Chitosan hydrogel dressing loaded with sustained-release exosome

Abstract

The invention discloses a chitosan hydrogel dressing loaded with a sustained-release exosome. The chitosan hydrogel dressing is prepared from graft modified GelMA, graft modified chitosan, poloxamer, hydroxyapatite, PEG (polyethylene glycol)-dopamine dual-modified black phosphorus quantum dots and the exosome. Quaternary ammonium salt, boric acid groups and the like are introduced through grafting modification, a multiple dynamic network containing Schiff base bonds, boric acid ester bonds and the like is formed, and the mechanical property and the self-healing capacity are improved by combining a nano reinforced phase. The dressing has ROS responsiveness and photo-thermal responsiveness, boric acid ester bonds can be broken to remove active oxygen and controllably release exosomes in a wound surface high ROS environment, quaternary ammonium salt and caffeoyl tartaric acid endow antibacterial activity, and black phosphorus quantum dots strengthen oxidation resistance and temperature regulation and control functions. The dressing overcomes the defects of poor exosome stability, uncontrollable release and natural chitosan, realizes antibacterial, antioxidant and exosome slow release synergy, and meets the complex wound repair requirements.

Description

Technical Field

[0001] This invention belongs to the field of hydrogel dressing technology, and particularly relates to a chitosan hydrogel dressing loaded with sustained-release exosomes. Background Technology

[0002] Exosomes are nanoscale membrane vesicles with a diameter of 40-160 nm secreted by cells. They are widely present in biological fluids such as blood, urine, and saliva. As biological messengers for precise intercellular communication, they play a key role in cell signal transduction and microenvironment regulation by carrying bioactive substances such as proteins, nucleic acids, and lipids. Studies have confirmed that exosomes derived from mesenchymal stem cells exhibit significant application potential in wound healing. The various active factors they carry can synergistically exert repair effects: Epidermal growth factor (EGF) at picomolar concentrations can effectively promote skin cell proliferation and migration, while simultaneously stimulating the synthesis and secretion of extracellular matrix components such as hyaluronic acid and glycoproteins, accelerating the re-epithelialization process of the wound; Hepatocyte growth factor (HGF) can enhance cell vitality, inhibit tyrosinase activity, and effectively lighten pigmentation and age spots formed after wound healing; Fibroblast growth factor (FGF) can deeply repair dermal tissue, reducing scar formation and acne scar residue; Vascular-associated microRNAs can target and promote vascular endothelial cell proliferation, activate angiogenesis, improve wound microcirculation, and ensure nutrient supply and metabolic waste removal. Furthermore, exosomes are rich in active ingredients such as insulin-like growth factor (IGF-1) and keratinocyte growth factor (KGF), which can optimize the repair microenvironment and enhance tissue regeneration by regulating signaling pathways such as Wnt and β-catenin.

[0003] However, the clinical application of exosomes still faces many technical bottlenecks: exosomes have a half-life of only 24-72 hours in vivo and are easily cleared by the liver and spleen, resulting in a short duration of effective concentration at the wound site and low bioavailability; the lipid bilayer membrane structure of exosomes is fragile and sensitive to temperature and enzymatic hydrolysis environments, the separation and purification process is complex and costly, and activity is easily lost during storage and transportation; direct application makes it difficult to achieve targeted retention and controlled release, and excessive use may also trigger immune responses or local inflammatory imbalances. These problems severely restrict the large-scale application of exosomes in wound treatment.

[0004] Chitosan, a natural alkaline polysaccharide derived from the shells of crustaceans such as shrimp and crab, is an ideal carrier material for wound dressings due to its good biocompatibility, biodegradability, weak antibacterial properties, and hemostatic effects. The amino groups in its molecular structure can interact with the negative charges on cell surfaces, promoting cell adhesion, and it also possesses a certain wound moisturizing ability, meeting the requirements of the moist wound healing theory. However, natural chitosan has significant drawbacks: poor water solubility, dissolving only under acidic conditions, limiting its application in physiological environments; insufficient mechanical strength, easily deformed and damaged in a moist state, and poor moisture barrier properties, making it difficult to meet the mechanical requirements of complex wounds; furthermore, its antibacterial activity is mild, with limited inhibitory effects on infected wounds, and it lacks the ability to scavenge damaging factors such as reactive oxygen species (ROS), failing to effectively alleviate oxidative stress damage to wounds.

[0005] Wound healing is a complex physiological process involving inflammation regulation, cell proliferation, angiogenesis, and tissue remodeling. Chronic and infected wounds, in particular, often suffer from delayed healing due to persistent inflammation, oxidative stress, and insufficient blood supply, imposing a heavy physiological and economic burden on patients. Current clinical dressings suffer from poor moisturizing properties, insufficient antibacterial activity, and a tendency to adhere to newly formed tissue. While existing hydrogel dressings offer good moisturizing and adhesion properties, they generally suffer from weak mechanical properties, lack of targeted responsiveness, and inability to achieve precise sustained release of bioactive ingredients. Although hydrogel-exosome composite systems have become a research hotspot, existing composite dressings still have shortcomings such as difficulty in controlling cross-linking density, uncontrollable exosome release rates, and a lack of synergistic antioxidant and antibacterial functions, making it difficult to meet the diverse repair needs of complex wounds. Summary of the Invention

[0006] In order to overcome the shortcomings of the prior art, this invention develops a composite hydrogel dressing that combines good mechanical properties, ROS responsiveness, antibacterial activity and exosome sustained-release function. The stability and controllable release of exosomes are solved by carrier modification, while making up for the performance defects of natural chitosan.

[0007] To achieve the above objectives, the following technical solution is adopted: This invention provides a chitosan hydrogel dressing loaded with sustained-release exosomes, composed of the following components: grafted modified GelMA, grafted modified chitosan, poloxamer, hydroxyapatite, PEG-dopamine dual-modified black phosphorus quantum dots, and exosomes; the mass ratio of the grafted modified GelMA, grafted modified chitosan, poloxamer, hydroxyapatite, and PEG-dopamine dual-modified black phosphorus quantum dots is 25-42:10-18:5-15:8-22:0.05-0.3; the final concentration of the exosomes in the hydrogel is 5×10⁻⁶. 7 -5×10 9 per mL.

[0008] Furthermore, the grafted modified GelMA is prepared through the following steps:

[0009] S1. Dissolve GelMA in PBS buffer solution at pH=7.4, add EDC·HCl and NHS, activate at room temperature for 15 min, add 4-dimethylaminobutyric acid, adjust pH to 7.0-7.5, react at 37°C in the dark for 12 h, dialyze with a dialysis bag with a molecular weight cutoff of 3500 Da for 3 days, freeze dry to obtain 4-dimethylaminobutyric acid grafted GelMA;

[0010] S2. Dissolve 4-dimethylaminobutyric acid (GelMA) in a PBS buffer solution with pH=6.5 containing 20% ​​DMF (v / v). Dissolve N-(3-bromopropyl)-3-boron benzamide and 5-(2-acetamido)-2-hydroxybenzaldehyde in a mixed solvent of anhydrous ethanol and DMF, respectively, and slowly add them dropwise to the 4-dimethylaminobutyric acid-grafted GelMA solution. Under nitrogen protection, stir at 45°C in the dark for 24 hours. Dialyze using a dialysis bag with a molecular weight cutoff of 3500 Da for 3 days, and freeze-dry to obtain grafted modified GelMA.

