is 5:1), gently invert and mix, let stand at 4℃ for 10 h, centrifuge at 12000×g for 30 min, discard the supernatant, and obtain the precipitate. Add 150 μL of sterile PBS buffer to the precipitate, and repeat the pipetting 13 times to obtain crude exosome extract solution. Filter the crude exosome extract solution through a 0.1 μm polycarbonate filter membrane to obtain active exosomes of traditional Chinese medicine. The preparation process of the coating solution is as follows: (a) Add 2% chitosan to 3% sodium alginate in a 2℃ water bath at 300 rpm, add 75% deionized water, and stir for 23 min to obtain the complex. (ii) 28% calf blood deproteinized extract was added to the complex and stirred in a water bath at 2°C for 15 min to obtain the coating solution; The preparation process of injectable thermosensitive hydrogel is as follows: (i) Take active exosomes of traditional Chinese medicine and add them into a sterile rolling coating machine. Set the rotation speed to 30 r / min and the temperature inside the chamber to 25℃. Use a spray gun with a droplet diameter of 40 μm (spray pressure of 0.2 MPa and spray rate of 0.8 mL / min) to spray the coating liquid into the coating machine. Continue for 13 min to allow the coating liquid to evenly coat the exosomes (mass-volume ratio of active exosomes of traditional Chinese medicine to coating liquid is 1 mg: 6 mL) to obtain a composite carrier. (ii) The composite carrier is added to the poloxamer 407 hydrogel and stirred for 15 minutes at 80 r / min. Then, hyaluronic acid is added (the mass-volume ratio of the composite carrier, the poloxamer 407 hydrogel aqueous solution and the hyaluronic acid is 1 mg: 13 mL: 10 mL). The mixture is stirred for 15 minutes at 130 r / min to obtain an injectable thermosensitive hydrogel.

[0022] Example 3 The preparation process of modified Astragalus extract is as follows: (a) Add 80 mL of anhydrous pyridine to a dry three-necked flask, place it in a 4°C water bath, stir at 300 rpm, and add 27 mL of citric acid dropwise at 0.2 mL / min. After the addition is complete, continue stirring at 120 rpm for 5 min in a 4°C water bath. Add astragalus polysaccharide in five batches, 2 g per batch, with a 2 min interval between each batch, and stir at 60 rpm until the addition is complete. After the addition is complete, stir at a constant temperature of 40°C water bath for 2 h to obtain the reaction solution. (ii) Cool the reaction solution to 6°C, add 25 mL of 65% sodium hydroxide aqueous solution to the reaction solution until the pH is 7.5, then add 250 mL of 75% ethanol aqueous solution to precipitate for 28 h, filter and collect the precipitate, and dialyze it four times with distilled water (1000 mL of distilled water per round of dialysis, and the molecular weight cutoff of the dialysis bag is 3500 Da) to obtain the modified Astragalus extract; The preparation process of active exosomes of traditional Chinese medicine is as follows: (a) Take 180 mL of α-MEM medium, add 75 μg of recombinant human fibroblast growth factor, 30 μg of recombinant human epidermal growth factor and 2.0 mL of penicillin-streptomycin antibiotics, mix well and sterilize with a 0.22 μm filter membrane to obtain stem cell culture medium, and store at 4°C in the dark for later use. (ii) Adipose-derived mesenchymal stem cells are prepared at a ratio of 3 × 10⁻⁶. 6 Each cell / flask was seeded into a T75 culture flask, and 20 mL of stem cell culture medium was added to the T75 culture flask. The flask was then placed in a 37°C, 5% CO2 saturated humidity incubator and cultured. The culture medium was changed every 2 days until the cell confluence reached 80% to obtain cultured cells. (III) Wash the cultured cells three times with 10 mL of sterile PBS buffer, add 15 mL of modified Astragalus extract aqueous solution with a concentration of 2 mg / mL, and return to the 37℃, 5% CO2 incubator for incubation for 72 h. After incubation, aspirate the supernatant A; centrifuge the supernatant A at 300×g for 10 min, discard the bottom cell pellet, and aspirate the supernatant B; centrifuge the supernatant B at 10000×g at 4℃ for 30 min, and aspirate the supernatant C. (iv) Add exosome enrichment agent to supernatant C (volume ratio of supernatant C to exosome enrichment agent is 5:1), gently invert and mix, let stand at 5℃ for 12h, centrifuge at 12000×g for 35min, discard the supernatant, and obtain the precipitate. Add 200μL of sterile PBS buffer to the precipitate and repeat the pipetting 15 times to obtain crude exosome extract solution. Filter the crude exosome extract solution through a 0.1μm polycarbonate filter membrane to obtain active exosomes of traditional Chinese medicine. The preparation process of the coating solution is as follows: (a) Add 3% chitosan to 5% sodium alginate in a 4℃ water bath with a stirring speed of 300rpm, add 80% deionized water, and stir for 30min to obtain the complex. (ii) 35% calf blood deproteinized extract was added to the complex and stirred in a water bath at 4°C for 20 min to obtain the coating solution; The preparation process of injectable thermosensitive hydrogel is as follows: (i) Take active exosomes of traditional Chinese medicine and add them into a sterile rolling coating machine. Set the rotation speed to 40 r / min and the temperature inside the chamber to 25℃. Use a spray gun with a droplet diameter of 50 μm (spray pressure of 0.3 MPa and spray rate of 1.0 mL / min) to spray the coating liquid into the coating machine. Continue for 15 min to allow the coating liquid to evenly coat the exosomes (the mass-volume ratio of active exosomes of traditional Chinese medicine to coating liquid is 1 mg: 8 mL) to obtain a composite carrier. (ii) The composite carrier is added to the poloxamer 407 hydrogel and stirred at 90 r / min for 20 min. Then, hyaluronic acid is added (the mass-volume ratio of the composite carrier, the poloxamer 407 hydrogel aqueous solution and the hyaluronic acid is 1 mg: 15 mL: 12 mL) and stirred at 150 r / min for 20 min to obtain an injectable thermosensitive hydrogel.

