Hydrogel component for transferring exosome and preparation method thereof

Abstract

The invention relates to the technical field of biology, in particular to a hydrogel component for transferring exosomes and a preparation method of the hydrogel component. The hydrogel component is of a core-shell structure and is composed of an outer shell and a core area, the outer shell comprises a carboxylated chondroitin sulfate-guar gum composite material, a disulfide bond-diacrylate modified heparin-chitosan oligosaccharide composite material and nanoscale hydroxyapatite, and the core area comprises exosome, sodium hyaluronate, polyethylene glycol and albumin. After the hydrogel enters a physiological environment, carboxylated chondroitin sulfate and water form a hydrogen bond to initiate swelling, and guar gum stabilizes a network. And the disulfide bond is fractured in a gradient manner under a reduction condition to form a micro-channel which is communicated with the swelling space to form a release path. The nano-hydroxyapatite delays degradation and provides ionic crosslinking stability. The exosome is slowly released under the assistance of sodium hyaluronate and polyethylene glycol, and albumin protects the activity of the exosome.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a hydrogel component for delivering exosomes and a preparation method thereof. Background Art

[0002] Although the existing exosome-delivering hydrogels on the market show certain application prospects in the fields of tissue engineering and regenerative medicine, they still face many technical bottlenecks that need to be solved urgently, which seriously restrict the process of their clinical translation.

[0003] One of the most prominent challenges is that the hydrogel components for delivering exosomes cannot respond well to the intracellular reducing environment, which severely limits their application potential as drug delivery systems. First of all, in the absence of reducing environment responsiveness, the hydrogel cannot achieve precise spatio-temporal controlled release, resulting in unpredictable and difficult-to-regulate release kinetics during the drug delivery process. This uncontrollability may cause the active drug to be prematurely released before reaching the target site or to stay in the cell for too long, both of which will reduce the therapeutic efficiency. Secondly, the hydrogel components that cannot respond to the intracellular specific reducing environment are difficult to distinguish normal cells from pathological cells, resulting in insufficient targeting and increasing the potential toxicity risk to healthy tissues. In addition, such non-responsive hydrogels often require additional complex modifications or co-delivery systems to compensate for functional defects, increasing the complexity and cost of the preparation process and possibly introducing new biocompatibility problems. Moreover, non-responsive hydrogels often exhibit limited biodegradability, which may accumulate in the body for a long time, triggering immune responses or inflammatory reactions. Finally, in the absence of an intracellular reduction-triggering mechanism, the bioactive molecules (such as proteins, nucleic acids, etc.) in exosomes are difficult to be efficiently released, and their therapeutic potential cannot be fully exerted. Summary of the Invention

[0004] (1) Technical Problems to be Solved

[0005] The purpose of the present invention is to provide a hydrogel component for delivering exosomes and a preparation method thereof, so as to solve the problem that the hydrogel component for delivering exosomes cannot be well released in the intracellular reducing environment.

[0006] (2) Technical Solutions

[0007] To achieve the above object, on the one hand, the present invention provides a hydrogel component for delivering exosomes, which has a core-shell structure and is composed of an outer shell and a core region. The outer shell includes a carboxylated chondroitin sulfate-guar gum composite material, a disulfide-diacrylate modified heparin-chitosan oligosaccharide composite material, and nano-hydroxyapatite. The core region includes exosomes, sodium hyaluronate, polyethylene glycol, albumin, sericin, plant extracts, and animal umbilical cord extracts. The plant extracts include centella asiatica extract, licorice extract, and chamomilla recutita extract.

[0008] Furthermore, for a hydrogel component for delivering exosomes, by weight fraction, the outer shell includes the following raw materials in parts by weight: 20-40 parts of a carboxylated chondroitin sulfate-guar gum composite material, 30-50 parts of a disulfide-diacrylate modified heparin-chitosan oligosaccharide composite material, and 10-30 parts of nano-hydroxyapatite; the core region includes the following raw materials in parts by weight: 1-5 parts of exosomes, 15-30 parts of sodium hyaluronate, 20-40 parts of polyethylene glycol, 20-35 parts of albumin, 10-25 parts of sericin, 5-15 parts of plant extracts, and 5-15 parts of animal umbilical cord extracts. The plant extracts include the following raw materials in parts by weight: 1-5 parts of centella asiatica extract, 2-5 parts of licorice extract, and 2-5 parts of chamomilla recutita extract.

[0009] Furthermore, the preparation method of the carboxylated chondroitin sulfate-guar gum composite material includes:

[0010] S11. Add chondroitin sulfate to deionized water to obtain a chondroitin sulfate solution, and add 1 mol / L NaOH solution dropwise to the chondroitin sulfate solution to adjust the pH to 8-8.5 to obtain the chondroitin sulfate solution with adjusted pH.

[0011] S12. Add chloroacetic acid to the chondroitin sulfate solution with adjusted pH at 0-4 °C and stir for 10-15 min to obtain a mixed solution A. Add 6 mol / L NaOH solution to the mixed solution A and stir for 30-40 min to obtain a mixed solution B. Heat the mixed solution B to 60-70 °C and keep it for 4-5 h to obtain a solution C. Cool the solution C to room temperature to obtain the cooled solution C. Add absolute ethanol to the cooled solution C to obtain a mixed solution D. Centrifuge the mixed solution D at 4000-5000 rpm for 10-15 min to obtain a precipitate. Wash the precipitate with 80% ethanol 3-4 times to obtain the washed precipitate. Vacuum dry the washed precipitate at 40-50 °C for 24-48 h to obtain carboxylated chondroitin sulfate.

[0012] S13. Add guar gum to deionized water and heat it to 70-80 °C and stir for 2-3 h to obtain a guar gum solution.

[0013] S14. Add carboxylated chondroitin sulfate to PBS buffer solution with pH 7.4 to obtain a mixed solution E. Add guar gum solution to the mixed solution E and stir for 2 - 3 h to obtain a mixed solution F. Pour the mixed solution F into a flat petri dish and control the thickness to be about 3 - 5 mm to obtain a mixed petri dish. Place the mixed petri dish in a refrigerator at -20 - -10 °C and freeze for 12 - 24 h to obtain a frozen sample. Put the frozen sample into a freeze dryer and freeze-dry it for 48 - 56 h under the conditions of -50 - -40 °C and 10 - 20 Pa to obtain a dried sample;

[0014] S15. Immerse the dried sample in 1% glutaraldehyde PBS solution with pH 7.4 and crosslink it at 4 - 6 °C for 12 - 24 h to obtain a crosslinked sample. Wash the crosslinked sample successively with a large amount of PBS and deionized water to obtain a washed sample. Freeze-dry the washed sample for 24 - 48 h to obtain a carboxylated chondroitin sulfate - guar gum composite material.