[0011] Furthermore, in the preparation process of the grafted modified GelMA, the mass ratio of GelMA, EDC·HCl, NHS, 4-dimethylaminobutyric acid, N-3-bromopropyl-3-boronbenzamide, and 5-2-acetamido-2-hydroxybenzaldehyde is 1.0:0.12-0.18:0.07-0.11:0.15-0.25:0.10-0.14:0.08-0.12.

[0012] Furthermore, the volume ratio of anhydrous ethanol to DMF in the mixed solvent of anhydrous ethanol and DMF is 1:1 to 1:2.

[0013] Furthermore, the grafted modified chitosan is prepared through the following steps:

[0014] Chitosan was dissolved in a 1% (v / v) aqueous acetic acid solution and bubbled with nitrogen for 15 min to remove oxygen. Caffeoyl tartaric acid was dissolved in a mixture of 1% aqueous acetic acid solution and DMF and stirred until completely dissolved. EDC·HCl and NHS were added and the caffeoyl tartaric acid was activated at room temperature for 30 min. Then it was slowly added dropwise to the chitosan solution and mixed evenly. The pH of the reaction solution was adjusted to 5.5-6.0 with triethylamine and the reaction was carried out at room temperature in the dark under nitrogen protection for 36 h. The reaction solution was poured into a large amount of acetone to precipitate the precipitate. The precipitate was collected and redissolved in a 1% aqueous acetic acid solution. The precipitate was dialyzed for 3 days using a dialysis bag with a molecular weight cutoff of 8000-14000 Da and then freeze-dried to obtain grafted modified chitosan.

[0015] Furthermore, in the preparation process of the grafted modified chitosan, the mass ratio of chitosan, caffeoyl tartaric acid, EDC·HCl, and NHS is 1.0:0.30-0.40:0.20-0.30:0.12-0.18.

[0016] Furthermore, the volume ratio of the aqueous acetic acid solution to DMF in the mixture of aqueous acetic acid and DMF is 3:1.

[0017] Furthermore, the gel dressing is prepared by the following steps:

[0018] P1. Disperse PEG-dopamine dual-modified black phosphorus quantum dots in PBS buffer solution at pH 7.4, add 0.01% (w / v) ascorbic acid, and sonicate for 10 min for later use;

[0019] P2. Grafted modified GelMA and grafted modified chitosan were added to dimethyl sulfoxide, heated to 100-120℃, and the pH was controlled at 4-6. The reaction was carried out for 12-24 hours. After removing the solvent dimethyl sulfoxide by vacuum distillation, the solution was dissolved again in a PBS buffer solution with pH=7.4 containing the photoinitiator LAP. The solution was stirred until completely dissolved to prepare a homogeneous mixed solution with a total concentration of 5% (w / v). Poloxamer, hydroxyapatite nanoparticles and PEG-dopamine dual-modified black phosphorus quantum dots treated in step P1 were added in sequence.

[0020] P3. Inject the uniformly mixed prepolymer obtained in step P2 into the mold, irradiate it under blue light at a wavelength of 405nm for 30s to initiate free radical polymerization, and then transfer it to a constant temperature oven at 37℃ for incubation for 2-4h.

[0021] P4. Preparation concentration is 10 10 -10 11 Mesenchymal stem cell-derived exosomes in PBS solution per mL were used to remove the basal gel formed in step P3 from the mold and completely immerse it in the exosome solution. The mixture was then incubated at 4°C for 12-24 hours.

[0022] P5. Remove the exosome-loaded gel and gently rinse the surface of unadsorbed exosomes with sterile PBS buffer solution to obtain the chitosan hydrogel dressing loaded with sustained-release exosomes.

[0023] Furthermore, in step P2, the mass fraction of the photoinitiator LAP in the PBS buffer solution is 0.2%-0.5%.

[0024] Furthermore, the exosomes are human umbilical cord mesenchymal stem cell exosomes.

[0025] Furthermore, the ultrasonic dispersion time is 8-18 minutes, and the irradiation intensity of 405nm blue light is 8-15mW / cm².2 The irradiation time is 6-35s, the constant temperature incubation time at 37℃ is 2.5-5h, and the exosome loading incubation time at 4℃ is 15-26h.

[0026] Furthermore, the PEG-dopamine dual-modified black phosphorus quantum dots are prepared through the following steps:

[0027] (1) Take 10-20 mg of block black phosphorus crystals and add them to a centrifuge tube containing 50 mL of N-methylpyrrolidone. Place the tube in an ice-water bath and sonicate for 6-10 h. Then, centrifuge the resulting suspension to collect the supernatant and filter it through a 0.22 μm microporous membrane. Concentrate the resulting liquid to 1 / 5 of the original volume to obtain a concentrated black phosphorus quantum dot NMP dispersion.

[0028] (2) Take 5-8 mL of concentrated black phosphorus quantum dot NMP dispersion, dissolve 20-40 mg of dopamine hydrochloride in 5 mL of Tris-HCl buffer solution with pH=8.5, and slowly add it dropwise to the black phosphorus quantum dot dispersion under nitrogen protection. React at room temperature in the dark for 12-18 h, dialyze with a dialysis bag with a molecular weight cutoff of 3500 Da for 24-48 h, freeze dry, and obtain dopamine modified black phosphorus quantum dots;

[0029] (3) Disperse the dopamine-modified black phosphorus quantum dots obtained in step (2) in 10 mL of PBS buffer solution with pH=7.4. Dissolve 50-100 mg of methoxy-polyethylene glycol-succinimide ester in 2 mL of PBS buffer solution and slowly add it dropwise to the above quantum dot dispersion under stirring. React in the dark for 24-48 h. Dialyze with ultrapure water for 36-72 h using a dialysis bag with a molecular weight cutoff of 14000 Da. Freeze dry to obtain PEG-dopamine dual-modified black phosphorus quantum dots.

[0030] The beneficial effects of this invention are:

[0031] This invention utilizes grafted modified GelMA, grafted modified chitosan, hydroxyapatite, poloxamer, PEG-dopamine dual-modified black phosphorus quantum dots, and exosomes to create a gel dressing with a three-dimensional structure. The exosomes are loaded into the gel through a combination of physical adsorption and network embedding, achieving slow and continuous release under ROS regulation, thus prolonging the effective action time and reducing the frequency of drug administration. This gel can efficiently scavenge reactive oxygen species and release exosomes in response to excess reactive oxygen species and temperature response effects, exhibiting better anti-inflammatory and wound-healing effects.