[0023] Comparative Example 1 The preparation process of active exosomes of traditional Chinese medicine is as follows: (a) Take 160 mL of α-MEM medium, add 70 μg of recombinant human fibroblast growth factor, 28 μg of recombinant human epidermal growth factor and 1.8 mL of penicillin-streptomycin antibiotics, mix well and sterilize with a 0.22 μm filter membrane to obtain stem cell culture medium, and store at 4°C in the dark for later use. (ii) Adipose-derived mesenchymal stem cells are prepared at a ratio of 3 × 10⁻⁶. 6 Each cell / flask was seeded into a T75 culture flask, and 20 mL of stem cell culture medium was added to the T75 culture flask. The flask was then placed in a 37°C, 5% CO2 saturated humidity incubator and cultured. The culture medium was changed every 2 days until the cell confluence reached 75% to obtain cultured cells. (III) Wash the cultured cells three times with 10 mL of sterile PBS buffer, add 15 mL of 2 mg / mL Astragalus polysaccharide aqueous solution (the raw materials for preparing Astragalus polysaccharide aqueous solution are Astragalus polysaccharide and distilled water), put it back into the 37℃, 5% CO2 incubator, and incubate for 60 h. After the incubation, aspirate the supernatant A; centrifuge the supernatant A at 300×g for 10 min, discard the bottom cell pellet, and aspirate the supernatant B; centrifuge the supernatant B at 10000×g at 4℃ for 30 min, and aspirate the supernatant C. (iv) Add exosome enrichment agent to supernatant C (volume ratio of supernatant C to exosome enrichment agent is 5:1), gently invert and mix, let stand at 4℃ for 10 h, centrifuge at 12000×g for 30 min, discard the supernatant, and obtain the precipitate. Add 150 μL of sterile PBS buffer to the precipitate, and repeat the pipetting 13 times to obtain crude exosome extract solution. Filter the crude exosome extract solution through a 0.1 μm polycarbonate filter membrane to obtain active exosomes of traditional Chinese medicine. The preparation process of the coating solution is as follows: (a) Add 2% chitosan to 3% sodium alginate in a 2℃ water bath at 300 rpm, add 75% deionized water, and stir for 23 min to obtain the complex. (ii) 28% calf blood deproteinized extract was added to the complex and stirred in a water bath at 2°C for 15 min to obtain the coating solution; The preparation process of injectable thermosensitive hydrogel is as follows: (i) Take active exosomes of traditional Chinese medicine and add them into a sterile rolling coating machine. Set the rotation speed to 30 r / min and the temperature inside the chamber to 25℃. Use a spray gun with a droplet diameter of 40 μm (spray pressure of 0.2 MPa and spray rate of 0.8 mL / min) to spray the coating liquid into the coating machine. Continue for 13 min to allow the coating liquid to evenly coat the exosomes (mass-volume ratio of active exosomes of traditional Chinese medicine to coating liquid is 1 mg: 6 mL) to obtain a composite carrier. (ii) The composite carrier is added to the poloxamer 407 hydrogel and stirred for 15 minutes at 80 r / min. Then, hyaluronic acid is added (the mass-volume ratio of the composite carrier, the poloxamer 407 hydrogel aqueous solution and the hyaluronic acid is 1 mg: 13 mL: 10 mL). The mixture is stirred for 15 minutes at 130 r / min to obtain an injectable thermosensitive hydrogel.