[0015] Furthermore, the preparation method of the disulfide bond - diacrylate modified heparin - chitosan oligosaccharide composite material includes:

[0016] S21. Dissolve sodium heparin in deionized water to obtain a sodium heparin solution. Pass the sodium heparin solution through a column filled with Dowex50WX8 ion exchange resin to obtain resin, and then wash the resin with deionized water until the pH is neutral to obtain a mixed solution A. Filter the mixed solution A to obtain a filtrate. Freeze-dry the filtrate for 24 - 48 h to obtain heparinic acid powder;

[0017] S22. Add heparinic acid powder to PBS buffer solution with pH 6.0 to obtain a mixed solution B. Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to the mixed solution B to obtain a mixed solution C. Stir the mixed solution C at room temperature for 1 - 2 h to obtain a stirred mixed solution C. Dissolve bis(2-methylacryloyl)ethoxydisulfide in dimethyl sulfoxide to obtain a mixed solution D. Add the mixed solution D to the mixed solution C to obtain a mixed solution E. Stir the mixed solution E under nitrogen protection at room temperature for 24 - 48 h to obtain a reaction mixture. Put the reaction mixture into a dialysis bag and dialyze it against deionized water for 48 - 56 h to obtain a dialyzed solution. Freeze-dry the dialyzed solution for 24 - 48 h to obtain disulfide bond - diacrylate modified heparin;

[0018] S23. Chitosan was added to acetic acid solution to obtain a chitosan solution. 30% hydrogen peroxide was added to the chitosan solution to obtain a mixed solution F. The mixed solution F was reacted at 60 - 70 °C for 3 - 4 h to obtain a mixed solution H. The mixed solution H was cooled to room temperature, and then the pH was adjusted to 7.0 with sodium hydroxide to obtain a mixture. The mixture was loaded into a dialysis bag and dialyzed against deionized water for 48 - 56 h to obtain a dialyzed mixed solution. The dialyzed mixed solution was freeze-dried for 24 - 48 h to obtain chitosan oligosaccharide;

[0019] S24. Weigh dithiol-diacrylate modified heparin and add it to PBS solution with pH 7.4 to obtain a dithiol-diacrylate modified heparin solution. Add chitosan oligosaccharide to PBS solution with pH 7.4 to obtain a chitosan oligosaccharide solution. Slowly drip the chitosan oligosaccharide solution into the dithiol-diacrylate modified heparin solution and stir at room temperature for 2 - 3 h to obtain a mixed solution I. Add azobisisobutyronitrile to the mixed solution I to obtain a mixed solution J. Transfer the mixed solution J to a mold. Under a nitrogen environment, heat the mold to 65 - 80 °C and react for 6 - 8 h to obtain a reaction solution. Cool the reaction solution to room temperature to obtain a cooled reactant. Immerse the cooled reactant in PBS for 24 - 48 h to obtain an immersed reactant. Freeze the immersed reactant in a refrigerator at -20 - -10 °C for 12 - 24 h to obtain a frozen reactant. Put the frozen reactant into a freeze dryer and freeze-dry it at -50 - -40 °C and 10 - 20 Pa for 48 - 56 h to obtain a dithiol-diacrylate modified heparin-chitosan oligosaccharide composite material.

[0020] On the other hand, based on the same inventive concept, the present invention also provides a preparation method of a hydrogel component for delivering exosomes, which is applied to the hydrogel component for delivering exosomes, and includes the following steps:

[0021] S31. Add sodium hyaluronate to deionized water to obtain a sodium hyaluronate solution. Add polyethylene glycol to PBS solution with pH 7.4 to obtain a polyethylene glycol solution. Add human albumin to PBS solution with pH 7.4 to obtain an albumin solution. Add sericin to PBS solution with pH 7.4 to obtain a sericin solution. Add asiatic pennywort extract, liquorice extract and chamomile extract to PBS solution with pH 7.4 to obtain a mixed solution of plant extracts; Add animal umbilical cord extract to PBS solution with pH 7.4 to obtain an animal umbilical cord extract solution;

[0022] S32. Mix the sodium hyaluronate solution, polyethylene glycol solution, albumin solution, sericin protein solution, plant extract mixed solution and animal umbilical cord extract solution, and then stir at room temperature for 1 - 2 h to obtain mixed solution A. Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to mixed solution A to obtain mixed solution B. Stir and react mixed solution B at pH 5.5 - 6.0 and 4 - 5 °C for 4 - 5 h to obtain mixed solution C. Adjust the pH of mixed solution C to 7.0 to obtain the mixed solution C with adjusted pH. Slowly add the exosome solution to the mixed solution C with adjusted pH and gently mix for 30 - 40 min to obtain a mixture. Let the mixture stand at 25 - 30 °C for 1 - 2 h to form a core hydrogel precursor;

[0023] S33. Add the carboxylated chondroitin sulfate - guar gum composite material to a PBS solution with pH 7.4 to obtain a carboxylated chondroitin sulfate - guar gum solution. Add the disulfide - diacrylate modified heparin - chitosan oligosaccharide composite material to a PBS solution with pH 7.4 to obtain a disulfide - diacrylate modified heparin - chitosan oligosaccharide solution. Add nano - hydroxyapatite to deionized water, and then ultrasonically treat for 30 - 40 min to obtain a nano - hydroxyapatite suspension;

[0024] S34. Slowly add the disulfide - diacrylate modified heparin - chitosan oligosaccharide solution to the carboxylated chondroitin sulfate - guar gum solution, and then stir for 2 - 3 h to obtain mixed solution D. Slowly add the nano - hydroxyapatite suspension to mixed solution D and continue to stir for 2 h to obtain mixed solution E. Add the photoinitiator Irgacure2959 to mixed solution E and stir in the dark for 30 - 40 min to obtain an outer layer material;

[0025] S35. Inject the core hydrogel precursor into the inner cavity of a concentric circle mold (inner diameter 5 mm, outer diameter 15 mm), inject the outer layer material into the outer cavity, and then irradiate with an ultraviolet lamp for 10 - 20 min to obtain a core - shell hydrogel. Immerse the core - shell hydrogel in PBS for 4 - 5 h to obtain the immersed core - shell hydrogel. Transfer the immersed core - shell hydrogel to a refrigerator at - 20 - - 10 °C and freeze for 12 - 24 h to obtain the frozen core - shell hydrogel. Place the frozen core - shell hydrogel in a freeze - dryer and freeze - dry at - 50 - - 40 °C and 10 - 20 Pa for 24 - 48 h to obtain a hydrogel component.

[0026] The mechanism of action of the above raw material components is as follows:

[0027] Carboxylated chondroitin sulfate - guar gum composite: This composite provides the basic framework structure in the hydrogel outer shell. Carboxylated chondroitin sulfate is rich in carboxyl and sulfate groups, which can form stable complexes with other components through electrostatic interactions. Meanwhile, guar gum enhances the viscoelasticity and hydration of the composite.

[0028] Disulfide - diacrylate modified heparin - chitosan oligosaccharide composite: This composite is the core responsive component of the hydrogel system. The disulfide bond structure has specific responsiveness to the intracellular reducing environment (such as high - concentration glutathione). When entering the intracellular reducing environment, it will be selectively cleaved, resulting in partial degradation of the hydrogel structure, thus realizing the controlled release of exosomes. At the same time, the highly sulfated structure of heparin can specifically bind a variety of growth factors and cytokines, helping to protect the bioactive molecules in exosomes from degradation. The diacrylate modification provides photo - crosslinking ability, enabling the hydrogel to form a stable network structure during the preparation process.

[0029] Nanoscale hydroxyapatite: This inorganic component mainly provides enhanced mechanical strength in the hydrogel outer shell, enhancing the mechanical properties of the hydrogel through composite with organic polymer materials. At the same time, it has excellent bioactivity and osteoconductivity, which can promote cell attachment and proliferation. In addition, it may participate in the formation of coordination cross - link points, cooperate with other components to construct a stable hydrogel network structure, and participate in the cell signal transduction process through the release of calcium ions.

[0030] Exosomes: As the main functional component in the core region of the hydrogel, exosomes are nanoscale membrane vesicles secreted by cells, containing abundant bioactive molecules such as proteins, nucleic acids (including mRNA, miRNA, etc.) and lipids. They can mediate inter - cellular communication and regulate a variety of biological processes, such as immune regulation, angiogenesis, anti - inflammation and tissue repair. In this hydrogel system, exosomes can be effectively released under the controlled - release mechanism triggered by the intracellular reducing environment, thus exerting their therapeutic effects.