[0032] This invention grafts GelMA with 4-dimethylaminobutyric acid (DMPA), and then reacts DMPA with N-(3-bromopropyl)-3-boronbenzamide and 5-(2-acetamido)-2-hydroxybenzaldehyde to form a quaternary ammonium salt structure, simultaneously introducing boric acid and aldehyde groups. Furthermore, it modifies chitosan by grafting caffeoyl tartaric acid, introducing catechol and polycarboxyl groups. Catechol can form dynamic borate esters with boric acid, while the carboxyl groups improve the dispersion of hydroxyapatite and black phosphorus quantum dots in the gel matrix, and the aldehyde groups... It can form Schiff bases with the amino groups on chitosan, forming a three-dimensional network structure through the above effects. The gel contains Schiff base bonds, borate ester bonds, hydrogen bonds, π-π stacking, and photo-initiated covalent crosslinking, forming multiple dynamic networks. This structure gives the hydrogel a certain self-healing ability and stress relaxation properties, enabling it to adapt to deformation during wound activity and reduce mechanical damage to new tissue. Hydroxyapatite and black phosphorus quantum dots, as nano-reinforcing phases, can disperse stress and improve the toughness and load-bearing capacity of the gel, making it less prone to breakage while maintaining softness.

[0033] The borate ester bonds in the dressing network are formed by the boric acid groups of modified GelMA and the catechol structure on chitosan. They are highly sensitive to reactive oxygen species. Under the high ROS environment of the wound, the borate ester bonds can undergo oxidative breakage, and the catechol structure is oxidized to a quinone structure, causing dynamic rearrangement of the network, effectively consuming local ROS and reducing oxidative stress damage. Furthermore, the network structure can be loosened to achieve controlled release of exosomes. In addition, the PEG-dopamine modified black phosphorus quantum dots also have ROS scavenging ability, further enhancing the antioxidant function of the dressing.

[0034] The quaternary ammonium salt structure introduced into the modified GelMA can disrupt bacterial cell membranes, giving the gel contact antibacterial ability; the caffeoyl tartaric acid grafted chitosan interferes with bacterial metabolism and inhibits biofilm formation through the synergistic effect of its phenolic hydroxyl and carboxyl groups. At the same time, the positively charged property of the gel as a whole can capture bacteria through electrostatic adsorption and limit their diffusion. Through ROS scavenging, it can inhibit the persistent inflammatory response caused by excessive ROS and indirectly weaken the microenvironment for bacterial proliferation.

[0035] Black phosphorus quantum dots with PEG-dopamine dual modification exhibit good photothermal conversion efficiency and can generate mild heat under near-infrared light irradiation, giving the gel temperature-responsive properties. By dual modification of black phosphorus quantum dots with PEG and dopamine, their dispersion stability and biocompatibility in the aqueous phase are significantly improved, and their potential toxicity is reduced. Attached Figure Description

[0036] Figure 1Infrared spectral images of GelMA, 4-dimethylaminobutyric acid-grafted GelMA, grafted modified GelMA, chitosan, and grafted modified chitosan prepared in Example 3.

[0037] Figure 2 The results of the free radical scavenging rate test of the hydrogel dressing extract of the present invention;

[0038] Figure 3 These are the results of in vitro antibacterial performance tests of the hydrogel dressing of this invention;

[0039] Figure 4 These are the test results of the exosome loading capacity of the hydrogel dressing of the present invention;

[0040] Figure 5 The results of the cell migration rate test for the hydrogel dressing of the present invention;

[0041] Figure 6 The results of the relative cell proliferation rate test for the hydrogel dressing of the present invention;

[0042] Figure 7 The results show the wound healing rate of the hydrogel dressing of this invention.

[0043] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof. Detailed Implementation

[0044] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention. The preferred embodiments and materials described herein are for illustrative purposes only and do not limit the scope of this application.

[0046] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all purchased from commercial channels. In the specific embodiments of the present invention, human umbilical cord mesenchymal stem cell exosomes were purchased from Shanghai Maclean Biochemical Technology Co., Ltd., and Staphylococcus aureus (BNCC381462), Escherichia coli (BNCC336435), human immortalized keratinocytes (BNCC339817), and L929 mouse fibroblasts (BNCC342604) were all purchased from Beina Chuanglian Biotechnology Co., Ltd.

[0047] Example 1:

[0048] A chitosan hydrogel dressing loaded with sustained-release exosomes comprises the following components: grafted modified GelMA, grafted modified chitosan, poloxamer, hydroxyapatite, PEG-dopamine dual-modified black phosphorus quantum dots, and exosomes; the mass ratio of the grafted modified GelMA, grafted modified chitosan, poloxamer, hydroxyapatite, and PEG-dopamine dual-modified black phosphorus quantum dots is 25:10:5:8:0.05; the final concentration of the exosomes in the hydrogel is 5 × 10⁻⁶. 7 The exosomes are human umbilical cord mesenchymal stem cell exosomes.

[0049] The grafted modified GelMA is prepared through the following steps:

[0050] S1. Dissolve GelMA in PBS buffer solution at pH=7.4, add EDC·HCl and NHS, activate at room temperature for 15 min, add 4-dimethylaminobutyric acid, adjust pH to 7.0, react at 37°C in the dark for 12 h, dialyze with a dialysis bag with a molecular weight cutoff of 3500 Da for 3 days, freeze dry to obtain 4-dimethylaminobutyric acid grafted GelMA;

[0051] S2. Dissolve 4-dimethylaminobutyric acid (GelMA) in a PBS buffer solution with pH=6.5 containing 20% ​​DMF (v / v). Dissolve N-(3-bromopropyl)-3-boron benzamide and 5-(2-acetamido)-2-hydroxybenzaldehyde in a mixed solvent of anhydrous ethanol and DMF, respectively, and slowly add them dropwise to the 4-dimethylaminobutyric acid-grafted GelMA solution. Under nitrogen protection, stir at 45°C in the dark for 24 hours. Dialyze using a dialysis bag with a molecular weight cutoff of 3500 Da for 3 days, and freeze-dry to obtain grafted modified GelMA.

[0052] In the preparation of the grafted modified GelMA, the mass ratio of GelMA, EDC·HCl, NHS, 4-dimethylaminobutyric acid, N-3-bromopropyl-3-boronbenzamide, and 5-2-acetamido-2-hydroxybenzaldehyde is 1.0:0.12:0.07:0.15:0.10:0.08; and the volume ratio of anhydrous ethanol to DMF in the mixed solvent of anhydrous ethanol and DMF is 1:1.

[0053] The grafted modified chitosan is prepared through the following steps:

[0054] Chitosan was dissolved in a 1% (v / v) aqueous acetic acid solution and bubbled with nitrogen for 15 min to remove oxygen. Caffeoyl tartaric acid was dissolved in a mixture of 1% aqueous acetic acid solution and DMF and stirred until completely dissolved. EDC·HCl and NHS were added and the caffeoyl tartaric acid was activated at room temperature for 30 min. Then it was slowly added dropwise to the chitosan solution and mixed evenly. The pH of the reaction solution was adjusted to 5.5 with triethylamine and the reaction was carried out at room temperature in the dark under nitrogen protection for 36 h. The reaction solution was poured into a large amount of acetone to precipitate the precipitate. The precipitate was collected and redissolved in a 1% aqueous acetic acid solution. The precipitate was dialyzed for 3 days using a dialysis bag with a molecular weight cutoff of 8000 Da and then freeze-dried to obtain grafted modified chitosan.

[0055] In the preparation of the grafted modified chitosan, the mass ratio of chitosan, caffeoyl tartaric acid, EDC·HCl, and NHS is 1.0:0.30:0.20:0.12; the volume ratio of acetic acid aqueous solution to DMF in the mixture of acetic acid aqueous solution and DMF is 3:1.