[0024] Comparative Example 2 The preparation process of modified Astragalus extract is as follows: (a) Add 60 mL of anhydrous pyridine to a dry three-necked flask, place it in a 2°C water bath, stir at 300 rpm, and add 20 mL of citric acid dropwise at 0.15 mL / min. After the addition is complete, continue stirring at 120 rpm for 5 min in a 2°C water bath. Add astragalus polysaccharide in five batches, 2 g per batch, with a 2 min interval between each batch, and stir at 60 rpm until the addition is complete. After the addition is complete, stir at a constant temperature of 35°C water bath for 1.5 h to obtain the reaction solution. (ii) Cool the reaction solution to 5°C, add 20 mL of 65% sodium hydroxide aqueous solution to the reaction solution until the pH is 7.5, then add 200 mL of 75% ethanol aqueous solution to precipitate for 24 h, filter and collect the precipitate, and dialyze it three times with distilled water (1000 mL of distilled water per round of dialysis, and the molecular weight cutoff of the dialysis bag is 3500 Da) to obtain the modified Astragalus extract; The preparation process of active exosomes of traditional Chinese medicine is as follows: (a) Take 160 mL of α-MEM medium, add 70 μg of recombinant human fibroblast growth factor, 28 μg of recombinant human epidermal growth factor and 1.8 mL of penicillin-streptomycin antibiotics, mix well and sterilize with a 0.22 μm filter membrane to obtain stem cell culture medium, and store at 4°C in the dark for later use. (ii) Adipose-derived mesenchymal stem cells are prepared at a ratio of 3 × 10⁻⁶. 6 Each cell / flask was seeded into a T75 culture flask, and 20 mL of stem cell culture medium was added to the T75 culture flask. The flask was then placed in a 37°C, 5% CO2 saturated humidity incubator and cultured. The culture medium was changed every 2 days until the cell confluence reached 75% to obtain cultured cells. (iii) Wash the cultured cells three times with 10 mL of sterile PBS buffer, add 15 mL of modified Astragalus extract aqueous solution with a concentration of 2 mg / mL, put them back into the 37℃, 5% CO2 incubator, and incubate for 60 h. After the incubation, remove the supernatant. (iv) Add exosome enrichment agent to the supernatant (the volume ratio of supernatant to exosome enrichment agent is 5:1), gently invert and mix, let stand at 4℃ for 10 h, centrifuge at 12000×g for 30 min, discard the supernatant, and obtain the precipitate. Add 150 μL of sterile PBS buffer to the precipitate and repeat the pipetting and refluxing 13 times to obtain the crude exosome extract solution. Filter the crude exosome extract solution through a 0.1 μm polycarbonate filter membrane to obtain the active exosomes of traditional Chinese medicine. The preparation process of the coating solution is as follows: (a) Add 2% chitosan to 3% sodium alginate in a 2℃ water bath at 300 rpm, add 75% deionized water, and stir for 23 min to obtain the complex. (ii) 28% calf blood deproteinized extract was added to the complex and stirred in a water bath at 2°C for 15 min to obtain the coating solution; The preparation process of injectable thermosensitive hydrogel is as follows: (i) Take active exosomes of traditional Chinese medicine and add them into a sterile rolling coating machine. Set the rotation speed to 30 r / min and the temperature inside the chamber to 25℃. Use a spray gun with a droplet diameter of 40 μm (spray pressure of 0.2 MPa and spray rate of 0.8 mL / min) to spray the coating liquid into the coating machine. Continue for 13 min to allow the coating liquid to evenly coat the exosomes (mass-volume ratio of active exosomes of traditional Chinese medicine to coating liquid is 1 mg: 6 mL) to obtain a composite carrier. (ii) The composite carrier is added to the poloxamer 407 hydrogel and stirred for 15 minutes at 80 r / min. Then, hyaluronic acid is added (the mass-volume ratio of the composite carrier, the poloxamer 407 hydrogel aqueous solution and the hyaluronic acid is 1 mg: 13 mL: 10 mL). The mixture is stirred for 15 minutes at 130 r / min to obtain an injectable thermosensitive hydrogel.