[0031] Sodium hyaluronate: As an important part of the core region, sodium hyaluronate has excellent moisturizing and viscoelastic properties, which can provide a highly hydrated micro - environment to protect the integrity and activity of exosomes. At the same time, as a natural component of the extracellular matrix, it can promote cell migration and proliferation and participate in the tissue repair process.

[0032] Polyethylene glycol: In the core region, polyethylene glycol mainly plays the role of enhancing hydration and immune stealth. Its hydrophilicity can enhance the water - absorption capacity and stability of the hydrogel.

[0033] Albumin: As the protein component in the core region, albumin has excellent biocompatibility and biodegradability, and can provide nutritional support and osmotic pressure regulation functions.

[0034] Silk fibroin: This natural protein mainly provides mechanical support and structural stability in the core region. Its unique amino acid composition and secondary structure endow the hydrogel with good mechanical properties and biocompatibility.

[0035] Animal umbilical cord extract: As a bioactive component in the core region, animal umbilical cord extract is rich in various growth factors, cytokines, collagen, hyaluronic acid and small molecule nutrients, and can promote cell proliferation, migration and differentiation.

[0036] Centella asiatica extract: As a bioactive ingredient of plant origin, Centella asiatica extract mainly contains triterpenoids (such as asiaticoside, madecassoside and madasiatic acid, etc.), and has significant antioxidant, anti-inflammatory and collagen synthesis promoting effects.

[0037] Licorice extract: This plant extract is rich in bioactive ingredients such as glycyrrhizin, licorice chalcone and licorice flavonoid, and mainly plays anti-inflammatory, antioxidant and immunomodulatory roles in the core region.

[0038] Chamomile flower extract: As one of the plant active ingredients in the core region of the hydrogel, chamomile flower extract contains various flavonoids such as apigenin, quercetin and rutin, as well as polyphenols, and has strong antioxidant ability, can scavenge free radicals and reduce the damage of oxidative stress to exosomes and cells.

[0039] (3) Beneficial effects

[0040] Compared with the prior art, the beneficial effects of the present invention are:

[0041] 1. The hydrogel component for delivering exosomes designed by the present invention solves the problem of insufficient exosome release of traditional hydrogels in the intracellular reducing environment through a unique core-shell structure and disulfide bond-responsive mechanism. Among them, the disulfide bond-diacrylate modified heparin-chitosan oligosaccharide composite material of the outer shell has specific responsiveness to reducing agents such as high-concentration glutathione in cells, realizing the controllable release of exosomes in a specific intracellular environment, and improving the therapeutic effect and targeting.

[0042] 2. The hydrogel component of the present invention forms a network structure that has both sufficient mechanical strength and maintains responsiveness through the carboxylated chondroitin sulfate-guar gum composite material and the disulfide bond-diacrylate modified heparin-chitosan oligosaccharide composite material. This structure not only provides a stable protective environment for exosomes, but also enhances the interaction with the positively charged proteins on the surface of exosomes through the rich negative charge distribution, improves the stability of exosomes, and solves the problem of reduced exosome activity in traditional carrier systems. Description of the drawings

[0043] Figure 1 SEM image of the hydrogel component for delivering exosomes in Example 1 of the present invention. Detailed implementation manners

[0044] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0045] The test equipment and preparations in the following embodiments are as follows:

[0046] Electronic balance (Sartorius, Germany), electrothermal blast drying oven (Shanghai Fuma Experimental Equipment Co., Ltd.), electrothermal constant temperature water bath (Jiangsu Keduo), grinder (Shandong Tianfang Machinery Co., Ltd.), magnetic stirrer (Shanghai Meiyingpu), vacuum drying oven (Jiangsu Wuxi Mary), high-speed centrifuge (Shanghai Jipu), freeze dryer (Shanghai Jipu), ultrasonic disperser (Shanghai Bilang); chemical drugs and reagents were purchased from Sigma-Aldrich.

[0047] Example 1: This example discloses a hydrogel component for delivering exosomes, which has a core-shell structure and consists of an outer shell and a core region. The outer shell includes a carboxylated chondroitin sulfate-guar gum composite material, a disulfide bond-diacrylate modified heparin-chitosan oligosaccharide composite material, and nano-hydroxyapatite. The core region includes exosomes, sodium hyaluronate, polyethylene glycol, albumin, sericin, plant extract, and animal umbilical cord extract. The plant extract includes centella asiatica extract, licorice extract, and chamomile flower extract.

[0048] For the hydrogel component for delivering exosomes described above, by weight fraction, the outer shell includes the following raw materials in parts by weight: 26 parts of carboxylated chondroitin sulfate-guar gum composite material, 39 parts of disulfide bond-diacrylate modified heparin-chitosan oligosaccharide composite material, and 18 parts of nano-hydroxyapatite; the core region includes the following raw materials in parts by weight: 3 parts of exosomes, 18 parts of sodium hyaluronate, 31 parts of polyethylene glycol, 27 parts of albumin, 12 parts of sericin, 8 parts of plant extract, and 10 parts of animal umbilical cord extract. The plant extract includes the following raw materials in parts by weight: 3 parts of centella asiatica extract, 3 parts of licorice extract, and 2 parts of chamomile flower extract.

[0049] The carboxylated chondroitin sulfate - guar gum composite material is rich in carboxyl and sulfate groups, which can initially form the basic framework of the hydrogel through electrostatic interaction with the amino groups of the disulfide - diacrylate modified heparin - chitosan oligosaccharide composite material. At the same time, responsive cross - link points are constructed on this framework. These cross - link points are mainly assisted by the photocrosslinking of diacrylate groups and the coordination with nanoscale hydroxyapatite to jointly construct a stable outer shell structure. It is worth noting that the combination of carboxylated chondroitin sulfate and guar gum not only integrates the respective advantages of the two, but also produces a synergistic enhancement effect, making the formed framework have better mechanical properties and spatial network structure, which cannot be achieved by using the two materials separately.

[0050] Similarly, the combination of disulfide - diacrylate modified heparin and chitosan oligosaccharide creates a unique interface with both reduction responsiveness and positive charge distribution, and the interaction formed with the carboxylated chondroitin sulfate - guar gum composite material is stronger and more stable than when each component is used alone. When the hydrogel enters the intracellular reducing environment, reducing agents such as high - concentration glutathione will selectively cleave the disulfide bonds, resulting in the breakage of cross - link points, thus achieving the controlled release of exosomes in the core region.

[0051] The core region contains exosomes, sodium hyaluronate, polyethylene glycol, albumin, sericin, and plant extracts (centella asiatica extract, licorice extract, and chamomile extract) and animal umbilical cord extract, forming a microenvironment rich in bioactive factors. In addition, when the hydrogel components enter the intracellular reducing environment, the breakage of disulfide bonds not only leads to the release of exosomes, but also releases bioactive molecules such as sericin and plant extracts. These molecules act together with the bioactive factors in exosomes to further enhance the therapeutic effect. At the same time, the carboxyl and sulfate groups of carboxylated chondroitin sulfate form a rich negative charge distribution with the sulfate groups of heparin. This negative charge distribution can interact with the positively charged proteins on the surface of exosomes through electrostatic interaction, improving the stability of exosomes. The protection network formed by this multiple interaction is denser and more uniform than when using any single component alone, providing a more comprehensive protection for exosomes. Sodium hyaluronate, polyethylene glycol, and albumin provide a good hydration environment to protect the exosomes and the bioactive factors they carry in the core region, while sericin enhances the mechanical strength and biocompatibility of the core region. It is worth emphasizing that the co - existence of these three components forms a complementary hydration network, which can more comprehensively prevent the leakage and degradation of exosome contents compared to using any single component alone.