[0056] The gel dressing is prepared by the following steps:

[0057] P1. Disperse PEG-dopamine dual-modified black phosphorus quantum dots in PBS buffer solution at pH 7.4, add 0.01% (w / v) ascorbic acid, and sonicate for 10 min for later use;

[0058] P2. Grafted modified GelMA and grafted modified chitosan were added to dimethyl sulfoxide, heated to 100℃, and the pH was controlled at 4. The reaction was carried out for 12 hours. After removing the solvent dimethyl sulfoxide by vacuum distillation, the solution was dissolved again in a PBS buffer solution with pH=7.4 containing the photoinitiator LAP. The solution was stirred until completely dissolved to prepare a homogeneous mixed solution with a total concentration of 5% (w / v). Poloxamer, hydroxyapatite nanoparticles and PEG-dopamine dual-modified black phosphorus quantum dots treated in step P1 were added in sequence.

[0059] P3. Inject the uniformly mixed prepolymer obtained in step P2 into the mold, irradiate it under blue light at a wavelength of 405nm for 30s to initiate free radical polymerization, and then transfer it to a constant temperature oven at 37℃ for 2h incubation.

[0060] P4. Preparation concentration is 10 10 Mesenchymal stem cell-derived exosomes were prepared in PBS solution at 1 / mL. The basal gel formed in step P3 was removed from the mold and completely immersed in the exosome solution, and incubated at 4°C for 12 h.

[0061] P5. Remove the exosome-loaded gel and gently rinse the surface of unadsorbed exosomes with sterile PBS buffer solution to obtain the chitosan hydrogel dressing loaded with sustained-release exosomes.

[0062] In step P2, the mass fraction of photoinitiator LAP in the PBS buffer solution is 0.2%; the ultrasonic dispersion time is 8 min; the irradiation intensity of 405 nm blue light is 8 mW / cm²; the irradiation time is 35 s; the constant temperature incubation time at 37℃ is 2.5 h; and the exosome loading incubation time at 4℃ is 15 h.

[0063] The PEG-dopamine dual-modified black phosphorus quantum dots were prepared by the following steps:

[0064] (1) Take 10 mg of block black phosphorus crystals and add them to a centrifuge tube containing 50 mL of N-methylpyrrolidone. Place the tube in an ice-water bath and sonicate for 6 h. Then, centrifuge the resulting suspension to collect the supernatant and filter it through a 0.22 μm microporous membrane. Concentrate the resulting liquid to 1 / 5 of the original volume to obtain a concentrated black phosphorus quantum dot NMP dispersion.

[0065] (2) Take 5 mL of concentrated black phosphorus quantum dot NMP dispersion, dissolve 20 mg of dopamine hydrochloride in 5 mL of Tris-HCl buffer solution with pH=8.5, and slowly add it dropwise to the black phosphorus quantum dot dispersion under nitrogen protection. React at room temperature in the dark for 12 h, dialyze with a dialysis bag with a molecular weight cutoff of 3500 Da for 24 h, freeze dry, and obtain dopamine modified black phosphorus quantum dots;

[0066] (3) The dopamine-modified black phosphorus quantum dots obtained in step (2) were dispersed in 10 mL of PBS buffer solution with pH=7.4. 50 mg of methoxy-polyethylene glycol-succinimide ester was dissolved in 2 mL of PBS buffer solution and slowly added dropwise to the above quantum dot dispersion under stirring. The reaction was carried out in the dark for 24 h. The mixture was dialyzed with ultrapure water for 36 h using a dialysis bag with a molecular weight cutoff of 14000 Da and then freeze-dried to obtain PEG-dopamine dual-modified black phosphorus quantum dots.

[0067] Example 2:

[0068] A chitosan hydrogel dressing loaded with sustained-release exosomes comprises the following components: grafted modified GelMA, grafted modified chitosan, poloxamer, hydroxyapatite, PEG-dopamine dual-modified black phosphorus quantum dots, and exosomes; the mass ratio of the grafted modified GelMA, grafted modified chitosan, poloxamer, hydroxyapatite, and PEG-dopamine dual-modified black phosphorus quantum dots is 42:18:15:22:0.3; the final concentration of the exosomes in the hydrogel is 5 × 10⁻⁶. 9 The exosomes are human umbilical cord mesenchymal stem cell exosomes.

[0069] The grafted modified GelMA is prepared through the following steps:

[0070] S1. Dissolve GelMA in PBS buffer solution at pH=7.4, add EDC·HCl and NHS, activate at room temperature for 15 min, add 4-dimethylaminobutyric acid, adjust pH to 7.5, react at 37°C in the dark for 12 h, dialyze in a dialysis bag with a molecular weight cutoff of 3500 Da for 3 days, freeze dry to obtain 4-dimethylaminobutyric acid grafted GelMA;

[0071] S2. Dissolve 4-dimethylaminobutyric acid (GelMA) in a PBS buffer solution with pH=6.5 containing 20% ​​DMF (v / v). Dissolve N-(3-bromopropyl)-3-boron benzamide and 5-(2-acetamido)-2-hydroxybenzaldehyde in a mixed solvent of anhydrous ethanol and DMF, respectively, and slowly add them dropwise to the 4-dimethylaminobutyric acid-grafted GelMA solution. Under nitrogen protection, stir at 45°C in the dark for 24 hours. Dialyze using a dialysis bag with a molecular weight cutoff of 3500 Da for 3 days, and freeze-dry to obtain grafted modified GelMA.

[0072] In the preparation of the grafted modified GelMA, the mass ratio of GelMA, EDC·HCl, NHS, 4-dimethylaminobutyric acid, N-3-bromopropyl-3-boronbenzamide, and 5-2-acetamido-2-hydroxybenzaldehyde is 1.0:0.18:0.11:0.25:0.14:0.12; and the volume ratio of anhydrous ethanol to DMF in the mixed solvent of anhydrous ethanol and DMF is 1:2.

[0073] The grafted modified chitosan is prepared through the following steps:

[0074] Chitosan was dissolved in a 1% (v / v) aqueous acetic acid solution and bubbled with nitrogen for 15 min to remove oxygen. Caffeoyl tartaric acid was dissolved in a mixture of 1% aqueous acetic acid solution and DMF and stirred until completely dissolved. EDC·HCl and NHS were added and the caffeoyl tartaric acid was activated at room temperature for 30 min. Then it was slowly added dropwise to the chitosan solution and mixed evenly. The pH of the reaction solution was adjusted to 6.0 with triethylamine and the reaction was carried out at room temperature in the dark under nitrogen protection for 36 h. The reaction solution was poured into a large amount of acetone to precipitate the precipitate. The precipitate was collected and redissolved in a 1% aqueous acetic acid solution. The precipitate was dialyzed for 3 days using a dialysis bag with a molecular weight cutoff of 14000 Da and then freeze-dried to obtain grafted modified chitosan.

[0075] In the preparation of the grafted modified chitosan, the mass ratio of chitosan, caffeoyl tartaric acid, EDC·HCl, and NHS is 1.0:0.40:0.30:0.18; the volume ratio of acetic acid aqueous solution to DMF in the mixture of acetic acid aqueous solution and DMF is 3:1.