[0025] Comparative Example 3 The preparation process of modified Astragalus extract is as follows: (a) Add 60 mL of anhydrous pyridine to a dry three-necked flask, place it in a 2°C water bath, stir at 300 rpm, and add 20 mL of citric acid dropwise at 0.15 mL / min. After the addition is complete, continue stirring at 120 rpm for 5 min in a 2°C water bath. Add astragalus polysaccharide in five batches, 2 g per batch, with a 2 min interval between each batch, and stir at 60 rpm until the addition is complete. After the addition is complete, stir at a constant temperature of 35°C water bath for 1.5 h to obtain the reaction solution. (ii) Cool the reaction solution to 5°C, add 20 mL of 65% sodium hydroxide aqueous solution to the reaction solution until the pH is 7.5, then add 200 mL of 75% ethanol aqueous solution to precipitate for 24 h, filter and collect the precipitate, and dialyze it three times with distilled water (1000 mL of distilled water per round of dialysis, and the molecular weight cutoff of the dialysis bag is 3500 Da) to obtain the modified Astragalus extract; The preparation process of active exosomes of traditional Chinese medicine is as follows: (a) Take 160 mL of α-MEM medium, add 70 μg of recombinant human fibroblast growth factor, 28 μg of recombinant human epidermal growth factor and 1.8 mL of penicillin-streptomycin antibiotics, mix well and sterilize with a 0.22 μm filter membrane to obtain stem cell culture medium, and store at 4°C in the dark for later use. (ii) Adipose-derived mesenchymal stem cells are prepared at a ratio of 3 × 10⁻⁶. 6 Each cell / flask was seeded into a T75 culture flask, and 20 mL of stem cell culture medium was added to the T75 culture flask. The flask was then placed in a 37°C, 5% CO2 saturated humidity incubator and cultured. The culture medium was changed every 2 days until the cell confluence reached 75% to obtain cultured cells. (III) Wash the cultured cells three times with 10 mL of sterile PBS buffer, add 15 mL of modified Astragalus extract aqueous solution with a concentration of 2 mg / mL, and return to the 37℃, 5% CO2 incubator for incubation for 60 h. After incubation, aspirate the supernatant A; centrifuge the supernatant A at 300×g for 10 min, discard the bottom cell pellet, and aspirate the supernatant B; centrifuge the supernatant B at 10000×g at 4℃ for 30 min, and aspirate the supernatant C. (iv) Add exosome enrichment agent to supernatant C (volume ratio of supernatant C to exosome enrichment agent is 5:1), gently invert and mix, let stand at 4℃ for 10 h, centrifuge at 12000×g for 30 min, discard the supernatant, and obtain the precipitate. Add 150 μL of sterile PBS buffer to the precipitate, and repeat the pipetting 13 times to obtain crude exosome extract solution. Filter the crude exosome extract solution through a 0.1 μm polycarbonate filter membrane to obtain active exosomes of traditional Chinese medicine. The preparation process of injectable thermosensitive hydrogel is as follows: The active exosomes of traditional Chinese medicine were added to poloxamer 407 hydrogel and stirred at 80 r / min for 15 min. Hyaluronic acid was then added (the mass-volume ratio of active exosomes of traditional Chinese medicine, poloxamer 407 hydrogel aqueous solution and hyaluronic acid was 1 mg: 13 mL: 10 mL), and stirred at 130 r / min for 15 min to obtain injectable thermosensitive hydrogel.