[0052] On the other hand, the disulfide bond-diacrylate modified heparin-chitosan oligosaccharide composite plays a crucial stimulus-responsive role in this system. The highly sulfated structure of heparin can specifically bind to a variety of growth factors and cytokines, helping to protect the bioactive molecules in exosomes from degradation. The diacrylate modification provides photocrosslinking ability, enabling the hydrogel to form a stable network structure during preparation. Plant extracts (including Centella asiatica extract, Glycyrrhiza glabra extract, and Matricaria chamomilla extract) and animal umbilical cord extract provide rich bioactive components, which act together with exosomes to further provide therapeutic effects. The combination of these extracts from different sources produces a complementary composition and synergistic efficacy, not only expanding the treatment spectrum but also enhancing the overall therapeutic effect through the mutual promotion among different bioactive components. In summary, this exosome-delivering hydrogel component with a core-shell structure, through the action of the outer shell (carboxylated chondroitin sulfate-guar gum composite, disulfide bond-diacrylate modified heparin-chitosan oligosaccharide composite, and nano-hydroxyapatite) and the core region (exosomes, sodium hyaluronate, polyethylene glycol, albumin, sericin, plant extracts, and animal umbilical cord extract), as Figure 1 shown, solves the problem of insufficient exosome release in the intracellular reducing environment of traditional hydrogels, and at the same time improves the therapeutic effect through the effects of various bioactive components.

[0053] The preparation method of the carboxylated chondroitin sulfate-guar gum composite includes:

[0054] S11. Add chondroitin sulfate to deionized water to obtain a chondroitin sulfate solution. Drop 1 mol / L NaOH solution into the chondroitin sulfate solution to adjust the pH to 8.5 to obtain the chondroitin sulfate solution with adjusted pH.

[0055] S12. Add chloroacetic acid to the chondroitin sulfate solution with adjusted pH at 2°C and stir for 15 min to obtain a mixed solution A. Add 6 mol / L NaOH solution to the mixed solution A and stir for 40 min to obtain a mixed solution B. Heat the mixed solution B to 70°C and keep it for 5 h to obtain a solution C. Cool the solution C to room temperature to obtain the cooled solution C. Add absolute ethanol to the cooled solution C to obtain a mixed solution D. Centrifuge the mixed solution D at 5000 rpm for 15 min to obtain a precipitate. Wash the precipitate 3 times with 80% ethanol to obtain the washed precipitate. Vacuum dry the washed precipitate at 40°C for 24 h to obtain carboxylated chondroitin sulfate.

[0056] S13. Add guar gum to deionized water and heat it to 70°C and stir for 2 h to obtain a guar gum solution.

[0057] S14. Add carboxylated chondroitin sulfate to PBS buffer solution with pH 7.4 to obtain mixed solution E. Add guar gum solution to mixed solution E and stir for 3 h to obtain mixed solution F. Pour mixed solution F into a flat petri dish, and control the thickness to be about 3 mm to obtain a mixed petri dish. Place the mixed petri dish in a -20 °C refrigerator and freeze for 24 h to obtain a frozen sample. Put the frozen sample into a freeze dryer and freeze-dry at -50 °C and 10 Pa for 56 h to obtain a dried sample;

[0058] S15. Immerse the dried sample in 1% glutaraldehyde PBS solution with pH 7.4 and crosslink at 6 °C for 24 h to obtain a crosslinked sample. Wash the crosslinked sample successively with a large amount of PBS and deionized water to obtain a washed sample. Freeze-dry the washed sample for 24 h to obtain a carboxylated chondroitin sulfate-guar gum composite material.

[0059] The preparation method of the disulfide bond-diacrylate modified heparin-chitosan oligosaccharide composite material comprises:

[0060] S21. Dissolve sodium heparin in deionized water to obtain a sodium heparin solution. Pass the sodium heparin solution through a column filled with Dowex50WX8 ion exchange resin to obtain resin, and then wash the resin with deionized water until the pH is neutral to obtain mixed solution A. Filter mixed solution A to obtain a filtrate. Freeze-dry the filtrate for 24 h to obtain heparinic acid powder;

[0061] S22. Add heparinic acid powder to PBS buffer solution with pH 6.0 to obtain mixed solution B. Add 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide to mixed solution B to obtain mixed solution C. Stir mixed solution C at room temperature for 2 h to obtain the stirred mixed solution C. Dissolve bis(2-methylallyl) ethoxydisulfide in dimethyl sulfoxide to obtain mixed solution D. Add mixed solution D to mixed solution C to obtain mixed solution E. Stir mixed solution E under nitrogen protection at room temperature for 48 h to obtain a reaction mixture. Load the reaction mixture into a dialysis bag and dialyze against deionized water for 56 h to obtain a dialyzed solution. Freeze-dry the dialyzed solution for 48 h to obtain disulfide bond-diacrylate modified heparin;

[0062] S23. Add chitosan to acetic acid solution to obtain a chitosan solution. Add 30% hydrogen peroxide to the chitosan solution to obtain mixed solution F. React mixed solution F at 70 °C for 3 h to obtain mixed solution H. Cool mixed solution H to room temperature, and then adjust the pH to 7.0 with sodium hydroxide to obtain a mixture. Load the mixture into a dialysis bag and dialyze against deionized water for 56 h to obtain a dialyzed mixed solution. Freeze-dry the dialyzed mixed solution for 48 h to obtain chitosan oligosaccharide;

[0063] S24. Weigh the disulfide bond - diacrylate modified heparin and add it to the PBS solution with pH 7.4 to obtain the disulfide bond - diacrylate modified heparin solution. Add chitosan oligosaccharide to the PBS solution with pH 7.4 to obtain the chitosan oligosaccharide solution. Slowly drip the chitosan oligosaccharide solution into the disulfide bond - diacrylate modified heparin solution and stir at room temperature for 3 h to obtain the mixed solution I. Add azobisisobutyronitrile to the mixed solution I to obtain the mixed solution J. Transfer the mixed solution J to a mold. Under a nitrogen environment, heat the mold to 80 °C and react for 8 h to obtain the reaction solution. Cool the reaction solution to room temperature to obtain the cooled reactant. Immerse the cooled reactant in PBS for 24 h to obtain the immersed reactant. Freeze the immersed reactant in a -20 °C refrigerator for 12 h to obtain the frozen reactant. Place the frozen reactant in a freeze dryer and freeze-dry it at -50 °C and 10 Pa for 48 h to obtain the disulfide bond - diacrylate modified heparin - chitosan oligosaccharide composite material.