[0076] The gel dressing is prepared by the following steps:

[0077] P1. Disperse PEG-dopamine dual-modified black phosphorus quantum dots in PBS buffer solution at pH 7.4, add 0.01% (w / v) ascorbic acid, and sonicate for 10 min for later use;

[0078] P2. Grafted modified GelMA and grafted modified chitosan were added to dimethyl sulfoxide, heated to 120°C, and the pH was controlled at 6. The reaction was carried out for 24 hours. After removing the solvent dimethyl sulfoxide by vacuum distillation, the solution was dissolved again in a PBS buffer solution with pH=7.4 containing the photoinitiator LAP. The solution was stirred until completely dissolved to prepare a homogeneous mixed solution with a total concentration of 5% (w / v). Poloxamer, hydroxyapatite nanoparticles and PEG-dopamine dual-modified black phosphorus quantum dots treated in step P1 were added in sequence.

[0079] P3. Inject the uniformly mixed prepolymer obtained in step P2 into the mold, irradiate it under blue light at a wavelength of 405nm for 30s to initiate free radical polymerization, and then transfer it to a constant temperature oven at 37℃ for 4h incubation.

[0080] P4. Preparation concentration is 10 11 Mesenchymal stem cell-derived exosomes in PBS solution were prepared by removing the basal gel formed in step P3 from the mold and completely immersing it in the exosome solution, and incubating it at 4°C for 24 h.

[0081] P5. Remove the exosome-loaded gel and gently rinse the surface of unadsorbed exosomes with sterile PBS buffer solution to obtain the chitosan hydrogel dressing loaded with sustained-release exosomes.

[0082] In step P2, the mass fraction of photoinitiator LAP in the PBS buffer solution is 0.5%; the ultrasonic dispersion time is 18 min; the irradiation intensity of 405 nm blue light is 15 mW / cm²; the irradiation time is 6 s; the constant temperature incubation time at 37℃ is 5 h; and the exosome loading incubation time at 4℃ is 26 h.

[0083] The PEG-dopamine dual-modified black phosphorus quantum dots were prepared by the following steps:

[0084] (1) Take 20 mg of block black phosphorus crystals and add them to a centrifuge tube containing 50 mL of N-methylpyrrolidone. Place the tube in an ice-water bath and sonicate for 10 h. Then, centrifuge the resulting suspension to collect the supernatant and filter it through a 0.22 μm microporous membrane. Concentrate the resulting liquid to 1 / 5 of the original volume to obtain a concentrated black phosphorus quantum dot NMP dispersion.

[0085] (2) Take 8 mL of concentrated black phosphorus quantum dot NMP dispersion, dissolve 40 mg of dopamine hydrochloride in 5 mL of Tris-HCl buffer solution with pH=8.5, and slowly add it dropwise to the black phosphorus quantum dot dispersion under nitrogen protection. React at room temperature in the dark for 18 h, dialyze with a dialysis bag with a molecular weight cutoff of 3500 Da for 48 h, freeze dry, and obtain dopamine modified black phosphorus quantum dots;

[0086] (3) The dopamine-modified black phosphorus quantum dots obtained in step (2) were dispersed in 10 mL of PBS buffer solution with pH=7.4. 100 mg of methoxy-polyethylene glycol-succinimide ester was dissolved in 2 mL of PBS buffer solution and slowly added dropwise to the above quantum dot dispersion under stirring. The reaction was carried out in the dark for 48 h. The mixture was dialyzed with ultrapure water for 72 h using a dialysis bag with a molecular weight cutoff of 14000 Da and then freeze-dried to obtain PEG-dopamine dual-modified black phosphorus quantum dots.

[0087] Example 3:

[0088] A chitosan hydrogel dressing loaded with sustained-release exosomes comprises the following components: grafted modified GelMA, grafted modified chitosan, poloxamer, hydroxyapatite, PEG-dopamine dual-modified black phosphorus quantum dots, and exosomes; the mass ratio of the grafted modified GelMA, grafted modified chitosan, poloxamer, hydroxyapatite, and PEG-dopamine dual-modified black phosphorus quantum dots is 35:15:10:15:0.15; the final concentration of the exosomes in the hydrogel is 5 × 10⁻⁶. 8 The exosomes are human umbilical cord mesenchymal stem cell exosomes.

[0089] The grafted modified GelMA is prepared through the following steps:

[0090] S1. Dissolve GelMA in PBS buffer solution at pH=7.4, add EDC·HCl and NHS, activate at room temperature for 15 min, add 4-dimethylaminobutyric acid, adjust pH to 7.2, react at 37°C in the dark for 12 h, dialyze with a dialysis bag with a molecular weight cutoff of 3500 Da for 3 days, freeze dry to obtain 4-dimethylaminobutyric acid grafted GelMA;

[0091] S2. Dissolve 4-dimethylaminobutyric acid (GelMA) in a PBS buffer solution with pH=6.5 containing 20% ​​DMF (v / v). Dissolve N-(3-bromopropyl)-3-boron benzamide and 5-(2-acetamido)-2-hydroxybenzaldehyde in a mixed solvent of anhydrous ethanol and DMF, respectively, and slowly add them dropwise to the 4-dimethylaminobutyric acid-grafted GelMA solution. Under nitrogen protection, stir at 45°C in the dark for 24 hours. Dialyze using a dialysis bag with a molecular weight cutoff of 3500 Da for 3 days, and freeze-dry to obtain grafted modified GelMA.

[0092] In the preparation of the grafted modified GelMA, the mass ratio of GelMA, EDC·HCl, NHS, 4-dimethylaminobutyric acid, N-3-bromopropyl-3-boronbenzamide, and 5-2-acetamido-2-hydroxybenzaldehyde is 1.0:0.15:0.09:0.20:0.12:0.10; and the volume ratio of anhydrous ethanol to DMF in the mixed solvent of anhydrous ethanol and DMF is 1:1.5.

[0093] The grafted modified chitosan is prepared through the following steps:

[0094] Chitosan was dissolved in a 1% (v / v) aqueous acetic acid solution and bubbled with nitrogen for 15 min to remove oxygen. Caffeoyl tartaric acid was dissolved in a mixture of 1% aqueous acetic acid solution and DMF and stirred until completely dissolved. EDC·HCl and NHS were added and the caffeoyl tartaric acid was activated at room temperature for 30 min. Then it was slowly added dropwise to the chitosan solution and mixed evenly. The pH of the reaction solution was adjusted to 5.8 with triethylamine and the reaction was carried out at room temperature in the dark under nitrogen protection for 36 h. The reaction solution was poured into a large amount of acetone to precipitate the precipitate. The precipitate was collected and redissolved in a 1% aqueous acetic acid solution. The precipitate was dialyzed for 3 days using a dialysis bag with a molecular weight cutoff of 12000 Da and then freeze-dried to obtain grafted modified chitosan.

[0095] In the preparation of the grafted modified chitosan, the mass ratio of chitosan, caffeoyl tartaric acid, EDC·HCl, and NHS is 1.0:0.35:0.25:0.15; the volume ratio of acetic acid aqueous solution to DMF in the mixture of acetic acid aqueous solution and DMF is 3:1.