[0026] Performance testing Simulation conditions for in vitro body fluids: Sample loading: Add 0.5 ml of injectable thermosensitive hydrogel to the dialysis bag (molecular weight cutoff of 300,000 Da); Release medium: Prepare sterile PBS buffer (1X concentration, pH 7.2) and sterile fetal bovine serum (FBS). In a sterile operating table (biosafety cabinet), prepare 100 mL of 10% FBS / PBS solution by mixing 10 mL of FBS with 90 mL of PBS. Temperature control: Immerse the dialysis bag after sample loading in the release medium and place it on a constant temperature shaker at 37°C (80 rpm). Sampling: Set time points at 24h, 48h, and 72h, collect all the release media for fluorescence detection, and immediately replenish with an equal amount of fresh preheated media, and record the release rate of the active exosomes of traditional Chinese medicine.

[0027] The injectable thermosensitive hydrogels prepared by the methods in Examples 1-3 and Comparative Examples 1-3 were used to detect the release rate of active exosomes of traditional Chinese medicine under simulated in vitro body fluid conditions. The results are shown in Table 1. Table 1 Item 24 h release rate / % 48 h release rate / % 72 h release rate / % Example 1 21 35 63 Example 2 22 35 64 Example 3 22 34 63 Comparative Example 1 31 48 76 Comparative Example 2 34 49 77 Comparative Example 3 37 51 89 As shown in Table 1, the cumulative release rates of the injectable thermosensitive hydrogels prepared by the methods in Examples 1-3 in simulated body fluids were 21%-22% at 24h, 34%-35% at 48h, and 63%-64% at 72h, respectively. These results indicate that the hydrogels prepared in Examples 1-3 have sustained-release properties, and the release time of the active exosomes of traditional Chinese medicine is relatively long, which helps to maintain the continuous drug effect during the healing process of refractory wounds such as diabetic foot.

[0028] In contrast, the hydrogels prepared by the methods in Comparative Examples 1-3 had higher cumulative release rates: 31%-37% at 24h, 48%-51% at 48h, and 76%-89% at 72h. This indicates that they release the active exosomes of traditional Chinese medicine more quickly and for a shorter time, which is not conducive to forming a stable and long-lasting delivery system at the wound site.

[0029] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. An injectable thermosensitive hydrogel loaded with active exosomes of traditional Chinese medicine, characterized in that, The injectable thermosensitive hydrogel is obtained by poloxamer 407 hydrogel and hyaluronic acid-loaded composite carrier; the composite carrier is formed by coating active exosomes of traditional Chinese medicine with a coating solution. The active exosomes of the traditional Chinese medicine were obtained by inducing adipose-derived mesenchymal stem cells with modified Astragalus extract.

2. The injectable thermosensitive hydrogel loaded with active exosomes of traditional Chinese medicine according to claim 1, characterized in that, The preparation process of the modified Astragalus extract is as follows: (a) Add 40-80 mL of anhydrous pyridine to a dry three-necked flask, place it in a water bath at 0-4℃, stir at 300 rpm, and add 13-27 mL of citric acid dropwise at 0.1-0.2 mL / min. After the addition is complete, continue stirring at 120 rpm for 5 min in a water bath at 0-4℃. Add 10 g of astragalus polysaccharide in batches and stir at 60 rpm until the addition is complete. After the addition is complete, stir at a constant temperature of 30-40℃ for 1-2 h to obtain the reaction solution. (ii) Cool the reaction solution to 3-6℃, add 15-25mL of 65% sodium hydroxide aqueous solution to the reaction solution until the pH is 7.5, then add 150-250mL of 75% ethanol aqueous solution to precipitate for 20-28h, filter and collect the precipitate, and dialyze it with distilled water 3-4 times to obtain the modified Astragalus extract.

3. The injectable thermosensitive hydrogel loaded with active exosomes of traditional Chinese medicine according to claim 2, characterized in that, During the preparation of the modified Astragalus extract, Astragalus polysaccharide was added in five batches, with 2g added in each batch and a 2min interval between each batch; during the distilled water dialysis, 1000ml of distilled water was used per round, and the molecular weight cutoff of the dialysis bag was 3500Da.