[0064] A preparation method of a hydrogel component for delivering exosomes, which is applied to the described hydrogel component for delivering exosomes, comprises the following steps:

[0065] S31. Add sodium hyaluronate to deionized water to obtain the sodium hyaluronate solution. Add polyethylene glycol to the PBS solution with pH 7.4 to obtain the polyethylene glycol solution. Add human albumin to the PBS solution with pH 7.4 to obtain the albumin solution. Add sericin to the PBS solution with pH 7.4 to obtain the sericin solution. Add the extracts of Centella asiatica, Glycyrrhiza glabra, and Matricaria chamomilla to the PBS solution with pH 7.4 to obtain the mixed plant extract solution; add the animal umbilical cord extract to the PBS solution with pH 7.4 to obtain the animal umbilical cord extract solution;

[0066] S32. Mix the sodium hyaluronate solution, polyethylene glycol solution, albumin solution, sericin solution, mixed plant extract solution, and animal umbilical cord extract solution, and then stir at room temperature for 2 h to obtain the mixed solution A. Add 1 - ethyl - (3 - dimethylaminopropyl) carbodiimide and N - hydroxysuccinimide to the mixed solution A to obtain the mixed solution B. React the mixed solution B at pH 5.5 and 4 °C for 5 h to obtain the mixed solution C. Adjust the pH of the mixed solution C to 7.0 to obtain the mixed solution C with adjusted pH. Slowly add the exosome solution to the mixed solution C with adjusted pH and gently mix for 40 min to obtain the mixture. Let the mixture stand at 30 °C for 2 h to form the core hydrogel precursor;

[0067] S33. Add the carboxylated chondroitin sulfate - guar gum composite material into PBS solution with pH 7.4 to obtain a carboxylated chondroitin sulfate - guar gum solution. Add the disulfide - diacrylate modified heparin - chitosan oligosaccharide composite material into PBS solution with pH 7.4 to obtain a disulfide - diacrylate modified heparin - chitosan oligosaccharide solution. Add nano - hydroxyapatite into deionized water, and then ultrasonically treat for 40 min to obtain a nano - hydroxyapatite suspension;

[0068] S34. Slowly add the disulfide - diacrylate modified heparin - chitosan oligosaccharide solution into the carboxylated chondroitin sulfate - guar gum solution, and then stir for 3 h to obtain a mixed solution D. Slowly add the nano - hydroxyapatite suspension into the mixed solution D, continue to stir for 2 hours to obtain a mixed solution E. Add the photoinitiator Irgacure 2959 into the mixed solution E, and stir in the dark for 40 min to obtain the outer layer material;

[0069] S35. Inject the core hydrogel precursor into the inner cavity of a concentric circle mold (inner diameter 5 mm, outer diameter 15 mm), inject the outer layer material into the outer cavity, and then irradiate with an ultraviolet lamp for 20 min to obtain a core - shell hydrogel. Immerse the core - shell hydrogel in PBS for 5 h to obtain the immersed core - shell hydrogel. Transfer the immersed core - shell hydrogel to a - 20 °C refrigerator and freeze for 12 h to obtain the frozen core - shell hydrogel. Place the frozen core - shell hydrogel in a freeze - dryer and freeze - dry at - 50 °C and 10 Pa for 24 h to obtain the hydrogel component.

[0070] Example 2: This example discloses a hydrogel component for delivering exosomes, which has a core - shell structure and consists of an outer shell and a core region. The outer shell includes a carboxylated chondroitin sulfate - guar gum composite material, a disulfide - diacrylate modified heparin - chitosan oligosaccharide composite material, and nano - hydroxyapatite. The core region includes exosomes, sodium hyaluronate, polyethylene glycol, albumin, sericin, plant extracts, and animal umbilical cord extracts. The plant extracts include centella asiatica extract, glycyrrhiza glabra extract, and chamomilla recutita extract.

[0071] For the hydrogel component for delivering exosomes described above, by weight fraction, the outer shell includes the following raw materials in parts by weight: 40 parts of carboxylated chondroitin sulfate - guar gum composite material, 50 parts of disulfide - diacrylate modified heparin - chitosan oligosaccharide composite material, and 30 parts of nano - hydroxyapatite; the core region includes the following raw materials in parts by weight: 5 parts of exosomes, 30 parts of sodium hyaluronate, 40 parts of polyethylene glycol, 35 parts of albumin, 25 parts of sericin, 15 parts of plant extracts, and 15 parts of animal umbilical cord extracts. The plant extracts include the following raw materials in parts by weight: 5 parts of centella asiatica extract, 5 parts of glycyrrhiza glabra extract, and 5 parts of chamomilla recutita extract.

[0072] The preparation method of the carboxylated chondroitin sulfate - guar gum composite material and the disulfide - diacrylate modified heparin - chitosan oligosaccharide composite material in this example is the same as that in Example 1. The preparation method of a hydrogel component for delivering exosomes in this example is the same as that in Example 1.

[0073] Example 3: This example discloses a hydrogel component for delivering exosomes, which has a core - shell structure and consists of an outer shell and a core region. The outer shell includes a carboxylated chondroitin sulfate - guar gum composite material, a disulfide - diacrylate modified heparin - chitosan oligosaccharide composite material, and nano - hydroxyapatite. The core region includes exosomes, sodium hyaluronate, polyethylene glycol, albumin, sericin, plant extracts, and animal umbilical cord extracts. The plant extracts include centella asiatica extract, glycyrrhiza glabra extract, and chamomilla recutita extract.

[0074] For the hydrogel component for delivering exosomes described above, by weight fraction, the outer shell includes the following raw materials in parts by weight: 20 parts of carboxylated chondroitin sulfate - guar gum composite material, 30 parts of disulfide - diacrylate modified heparin - chitosan oligosaccharide composite material, and 10 parts of nano - hydroxyapatite; the core region includes the following raw materials in parts by weight: 1 part of exosomes, 15 parts of sodium hyaluronate, 20 parts of polyethylene glycol, 20 parts of albumin, 10 parts of sericin, 5 parts of plant extracts, and 5 parts of animal umbilical cord extracts. The plant extracts include the following raw materials in parts by weight: 1 part of centella asiatica extract, 2 parts of glycyrrhiza glabra extract, and 2 parts of chamomilla recutita extract.

[0075] The preparation method of the carboxylated chondroitin sulfate - guar gum composite material and the disulfide - diacrylate modified heparin - chitosan oligosaccharide composite material in this example is the same as that in Example 1. The preparation method of a hydrogel component for delivering exosomes in this example is the same as that in Example 1.

[0076] Example 4: This example discloses a hydrogel component for delivering exosomes, which has a core - shell structure and consists of an outer shell and a core region. The outer shell includes a carboxylated chondroitin sulfate - guar gum composite material, a disulfide - diacrylate modified heparin - chitosan oligosaccharide composite material, and nano - hydroxyapatite. The core region includes exosomes, sodium hyaluronate, polyethylene glycol, and albumin.

[0077] The described hydrogel component for delivering exosomes, by weight fraction, the outer shell comprises the following raw materials in parts by weight: 30 parts of carboxylated chondroitin sulfate - guar gum composite, 40 parts of disulfide - diacrylate modified heparin - chitosan oligosaccharide composite, and 20 parts of nano - hydroxyapatite; the core region comprises the following raw materials in parts by weight: 3 parts of exosomes, 22.5 parts of sodium hyaluronate, 30 parts of polyethylene glycol, 27.5 parts of albumin, 17.5 parts of sericin, 10 parts of plant extract, and 10 parts of animal umbilical cord extract, and the plant extract comprises the following raw materials in parts by weight: 3 parts of centella asiatica extract, 3 parts of glycyrrhiza glabra extract, and 3 parts of chamomilla recutita extract.

[0078] The preparation method of the carboxylated chondroitin sulfate - guar gum composite and the disulfide - diacrylate modified heparin - chitosan oligosaccharide composite in this example is the same as that in Example 1. The preparation method of the hydrogel component for delivering exosomes in this example is the same as that in Example 1.

[0079] Control Group 1: This example discloses a hydrogel component for delivering exosomes, which is a core - shell structure and consists of an outer shell and a core region. The outer shell comprises a disulfide - diacrylate modified heparin - chitosan oligosaccharide composite and nano - hydroxyapatite, and the core region comprises exosomes, sodium hyaluronate, polyethylene glycol, albumin, sericin, plant extract, and animal umbilical cord extract, and the plant extract comprises centella asiatica extract, glycyrrhiza glabra extract, and chamomilla recutita extract.