[0096] The gel dressing is prepared by the following steps:

[0097] P1. Disperse PEG-dopamine dual-modified black phosphorus quantum dots in PBS buffer solution at pH 7.4, add 0.01% (w / v) ascorbic acid, and sonicate for 10 min for later use;

[0098] P2. Grafted modified GelMA and grafted modified chitosan were added to dimethyl sulfoxide, heated to 110°C, and the pH was controlled at 5. The reaction was carried out for 18 hours. After removing the solvent dimethyl sulfoxide by vacuum distillation, the solution was dissolved again in a PBS buffer solution with pH=7.4 containing the photoinitiator LAP. The solution was stirred until completely dissolved to prepare a homogeneous mixed solution with a total concentration of 5% (w / v). Poloxamer, hydroxyapatite nanoparticles and PEG-dopamine dual-modified black phosphorus quantum dots treated in step P1 were added in sequence.

[0099] P3. Inject the uniformly mixed prepolymer obtained in step P2 into the mold, irradiate it under blue light at a wavelength of 405nm for 30s to initiate free radical polymerization, and then transfer it to a constant temperature oven at 37℃ for 3h incubation.

[0100] P4. The prepared concentration is 5 × 10⁻⁶. 10 Mesenchymal stem cell-derived exosomes in PBS solution were prepared by removing the basal gel formed in step P3 from the mold and immersing it completely in the exosome solution for 18 h at 4 °C.

[0101] P5. Remove the exosome-loaded gel and gently rinse the surface of unadsorbed exosomes with sterile PBS buffer solution to obtain the chitosan hydrogel dressing loaded with sustained-release exosomes.

[0102] In step P2, the mass fraction of photoinitiator LAP in the PBS buffer solution is 0.35%; the ultrasonic dispersion time is 12 min, the irradiation intensity of 405 nm blue light is 12 mW / cm², the irradiation time is 20 s, the constant temperature incubation time at 37℃ is 3.5 h, and the exosome loading incubation time at 4℃ is 20 h.

[0103] The PEG-dopamine dual-modified black phosphorus quantum dots were prepared by the following steps:

[0104] (1) Take 15 mg of block black phosphorus crystals and add them to a centrifuge tube containing 50 mL of N-methylpyrrolidone. Place the tube in an ice-water bath and sonicate for 8 h. Then, centrifuge the resulting suspension to collect the supernatant and filter it through a 0.22 μm microporous membrane. Concentrate the resulting liquid to 1 / 5 of the original volume to obtain a concentrated black phosphorus quantum dot NMP dispersion.

[0105] (2) Take 6.5 mL of concentrated black phosphorus quantum dot NMP dispersion, dissolve 30 mg of dopamine hydrochloride in 5 mL of Tris-HCl buffer solution with pH=8.5, and slowly add it dropwise to the black phosphorus quantum dot dispersion under nitrogen protection. React at room temperature in the dark for 15 h, dialyze with a dialysis bag with a molecular weight cutoff of 3500 Da for 36 h, freeze dry, and obtain dopamine modified black phosphorus quantum dots;

[0106] (3) The dopamine-modified black phosphorus quantum dots obtained in step (2) were dispersed in 10 mL of PBS buffer solution with pH=7.4. 75 mg of methoxy-polyethylene glycol-succinimide ester was dissolved in 2 mL of PBS buffer solution and slowly added dropwise to the above quantum dot dispersion under stirring. The reaction was carried out in the dark for 36 h. The mixture was dialyzed with ultrapure water for 54 h using a dialysis bag with a molecular weight cutoff of 14000 Da and then freeze-dried to obtain PEG-dopamine dual-modified black phosphorus quantum dots.

[0107] Comparative Example 1:

[0108] In this comparative example, unmodified GelMA was used to replace grafted modified GelMA, and the remaining components and proportions were the same as in Example 3.

[0109] Comparative Example 2:

[0110] In this comparative example, unmodified chitosan was used to replace grafted modified chitosan, while the remaining components and proportions remained the same as in Example 3.

[0111] Comparative Example 3:

[0112] In this comparative example, unmodified black phosphorus quantum dots were used to replace PEG-dopamine dual-modified black phosphorus quantum dots, while the remaining components and proportions remained the same as in Example 3.

[0113] Comparative Example 4:

[0114] In this comparative example, PEG-dopamine dual-modified black phosphorus quantum dots were removed, while the remaining components and proportions remained the same as in Example 3.

[0115] Comparative Example 5:

[0116] In this comparative example, traditional chitosan-GelMA hydrogels (none of which were grafted or modified, and none of which were PEG-dopamine-modified black phosphorus quantum dots) were used to load exosomes of the same concentration (the preparation process only involved blue light polymerization), and the remaining components and proportions were consistent with those in Example 3.

[0117] Results Analysis

[0118] Test Example 1: Infrared Image Characterization of the Synthesis Process

[0119] The GelMA, 4-dimethylaminobutyric acid-grafted GelMA, grafted modified GelMA, chitosan, and grafted modified chitosan prepared in Example 3 of this invention were characterized by infrared-visible spectroscopy. The images are shown below. Figure 1 As shown, a comparison of images of GelMA grafted with 4-dimethylaminobutyric acid and GelMA is presented, at 2774, 2778, and 2787 cm⁻¹. -1 The absorbance at 2809, 2906, 80101, and 2928 significantly increased, proving the successful introduction of the tertiary amine group. The absorbance at 2800–3000 cm⁻¹ was also observed. -1 The generally increased absorbance in the region proves the successful alkyl linking of 4-dimethylaminobutyric acid, at 3250 and 3255 cm⁻¹. -1 The decrease in absorbance at 1028 and 1073 cm⁻¹ indicates that the amino groups of GelMA were consumed in the reaction; compared with the previous two images, the absorbance of the grafted GelMA decreased at 1028 and 1073 cm⁻¹. -1 The position is quaternary ammonium CN. + The stretching vibration peaks and the significant increase in absorbance at two locations indicate the formation of quaternary ammonium salts (1335 and 1355 cm⁻¹). -1 The peak at 3744 cm⁻¹ corresponds to the BO stretching vibration of borate, and the absorbance increases slightly, indicating the successful introduction of the borate group. (Cyclophorus values: 3744, 33249 cm⁻¹) -1 The CO stretching peak of the phenolic hydroxyl group is indicated by the increased absorbance, which proves the introduction of the phenolic hydroxyl group. In summary, this proves that the grafted modified GelMA was successfully synthesized. Figure 1 Image comparison of chitosan and grafted modified chitosan, 1530 and 1545 cm⁻¹ -1 The absorbance at 1661 and 1591 cm⁻¹ represents the amino bending vibration peak, and the significant decrease in absorbance indicates that the amino group has been consumed and the grafting reaction has occurred. -1 These are characteristic peaks of amide I and amide II, respectively. Chitosan does not show a clear signal of these peaks. The modified peaks retain their shape, and the absorbance changes conform to the formation rules of amide bonds, confirming the presence of amide bonds in the grafted product. (3032, 3039 cm⁻¹) -1 The peaks at 1707 and 1726 cm⁻¹ represent the CH stretching vibrations of the benzene ring, confirming the introduction of the benzene ring structure. -1 The peak at the C=O stretching vibration of the ester carbonyl group indicates that the ester group of caffeoyl tartaric acid has been introduced, thus proving the successful synthesis of grafted chitosan.