4. The injectable thermosensitive hydrogel loaded with active exosomes of traditional Chinese medicine according to claim 3, characterized in that, The preparation process of the active exosomes of the traditional Chinese medicine is as follows: (a) Take 150-180mL of α-MEM medium, add 65-75μg of recombinant human fibroblast growth factor and 25-30μg of recombinant human epidermal growth factor and 1.5-2.0ml of penicillin-streptomycin antibiotics, mix well and sterilize with a 0.22μm filter membrane to obtain stem cell culture medium, and store at 4℃ in the dark for later use. (ii) Adipose-derived mesenchymal stem cells are prepared at a ratio of 3 × 10⁻⁶. 6 Each cell / flask was seeded into a T75 culture flask, and 20 mL of stem cell culture medium was added to the T75 culture flask. The flask was then placed in a 37°C, 5% CO2 saturated humidity incubator and cultured. The culture medium was changed every 2 days until the cell confluence reached 70%-80% to obtain cultured cells. (III) Wash the cultured cells 2-3 times with 10 mL of sterile PBS buffer, add 15 mL of 2 mg / mL modified Astragalus extract aqueous solution, return to 37℃, 5% CO2 incubator, and incubate for 48-72 h. After incubation, aspirate supernatant A; centrifuge supernatant A at 300×g for 10 min, discard the bottom cell pellet, and aspirate supernatant B; centrifuge supernatant B at 10000×g at 4℃ for 30 min, and aspirate supernatant C. (iv) Add exosome enrichment agent to supernatant C, gently invert and mix, let stand at 3-5℃ for 8-12h, centrifuge at 12000×g for 25-35min, discard the supernatant to obtain precipitate, add 100-200μL sterile PBS buffer to precipitate, repeat pipetting 10-15 times to obtain crude exosome extract solution; filter the crude exosome extract solution through a 0.1μm polycarbonate filter membrane to obtain active exosomes of traditional Chinese medicine.

5. The injectable thermosensitive hydrogel loaded with active exosomes of traditional Chinese medicine according to claim 4, characterized in that, The volume ratio of the supernatant C to the exosome enrichment agent is 5:

1.

6. The injectable thermosensitive hydrogel loaded with active exosomes of traditional Chinese medicine according to claim 1, characterized in that, The raw materials for preparing the coating solution, by percentage, include: 2-5% sodium alginate, 1-3% chitosan, 20-35% calf blood deproteinized extract, and 65-80% deionized water.

7. The injectable thermosensitive hydrogel loaded with active exosomes of traditional Chinese medicine according to claim 6, characterized in that, The preparation process of the coating solution is as follows: (a) Add chitosan to sodium alginate in a 0-4℃ water bath at 300 rpm, add deionized water, and stir for 15-30 min to obtain the complex. (ii) Add the deproteinized calf blood extract to the complex and stir in a water bath at 0-4℃ for 10-20 min to obtain the coating solution.

8. The injectable thermosensitive hydrogel loaded with active exosomes of traditional Chinese medicine according to claim 7, characterized in that, The preparation process of the injectable thermosensitive hydrogel is as follows: (a) Take active exosomes of traditional Chinese medicine and add them into a sterile rolling coating machine. Set the rotation speed to 20-40 r / min and the temperature inside the chamber to 25℃. Use a spray gun with a droplet diameter of 30-50 μm to spray the coating liquid into the coating machine. Continue for 12-15 minutes to allow the coating liquid to evenly coat the exosomes and obtain a composite carrier. (ii) The composite carrier is added to the poloxamer 407 hydrogel and stirred at a speed of 60-90 r / min for 10-20 min. Hyaluronic acid is added and stirred at a speed of 120-150 r / min for 10-20 min to obtain an injectable thermosensitive hydrogel.

9. The injectable thermosensitive hydrogel loaded with active exosomes of traditional Chinese medicine according to claim 8, characterized in that, The mass-to-volume ratio of the active exosomes of the traditional Chinese medicine to the coating solution is 1 mg: 5-8 mL; the mass-to-volume ratio of the composite carrier, poloxamer 407 hydrogel aqueous solution and hyaluronic acid is 1 mg: 10-15 mL: 8-12 mL.

10. The injectable thermosensitive hydrogel loaded with active exosomes of traditional Chinese medicine according to claim 8, characterized in that, The spray gun has a spray pressure of 0.2-0.3 MPa and a spray rate of 0.5-1.0 mL / min.