[0080] The described hydrogel component for delivering exosomes, by weight fraction, the outer shell comprises the following raw materials in parts by weight: 39 parts of disulfide - diacrylate modified heparin - chitosan oligosaccharide composite and 18 parts of nano - hydroxyapatite; the core region comprises the following raw materials in parts by weight: 3 parts of exosomes, 18 parts of sodium hyaluronate, 31 parts of polyethylene glycol, 27 parts of albumin, 12 parts of sericin, 8 parts of plant extract, and 10 parts of animal umbilical cord extract, and the plant extract comprises the following raw materials in parts by weight: 3 parts of centella asiatica extract, 3 parts of glycyrrhiza glabra extract, and 2 parts of chamomilla recutita extract.

[0081] The preparation method of the disulfide - diacrylate modified heparin - chitosan oligosaccharide composite in this example is the same as that in Example 1. The preparation method of the hydrogel component for delivering exosomes in this example is the same as that in Example 1.

[0082] Control group 2: This example discloses a hydrogel component for delivering exosomes, which has a core-shell structure and consists of an outer shell and a core region. The outer shell includes carboxylated chondroitin sulfate, disulfide-diacrylate modified heparin-chitosan oligosaccharide composite, and nano-hydroxyapatite. The core region includes exosomes, sodium hyaluronate, polyethylene glycol, albumin, sericin, plant extract, and animal umbilical cord extract. The plant extract includes centella asiatica extract, licorice extract, and chamomile flower extract.

[0083] For the hydrogel component for delivering exosomes described above, by weight fraction, the outer shell includes the following raw materials in parts by weight: 26 parts of carboxylated chondroitin sulfate, 39 parts of disulfide-diacrylate modified heparin-chitosan oligosaccharide composite, and 18 parts of nano-hydroxyapatite; the core region includes the following raw materials in parts by weight: 3 parts of exosomes, 18 parts of sodium hyaluronate, 31 parts of polyethylene glycol, 27 parts of albumin, 12 parts of sericin, 8 parts of plant extract, and 10 parts of animal umbilical cord extract. The plant extract includes the following raw materials in parts by weight: 3 parts of centella asiatica extract, 3 parts of licorice extract, and 2 parts of chamomile flower extract.

[0084] The preparation method of carboxylated chondroitin sulfate and disulfide-diacrylate modified heparin-chitosan oligosaccharide composite in this example is the same as that in Example 1. The preparation method of the hydrogel component for delivering exosomes in this example is the same as that in Example 1.

[0085] Control group 3: This example discloses a hydrogel component for delivering exosomes, which has a core-shell structure and consists of an outer shell and a core region. The outer shell includes guar gum, disulfide-diacrylate modified heparin-chitosan oligosaccharide composite, and nano-hydroxyapatite. The core region includes exosomes, sodium hyaluronate, polyethylene glycol, albumin, sericin, plant extract, and animal umbilical cord extract. The plant extract includes centella asiatica extract, licorice extract, and chamomile flower extract.

[0086] For the hydrogel component for delivering exosomes described above, by weight fraction, the outer shell includes the following raw materials in parts by weight: 26 parts of guar gum, 39 parts of disulfide-diacrylate modified heparin-chitosan oligosaccharide composite, and 18 parts of nano-hydroxyapatite; the core region includes the following raw materials in parts by weight: 3 parts of exosomes, 18 parts of sodium hyaluronate, 31 parts of polyethylene glycol, 27 parts of albumin, 12 parts of sericin, 8 parts of plant extract, and 10 parts of animal umbilical cord extract. The plant extract includes the following raw materials in parts by weight: 3 parts of centella asiatica extract, 3 parts of licorice extract, and 2 parts of chamomile flower extract.

[0087] The preparation method of the guar gum and disulfide-diacrylate modified heparin-chitosan oligosaccharide composite material in this example is the same as that in Example 1. The preparation method of a hydrogel component for delivering exosomes in this example is the same as that in Example 1.

[0088] Control group 4: This example discloses a hydrogel component for delivering exosomes, which is a core-shell structure and consists of an outer shell and a core region. The outer shell includes a carboxylated chondroitin sulfate-guar gum composite material and nano-hydroxyapatite. The core region includes exosomes, sodium hyaluronate, polyethylene glycol, albumin, sericin, plant extracts, and animal umbilical cord extracts. The plant extracts include centella asiatica extract, licorice extract, and chamomile flower extract.

[0089] For the described hydrogel component for delivering exosomes, by weight fraction, the outer shell includes the following raw materials in parts by weight: 26 parts of carboxylated chondroitin sulfate-guar gum composite material and 18 parts of nano-hydroxyapatite; the core region includes the following raw materials in parts by weight: 3 parts of exosomes, 18 parts of sodium hyaluronate, 31 parts of polyethylene glycol, 27 parts of albumin, 12 parts of sericin, 8 parts of plant extracts, and 10 parts of animal umbilical cord extracts. The plant extracts include the following raw materials in parts by weight: 3 parts of centella asiatica extract, 3 parts of licorice extract, and 2 parts of chamomile flower extract.

[0090] The preparation method of the carboxylated chondroitin sulfate-guar gum composite material in this example is the same as that in Example 1. The preparation method of a hydrogel component for delivering exosomes in this example is the same as that in Example 1.

[0091] Control group 5: This example discloses a hydrogel component for delivering exosomes, which is a core-shell structure and consists of an outer shell and a core region. The outer shell includes a carboxylated chondroitin sulfate-guar gum composite material, chitosan oligosaccharide, and nano-hydroxyapatite. The core region includes exosomes, sodium hyaluronate, polyethylene glycol, albumin, sericin, plant extracts, and animal umbilical cord extracts. The plant extracts include centella asiatica extract, licorice extract, and chamomile flower extract.

[0092] For the described hydrogel component for delivering exosomes, by weight fraction, the outer shell includes the following raw materials in parts by weight: 26 parts of carboxylated chondroitin sulfate-guar gum composite material, 39 parts of disulfide-diacrylate modified heparin-chitosan oligosaccharide composite material, and 18 parts of nano-hydroxyapatite; the core region includes the following raw materials in parts by weight: 3 parts of exosomes, 18 parts of sodium hyaluronate, 31 parts of polyethylene glycol, 27 parts of albumin, 12 parts of sericin, 8 parts of plant extracts, and 10 parts of animal umbilical cord extracts. The plant extracts include the following raw materials in parts by weight: 3 parts of centella asiatica extract, 3 parts of licorice extract, and 2 parts of chamomile flower extract.

[0093] The preparation method of the carboxylated chondroitin sulfate-guar gum composite material and chitosan oligosaccharide in this example is the same as that in Example 1. The preparation method of a hydrogel component for delivering exosomes in this example is the same as that in Example 1.

[0094] This example discloses a hydrogel component for delivering exosomes, which has a core-shell structure and consists of an outer shell and a core region. The outer shell includes a carboxylated chondroitin sulfate-guar gum composite material, disulfide bond-diacrylate modified heparin, and nano-hydroxyapatite. The core region includes exosomes, sodium hyaluronate, polyethylene glycol, albumin, sericin, plant extracts, and animal umbilical cord extracts. The plant extracts include centella asiatica extract, licorice extract, and chamomile flower extract.

[0095] For the described hydrogel component for delivering exosomes, by weight fraction, the outer shell includes the following raw materials in parts by weight: 26 parts of carboxylated chondroitin sulfate-guar gum composite material, 39 parts of disulfide bond-diacrylate modified heparin, and 18 parts of nano-hydroxyapatite; the core region includes the following raw materials in parts by weight: 3 parts of exosomes, 18 parts of sodium hyaluronate, 31 parts of polyethylene glycol, 27 parts of albumin, 12 parts of sericin, 8 parts of plant extracts, and 10 parts of animal umbilical cord extracts. The plant extracts include the following raw materials in parts by weight: 3 parts of centella asiatica extract, 3 parts of licorice extract, and 2 parts of chamomile flower extract.