[0120] Test Example 2: Physical and Mechanical Properties Testing of Hydrogel Dressings

[0121] The hydrogel dressings prepared in Example 3 and the comparative examples were subjected to the following tests and characterizations:

[0122] Rheological testing: Measure the storage modulus and loss modulus of the hydrogel to evaluate its mechanical strength, elasticity, and self-healing ability.

[0123] Compression / Tension Test: Determine the compressive modulus and fracture stress / strain of the hydrogel to evaluate its toughness.

[0124] Swelling rate and degradation rate: Weigh a certain mass (W0) of freeze-dried hydrogel sample and immerse the dried gel sample in 20 times its volume of the following two solutions: a) PBS (pH 7.4); b) PBS (pH 7.4) containing 5 mg H2O2. Place in a shaker at 37°C (50 rpm). After 14 days, remove the sample, gently blot off the surface moisture with filter paper, and weigh immediately (W0). t The swelling ratio (SR) is calculated using the following formula: SR(%) = [W t -W0) / W0]×100%.

[0125] The solution was replaced regularly with fresh solution to maintain enzyme activity and ROS concentration. After 14 days, the sample was removed, washed with PBS, freeze-dried, and weighed (W). t, dry).

[0126] Remaining mass percentage (Rm) formula: Rm(%) = (W t, dry / W0)×100%.

[0127] The test results are shown in Table 1.

[0128] Table 1 Comparison of test results for rheological, compressive properties, swelling, and degradation behavior of hydrogels

[0129] project Example 3 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Comparative Example 5 Energy storage modulus G' (kPa, 1Hz) 8.5 3.2 4.1 7.8 7.0 2.0 Compression modulus (kPa) 45.2 18.5 22.7 42.8 40.1 12.3 Fracture stress (kPa) 120.5 52.3 61.8 115.0 105.3 35.5 Fracture strain (%) 85 65 70 82 80 55 Swelling rate (%, PBS) 580 1050 920 590 640 1250 <![CDATA[Swelling ratio (%, 5 mM H2O2)]]> 1250 1100 950 1180 980 1280 Remaining mass (%, PBS) 65 40 48 63 62 30 <![CDATA[Residual mass (%, 5 mM H2O2)]]> 35 38 46 38 50 28

[0130] As shown in Table 1, the storage modulus and compressive properties (modulus, fracture stress / strain) of Example 3 were significantly higher than those of all comparative examples. This confirms that the multi-dynamic cross-linked network constructed through graft modification and nano-reinforcement effectively improves the stiffness and toughness of the hydrogel.

[0131] In the PBS buffer solution containing H2O2, only Example 3 and Comparative Example 3 showed significantly higher swelling rates than the PBS environment, with Example 3 exhibiting more severe swelling, indicating that both borate ester bonds and black phosphorus quantum dots contribute to the ROS responsiveness. Comparative Example 4 showed a significantly lower ROS-responsive swelling rate. Regarding degradation, Example 3 degraded fastest in the H2O2 environment, significantly faster than in the PBS environment and other comparative examples, further demonstrating its ROS-responsive cleavage characteristics.

[0132] Test Example 3: Biofunctional Activity Test

[0133] The free radical scavenging rate of the hydrogel dressing extracts from Example 3 and each comparative example was determined by the DPPH / ABTS free radical scavenging experiment. The test results are shown below. Figure 2 .

[0134] like Figure 2 As shown, Example 3 of the present invention exhibits a stronger free radical scavenging ability, proving that the modified GelMA, modified chitosan, and PEG-dopamine dual-modified black phosphorus quantum dots in the present invention all contribute to the free radical scavenging ability.

[0135] Test Example 4: In vitro antibacterial performance test:

[0136] For Staphylococcus aureus and Escherichia coli, the inhibition zone method was used for testing. The diameter of the inhibition zone was observed and measured. The test results are shown below. Figure 3 .like Figure 3 As shown, Example 3 produced obvious inhibition zones against both bacteria, demonstrating broad-spectrum antibacterial activity.

[0137] Test Example 5: Exosome Loading, Release, and Bioactivity Validation

[0138] The change in protein content in the solution before and after loading was quantified using the BCA protein method, and the exosome loading was calculated.

[0139] The cell migration assay was used to test the scratch closure rate of human immortalized keratinocytes (HaCaT) treated with different groups of hydrogel release media.

[0140] The hydrogel loaded with exosomes (100 mg) was immersed in 2 mL of serum-free culture medium and placed in a shaker at 37°C. The release media were collected after 24 hours of incubation under normal conditions (no stimulation) and under simulated ROS conditions containing 100 μM H2O2.

[0141] HaCaT cells were used at a rate of 5 × 10 5 Seeds were planted at a density of / wells in 24-well plates and cultured until a dense monolayer formed. Using a 200 μL sterile pipette tip, a straight line was drawn perpendicular to the bottom of the plate across the monolayer of cells. The cells were then gently washed twice with PBS to remove any floating cells.

[0142] Experimental groups: serum-free culture medium (negative control); culture medium containing 10% FBS (positive control); release medium collected under normal conditions; and release medium collected under ROS conditions.

[0143] Add 500 μL of the appropriate treatment medium to each well. Take images of the scratches at the same location under a microscope at 0 and 24 hours. Measure the scratch area using ImageJ software and calculate the cell migration rate (%).

[0144] The test results are shown below. Figure 4 and Figure 5 .like Figure 4 Example 3 showed the highest exosome loading, thanks to its dense network structure with multiple adsorption sites. Figure 5As shown, in the normal release medium, all groups loaded with exosomes were able to promote cell migration; under ROS stimulation, the migration-promoting effect of the release medium in Example 3 was significantly enhanced, with the healing rate increasing from 68.5% to 85.3%, indicating that it can responsively accelerate the release of biologically active exosomes under ROS conditions.

[0145] Test Example 6:

[0146] In vitro cell compatibility and wound healing evaluation

[0147] Sterile hydrogel samples were immersed in DMEM medium containing 10% FBS (extraction ratio 0.1 g / mL) at 37°C and 5% CO2 for 24 hours. The supernatant was collected, filtered through a 0.22 μm filter for sterilization, and then co-cultured with L929 mouse fibroblasts using the CCK-8 assay. Cytotoxicity was assessed by relative cell proliferation rate (RGR, %). Results are as follows: Figure 6 As shown, the RGR of all hydrogel groups is higher than 90%, indicating that the present invention and the comparative materials have good biocompatibility.

[0148] Forty-two SD rats were used and acclimatized for one week before the experiment. The rats were anesthetized by intraperitoneal injection of 3% sodium pentobarbital (40 mg / kg). The backs were shaved and disinfected. Using a sterile biopsy punch, an 8 mm diameter full-thickness skin defect was created on each side of the spine on the rat's back. The rats were randomly divided into seven groups: comparative groups 1-5, example group 3, and a control group. The control group received no treatment or was covered with sterile gauze. Immediately after surgery, the corresponding group's dressing was applied to the wound and secured with transparent dressings and elastic bandages. The dressings were changed every two days, and the wound condition was observed and recorded. On postoperative days 7 and 14, the wounds were photographed using a digital camera at a fixed distance and under fixed lighting conditions. The outline of the unhealed wound in each photograph was delineated using ImageJ software, and the wound healing rate (%) was calculated. The results are as follows: Figure 7 As shown, Example 3 exhibited the fastest wound healing speed, with a near-complete healing rate by day 14, significantly outperforming all comparative examples and the control group.