[0096] The preparation method of the carboxylated chondroitin sulfate-guar gum composite material and disulfide bond-diacrylate modified heparin in this example is the same as that in Example 1. The preparation method of a hydrogel component for delivering exosomes in this example is the same as that in Example 1.

[0097] Experimental verification:

[0098] 1. Exosome labeling: Mix the purified exosomes with DiI fluorescent dye at a ratio of 1:200, incubate at 37 °C in the dark for 30 minutes, wash 3 times with PBS, and remove the free dye by ultra-high speed centrifugation (100,000 g, 70 minutes).

[0099] 2. Degradation kinetics study: Place the same-sized hydrogel samples (200 μL) of different examples and the control group in PBS, PBS containing glutathione, and enzyme solution respectively, incubate at 37 °C, take them out at regular time points (0 h, 6 h, 12 h, 24 h, 48 h, 72 h, 7 d), freeze-dry and weigh them, and calculate the mass loss rate.

[0100] 3. Exosome release kinetics: Place the hydrogel containing fluorescently labeled exosomes into a dialysis bag, immerse the dialysis bag in PBS, PBS containing glutathione, and enzyme solution, and shake it at a constant temperature of 37°C (60 rpm). Collect the external medium at preset time points (0 h, 3 h, 6 h, 12 h, 24 h, 48 h, 72 h, 7 d, 14 d), and measure the amount of exosomes released by fluorescence measurement.

[0101] 4. Rheological tests: Use a parallel plate rheometer to measure the storage modulus (G') and loss modulus (G") of different examples and control groups. Frequency sweep: 0.1 - 100 Hz, strain 1%, operating conditions: 37°C, parallel plate diameter 25 mm, gap 1 mm.

[0102] Table 1 Hydrogel degradation kinetics - Mass loss rate (%) in PBS

[0103]

[0104] Table 2 Hydrogel degradation kinetics - Mass loss rate (%) in PBS containing glutathione

[0105]

[0106] Table 3 Hydrogel degradation kinetics - Mass loss rate (%) in enzyme solution

[0107]

[0108] Table 4 Exosome release kinetics - Cumulative release rate (%) in PBS

[0109]

[0110] Table 5 Exosome release kinetics - Cumulative release rate (%) in PBS containing glutathione

[0111]

[0112] Table 6 Exosome release kinetics - Cumulative release rate (%) in enzyme solution

[0113]

[0114] Table 7 Rheological tests - Storage modulus G' (Pa) frequency sweep results

[0115]

[0116] Table 8 Rheological tests - Loss modulus G" (Pa) frequency sweep results

[0117]

[0118] The degradation kinetics, exosome release kinetics tests of Examples 1-4 and Control Groups 1-6 in PBS, glutathione-containing PBS and enzyme solution environments, as well as the results of rheological property tests all showed obvious regular differences. Example 1 performed best under all test conditions, with the lowest degradation rate, the slowest exosome release rate and the optimal mechanical properties. After 7 days in the PBS environment, Example 1 only degraded by 24.5%, while Control Group 4 degraded up to 56.1%; although the degradation accelerated in the glutathione-containing environment and enzyme solution, Example 1 still maintained relative stability. In the exosome release test, the cumulative release rate of Example 1 was 57.2% after 14 days in the PBS environment, significantly lower than that of the control group (>90%). Rheological data further confirmed this conclusion. Example 1 showed the highest storage modulus (G' = 485-943 Pa) in the frequency range of 0.1-100 Hz, indicating its best mechanical strength and network structure stability. The data trend shows that the synergistic effect of carboxylated chondroitin sulfate-guar gum composite and disulfide-diacrylate modified heparin-chitosan oligosaccharide composite significantly enhanced the stability, controlled release ability and mechanical properties of the hydrogel. The performance of Control Group 1 (lacking carboxylated chondroitin sulfate-guar gum composite) and Control Group 4 (lacking disulfide-diacrylate modified heparin-chitosan oligosaccharide composite) decreased significantly, further confirming the necessity of the two composites. Environmental sensitivity analysis showed that the degradation of all samples accelerated in glutathione-containing PBS (due to glutathione promoting the cleavage of disulfide bonds) and was the fastest in enzyme solution (biological enzyme degradation mechanism), but Example 1 still maintained relatively the best stability. Generally speaking, the experimental data confirmed that Example 1 (the composite with the optimal ratio) performed excellently in all indicators, providing an ideal formula for the development of biomedical hydrogels with long-acting controlled release, appropriate degradation rate and good mechanical properties.

[0119] Finally, it should be noted that although the present invention has been described in detail with reference to the foregoing embodiments, for those skilled in the art, they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent substitutions on some of the technical features. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A hydrogel component for delivering exosomes, characterized in that: It is a core-shell structure, consisting of an outer shell and a core region, wherein the outer shell comprises a carboxylated chondroitin sulfate-guar gum composite material, a disulfide bond-diacrylate modified heparin-chitooligosaccharide composite material and nano-hydroxyapatite, and the core region comprises exosomes, sodium hyaluronate, polyethylene glycol, albumin, silk colloid, plant extracts and animal umbilical cord extracts, wherein the plant extracts comprise Centella asiatica extract, licorice extract and chamomile extract.

2. A hydrogel component for delivering exosomes according to claim 1, characterized in that In terms of weight fraction, the outer shell comprises the following raw materials in parts by weight: 20 to 40 parts of carboxylated chondroitin sulfate-guar gum composite material, 30 to 50 parts of disulfide bond-diacrylate modified heparin-chitooligosaccharide composite material and 10 to 30 parts of nano-hydroxyapatite; the core region comprises the following raw materials in parts by weight: 1 to 5 parts of exosomes, 15 to 30 parts of sodium hyaluronate, 20 to 40 parts of polyethylene glycol, 20 to 35 parts of albumin, 10 to 25 parts of silk colloid protein, 5 to 15 parts of plant extract and 5 to 15 parts of animal umbilical cord extract, and the plant extract comprises the following raw materials in parts by weight: 1 to 5 parts of Centella asiatica extract, 2 to 5 parts of licorice extract and 2 to 5 parts of chamomile extract.

3. A hydrogel component for delivering exosomes as claimed in claim 2, characterized in that: The preparation method of the carboxylated chondroitin sulfate-guar and soy gum composite material comprises: S11. adding chondroitin sulfate to deionized water to obtain a chondroitin sulfate solution, and adding 1 mol / L NaOH solution to the chondroitin sulfate solution to adjust the pH to 8 to 8.5 to obtain a chondroitin sulfate solution after adjusting the pH; S12. Add chloroacetic acid at 0-4°C to the pH-adjusted chondroitin sulfate solution, and stir for 10-15 min to obtain a mixed solution A, add 6 mol / L NaOH solution to the mixed solution A, and stir for 30-40 min to obtain a mixed solution B, heat the mixed solution B to 60-70°C, and keep it for 4-5 h to obtain a solution C, cool the solution C to room temperature to obtain a cooled solution C, add anhydrous ethanol to the cooled solution C to obtain a mixed solution D, centrifuge the mixed solution D at 4000-5000 rpm for 10-15 min to obtain a precipitate, wash the precipitate with 80% ethanol for 3-4 times to obtain a washed precipitate, and vacuum dry the washed precipitate at 40-50°C for 24-48 h to obtain carboxylated chondroitin sulfate; S13. Add guar gum to deionized water, raise the temperature to 70-80° C. and stir for 2-3 h to obtain a guar gum solution; S14. Add carboxylated chondroitin sulfate to a PBS buffer solution of pH 7.4 to obtain a mixed solution E, add a guar gum solution to the mixed solution E, and stir for 2 to 3 hours to obtain a mixed solution F, pour the mixed solution F into a flat-bottomed culture dish with a thickness of about 3 to 5 mm to obtain a mixed culture dish, place the mixed culture dish in a -20 to -10°C refrigerator to freeze for 12 to 24 hours to obtain a frozen sample, place the frozen sample in a freeze dryer, and freeze-dry for 48 to 56 hours at -50 to -40°C and 10 to 20 Pa to obtain a dried sample; S15. The dried sample is immersed in a 1% glutaraldehyde PBS solution with a pH of 7.4, and cross-linked at 4 to 6°C for 12 to 24 hours to obtain a cross-linked sample, and the cross-linked sample is washed with a large amount of PBS and deionized water in sequence to obtain a washed sample, and the washed sample is freeze-dried for 24 to 48 hours to obtain a carboxylated chondroitin sulfate-guar gum composite material.