[0149] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

[0150] The present invention and its embodiments have been described above. This description is not restrictive, and the embodiments shown are only one of the embodiments of the present invention. The actual application is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar methods and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. A chitosan hydrogel dressing loaded with sustained-release exosomes, characterized in that: The product is composed of the following components: grafted modified GelMA, grafted modified chitosan, poloxamer, hydroxyapatite, PEG-dopamine dual-modified black phosphorus quantum dots, and exosomes; the mass ratio of the grafted modified GelMA, grafted modified chitosan, poloxamer, hydroxyapatite, and PEG-dopamine dual-modified black phosphorus quantum dots is 25-42:10-18:5-15:8-22:0.05-0.3; the final concentration of the exosomes in the hydrogel is 5 × 10⁻⁶. 7 -5×10 9 cells / mL; The grafted modified GelMA is prepared through the following steps: S1. Dissolve GelMA in PBS buffer solution at pH=7.4, add EDC·HCl and NHS, activate at room temperature for 15 min, add 4-dimethylaminobutyric acid, adjust pH to 7.0-7.5, react at 37°C in the dark for 12 h, dialyze with a dialysis bag with a molecular weight cutoff of 3500 Da for 3 days, freeze dry to obtain 4-dimethylaminobutyric acid grafted GelMA; S2. Dissolve 4-dimethylaminobutyric acid (GelMA) in a PBS buffer solution with pH=6.5 containing 20% ​​DMF (v / v). Dissolve N-(3-bromopropyl)-3-boron benzamide and 5-(2-acetamido)-2-hydroxybenzaldehyde in a mixed solvent of anhydrous ethanol and DMF, respectively, and slowly add them dropwise to the 4-dimethylaminobutyric acid-grafted GelMA solution. Under nitrogen protection, stir at 45°C in the dark for 24 hours. Dialyze using a dialysis bag with a molecular weight cutoff of 3500 Da for 3 days, and freeze-dry to obtain grafted modified GelMA.

2. The chitosan hydrogel dressing loaded with sustained-release exosomes according to claim 1, characterized in that: In the preparation of the grafted modified GelMA, the mass ratio of GelMA, EDC·HCl, NHS, 4-dimethylaminobutyric acid, N-3-bromopropyl-3-boronbenzamide, and 5-2-acetamido-2-hydroxybenzaldehyde is 1.0:0.12-0.18:0.07-0.11:0.15-0.25:0.10-0.14:0.08-0.

12.

3. The chitosan hydrogel dressing loaded with sustained-release exosomes according to claim 2, characterized in that: The volume ratio of anhydrous ethanol to DMF in the mixed solvent of anhydrous ethanol and DMF is 1:1 to 1:

2.

4. The chitosan hydrogel dressing loaded with sustained-release exosomes according to claim 3, characterized in that: The grafted modified chitosan is prepared through the following steps: Chitosan was dissolved in a 1% (v / v) aqueous acetic acid solution and bubbled with nitrogen for 15 min to remove oxygen. Caffeoyl tartaric acid was dissolved in a mixture of 1% aqueous acetic acid solution and DMF and stirred until completely dissolved. EDC·HCl and NHS were added and the caffeoyl tartaric acid was activated at room temperature for 30 min. Then it was slowly added dropwise to the chitosan solution and mixed evenly. The pH of the reaction solution was adjusted to 5.5-6.0 with triethylamine and the reaction was carried out at room temperature in the dark under nitrogen protection for 36 h. The reaction solution was poured into a large amount of acetone to precipitate the precipitate. The precipitate was collected and redissolved in a 1% aqueous acetic acid solution. The precipitate was dialyzed for 3 days using a dialysis bag with a molecular weight cutoff of 8000-14000 Da and then freeze-dried to obtain grafted modified chitosan.

5. The chitosan hydrogel dressing loaded with sustained-release exosomes according to claim 4, characterized in that: In the preparation of the grafted modified chitosan, the mass ratio of chitosan, caffeoyl tartaric acid, EDC·HCl and NHS is 1.0:0.30-0.40:0.20-0.30:0.12-0.

18.

6. The chitosan hydrogel dressing loaded with sustained-release exosomes according to claim 5, characterized in that: The volume ratio of the aqueous acetic acid solution to DMF in the mixture of the aqueous acetic acid solution and DMF is 3:

1.

7. The chitosan hydrogel dressing loaded with sustained-release exosomes according to claim 6, characterized in that: The gel dressing is prepared by the following steps: P1. Disperse PEG-dopamine dual-modified black phosphorus quantum dots in PBS buffer solution at pH 7.4, add 0.01% (w / v) ascorbic acid, and sonicate for 10 min for later use; P2. Grafted modified GelMA and grafted modified chitosan were added to dimethyl sulfoxide, heated to 100-120℃, and the pH was controlled at 4-6. The reaction was carried out for 12-24 hours. After removing the solvent dimethyl sulfoxide by vacuum distillation, the solution was dissolved again in a PBS buffer solution with pH=7.4 containing the photoinitiator LAP. The solution was stirred until completely dissolved to prepare a homogeneous mixed solution with a total concentration of 5% (w / v). Poloxamer, hydroxyapatite nanoparticles and PEG-dopamine dual-modified black phosphorus quantum dots treated in step P1 were added in sequence. P3. Inject the uniformly mixed prepolymer obtained in step P2 into the mold, irradiate it under blue light at a wavelength of 405nm for 30s to initiate free radical polymerization, and then transfer it to a constant temperature oven at 37℃ for incubation for 2-4h. P4. Preparation concentration is 10 10 -10 11 Mesenchymal stem cell-derived exosomes in PBS solution per mL were used to remove the basal gel formed in step P3 from the mold and completely immerse it in the exosome solution. The mixture was then incubated at 4°C for 12-24 hours. P5. Remove the exosome-loaded gel and gently rinse the surface of unadsorbed exosomes with sterile PBS buffer solution to obtain the chitosan hydrogel dressing loaded with sustained-release exosomes.

8. The chitosan hydrogel dressing loaded with sustained-release exosomes according to claim 7, characterized in that: In step P2, the mass fraction of the photoinitiator LAP in the PBS buffer solution is 0.2%-0.5%.

9. A chitosan hydrogel dressing loaded with sustained-release exosomes according to claim 8, characterized in that: The exosomes are human umbilical cord mesenchymal stem cell exosomes.

10. A chitosan hydrogel dressing loaded with sustained-release exosomes according to claim 9, characterized in that: The ultrasonic dispersion time is 8-18 minutes, and the irradiation intensity of 405nm blue light is 8-15 mW / cm². 2 The irradiation time is 6-35s, the constant temperature incubation time at 37℃ is 2.5-5h, and the exosome loading incubation time at 4℃ is 15-26h.

Images