4. A hydrogel component for delivering exosomes as claimed in claim 2, characterized in that: The preparation method of the disulfide bond-diacrylate modified heparin-chitosan oligosaccharide composite material comprises: S21. dissolving heparin sodium in deionized water to obtain a heparin sodium solution, passing the heparin sodium solution through a column loaded with Dowex 50WX8 ion exchange resin to obtain a resin, and then washing the resin with deionized water to a neutral pH to obtain a mixed solution A, filtering the mixed solution A to obtain a filtrate, and freeze-drying the filtrate for 24 to 48 hours to obtain a heparin acid powder; S22. Add heparin acid powder to a PBS buffer solution of pH 6.0 to obtain a mixed solution B, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to the mixed solution B to obtain a mixed solution C, stir the mixed solution C at room temperature for 1 to 2 hours to obtain a stirred mixed solution C, dissolve bis(2-methylpropylene)ethoxy disulfide in dimethyl sulfoxide to obtain a mixed solution D, add the mixed solution D to the mixed solution C to obtain a mixed solution E, stir the mixed solution E at room temperature for 24 to 48 hours under nitrogen protection to obtain a reaction mixture, put the reaction mixture into a dialysis bag, dialyze against deionized water for 48 to 56 hours to obtain a dialyzed solution, freeze-dry the dialyzed solution for 24 to 48 hours to obtain disulfide bond-diacrylate modified heparin; S23. chitosan is added to an acetic acid solution to obtain a chitosan solution, 30% hydrogen peroxide is added to the chitosan solution to obtain a mixed solution F, the mixed solution F is reacted at 60 to 70° C. for 3 to 4 hours to obtain a mixed solution H, the mixed solution H is cooled to room temperature, and then the pH is adjusted to 7.0 with sodium hydroxide to obtain a mixture, the mixture is placed in a dialysis bag, and dialyzed against deionized water for 48 to 56 hours to obtain a dialyzed mixed solution, and the dialyzed mixed solution is freeze-dried for 24 to 48 hours to obtain chitosan oligosaccharide; S24. Weigh disulfide-diacrylate modified heparin and add it to a PBS solution at pH 7.4 to obtain a disulfide-diacrylate modified heparin solution. Add chitosan oligosaccharide to pH 7.4 PBS solution to obtain a chitosan oligosaccharide solution, slowly add the chitosan oligosaccharide solution dropwise to the disulfide bond-diacrylate modified heparin solution, and stir at room temperature for 2 to 3 hours to obtain a mixed solution I, add azobisisobutyronitrile to the mixed solution I to obtain a mixed solution J, transfer the mixed solution J to a mold, heat the mold to 65 to 80° C. under a nitrogen environment, react for 6 to 8 hours to obtain a reaction solution, cool the reaction solution to room temperature to obtain a cooled reactant, soak the cooled reactant in PBS for 24 to 48 hours to obtain a soaked reactant, freeze the soaked reactant in a -20 to -10° C. refrigerator for 12 to 24 hours to obtain a frozen reactant, put the frozen reactant into a freeze dryer, and freeze-dry for 48 to 56 hours at -50 to -40° C. and 10 to 20 Pa to obtain a disulfide bond-diacrylate modified heparin-chitosan oligosaccharide composite material.

5. A method for preparing a hydrogel component for delivering exosomes, which is used to prepare the hydrogel component for delivering exosomes as claimed in any one of claims 1 to 4, characterized in that: The method comprises the following steps: S31. Sodium hyaluronate is added to deionized water to obtain a sodium hyaluronate solution, polyethylene glycol is added to a PBS solution at pH 7.4 to obtain a polyethylene glycol solution, human albumin is added to a PBS solution at pH 7.4 to obtain an albumin solution, sericin is added to a PBS solution at pH 7.4 to obtain a sericin solution, Centella asiatica extract, licorice extract and chamomile extract are added to a PBS solution at pH 7.4 to obtain a mixed plant extract solution; an animal umbilical cord extract is added to a PBS solution at pH 7.4 to obtain an animal umbilical cord extract solution; S32. Sodium hyaluronate solution, polyethylene glycol solution, albumin solution, sericin solution, plant extract mixed solution and animal umbilical cord extract solution are mixed, and then stirred at room temperature for 1 to 2 hours to obtain a mixed solution A, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide are added to the mixed solution A to obtain a mixed solution B, the mixed solution B is stirred at pH 5.5 to 6.0 and 4 to 5° C. for 4 to 5 hours to obtain a mixed solution C, the pH of the mixed solution C is adjusted to 7.0 to obtain a pH-adjusted mixed solution C, the exosome solution is slowly added to the pH-adjusted mixed solution C, and gently mixed for 30 to 40 minutes to obtain a mixture, and the mixture is allowed to stand at 25 to 30° C. for 1 to 2 hours to form a core hydrogel precursor; S33. adding the carboxylated chondroitin sulfate-guar gum composite material to a PBS solution at pH 7.4 to obtain a carboxylated chondroitin sulfate-guar gum solution, adding the disulfide bond-diacrylate modified heparin-chitosan oligosaccharide composite material to a PBS solution at pH 7.4 to obtain a disulfide bond-diacrylate modified heparin-chitosan oligosaccharide solution, adding nano-hydroxyapatite to deionized water, and then ultrasonically treating for 30 to 40 minutes to obtain a nano-hydroxyapatite suspension; S34. Slowly add the disulfide bond-diacrylate modified heparin-chitosan oligosaccharide solution to the carboxylated chondroitin sulfate-guar gum solution, and then stir for 2 to 3 hours to obtain a mixed solution D, slowly add the nano-hydroxyapatite suspension to the mixed solution D, continue stirring for 2 hours to obtain a mixed solution E, add the photoinitiator Irgacure 2959 to the mixed solution E, and stir in the dark for 30 to 40 minutes to obtain an outer layer material; S35. Inject the core hydrogel precursor into the inner cavity of a concentric circle mold (inner diameter 5 mm, outer diameter 15 mm), inject the outer layer material into the outer cavity, and then irradiate with ultraviolet light for 10 to 20 minutes to obtain a core-shell hydrogel, soak the core-shell hydrogel in PBS for 4 to 5 hours to obtain the soaked core-shell hydrogel, transfer the soaked core-shell hydrogel to a -20 to -10°C refrigerator and freeze it for 12 to 24 hours to obtain a frozen core-shell hydrogel, place the frozen core-shell hydrogel in a freeze dryer, and freeze-dry it at -50 to -40°C and 10 to 20 Pa for 24 to 48 hours to obtain a hydrogel component.

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