Nampt-enriched wound repair exosome hydrogel and preparation method thereof

Abstract

The application relates to the field of biomedical materials, and particularly discloses a nampt-rich wound repair exosome hydrogel and a preparation method thereof, which comprises the following steps: inducing human umbilical cord mesenchymal stem cells under low-oxygen conditions and in a nampt-rich culture medium containing nicotinamide and deferoxamine to obtain a nampt-rich exosome suspension; mixing oxidized sodium alginate, carboxymethyl chitosan and a gelatin-tannic acid solution, adding the nampt-rich exosome suspension, zinc-modified montmorillonite and quercetin-chitosan particles, removing bubbles through centrifugation, and solidifying at room temperature to obtain the hydrogel. The nampt protein is enriched, the zinc-modified montmorillonite and the quercetin-chitosan particles are introduced, a hydrogel system with antibacterial, antioxidant and repair triple synergistic effects is constructed, the stability and biological activity of the exosome are significantly improved, the preparation process is mild, the hydrogel system is suitable for repairing chronic non-healing wounds, and has a good clinical application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials, specifically to a wound repair exosome hydrogel rich in nampt and its preparation method. Background Technology

[0002] Repair of chronic, refractory wounds (such as diabetic foot ulcers and pressure sores) is hindered by factors such as persistent inflammation, oxidative stress, and impaired cellular energy metabolism, resulting in poor clinical treatment outcomes. While exosomes, as intercellular communication carriers, can promote wound healing, natural exosomes have poor stability and limited content of functional active ingredients, making it difficult to meet therapeutic needs.

[0003] Nicotinamide phosphoribosyltransferase (nampt) is nicotinamide adenine dinucleotide (NAD) + nampt, a rate-limiting enzyme in NAD+ synthesis, plays a crucial role in regulating cellular energy metabolism, combating oxidative stress, and promoting tissue repair. It can upregulate NAD+. + Activating deacetylases such as SIRT1 at a certain level promotes macrophage polarization towards the M2 type, reduces inflammation, and accelerates wound healing. However, directly applying free nampt to wound healing presents challenges such as poor stability, easy degradation, and potential immunogenicity, limiting its clinical application.

[0004] Hydrogels, as wound dressings, possess excellent biocompatibility, controllable physicochemical properties, and sustained drug release capabilities, making them one of the main carriers for exosome delivery. However, existing exosome hydrogels mostly employ single thermosensitive or photocrosslinking systems, which suffer from drawbacks such as complex preparation processes, the need for additional crosslinking agents or specific triggering conditions (e.g., temperature changes, ultraviolet irradiation), and difficulty in achieving stable exosome loading and controllable release. Furthermore, existing technologies largely focus on the physical encapsulation of exosomes, neglecting to enhance the biological effects of exosomes through induction strategies to enrich specific functional proteins (such as nampt). Based on the above, this invention provides a nampt-rich wound repair exosome hydrogel and its preparation method. Summary of the Invention

[0005] To address the problems of complex preparation processes, limited content of functional active ingredients, and poor stability of free nampt in existing exosome hydrogels, this invention provides a wound repair exosome hydrogel rich in nampt and its preparation method.

[0006] In a first aspect, the present invention provides a method for preparing a wound repair exosome hydrogel rich in nampt, using the following technical solution: A method for preparing a nampt-rich exosome hydrogel for wound repair includes the following steps: S1. Human umbilical cord mesenchymal stem cells were seeded into the basic culture medium and cultured to 80-90% confluence. The medium was then replaced with enrichment medium and induced to culture under hypoxic conditions. The cell supernatant was collected, centrifuged, filtered, and ultrafiltered to obtain an exosome suspension rich in nampt. S2. Dissolve sodium oxidized alginate in PBS buffer to obtain sodium oxidized alginate solution; dissolve carboxymethyl chitosan in PBS buffer to obtain carboxymethyl chitosan solution; S3. Mix sodium alginate oxidized solution, carboxymethyl chitosan solution and gelatin-tannic acid solution evenly, adjust pH to obtain mixed solution A; S4. Add the exosome suspension rich in nampt, zinc-modified montmorillonite and quercetin-chitosan particles to mixed solution A, mix well, centrifuge to remove air bubbles, and obtain the prepolymer solution. S5. Inject the prepolymer into the mold and allow it to solidify at room temperature to obtain a wound repair exosome hydrogel rich in nampt.

[0007] Preferably, in step S1, the human umbilical cord mesenchymal stem cells are P3-P5 generation, and the seeding density is 1×10⁻⁶. 4 pcs / cm² - 5×10 4 pcs / cm²

[0008] Preferably, the basal culture medium in step S1 is DMEM / F12 medium containing 10% exosome-free fetal bovine serum.

[0009] Preferably, the enrichment medium in step S1 consists of DMEM / F12 medium, nicotinamide, and deferoxamine, with the nicotinamide concentration being 1-5 mM and the deferoxamine concentration being 50-200 μM.

[0010] Preferably, the low-oxygen condition in step S1 refers to an oxygen concentration of 1-5%.

[0011] Preferably, the method for preparing oxidized sodium alginate in step S2 is as follows: Sodium periodate solution was added dropwise to sodium alginate solution and stirred to react; ethylene glycol was added and stirred to terminate the reaction; after dialysis and freeze-drying, sodium alginate oxide was obtained.

[0012] Preferably, the specific preparation method of sodium alginate oxidation in step S2 is as follows: With a molar ratio of sodium alginate to sodium periodate of 1:0.2-0.5, add 0.05-0.2 mol / L sodium periodate solution dropwise to 1-3 wt% sodium alginate solution, controlling the addition time to 1-2 h. Stir the reaction at room temperature and 100-200 rpm for 4-8 h, then add ethylene glycol with a molar ratio of ethylene glycol to sodium periodate of 1:1-2, and continue stirring for 20-40 min to terminate the reaction. Transfer the reaction solution to an 8-14 kDa dialysis bag and dialyze with 100-200 times the volume of deionized water at room temperature for 36-48 h, changing the deionized water every 6-8 h. Pre-freeze the dialysis product at -80℃ for 1-2 h, then freeze-dry at -50℃ to -60℃ and 10-20 Pa for 24-48 h to obtain oxidized sodium alginate.

[0013] Preferably, in step S2, the mass fraction of the oxidized sodium alginate solution is 1-5%; and the mass fraction of the carboxymethyl chitosan solution is 2-5%.

[0014] Preferably, in step S3, the volume ratio of sodium alginate solution, carboxymethyl chitosan solution and gelatin-tannic acid solution is 1-2:1-2:0.5-1; and the pH is adjusted to 7.2-7.5.

[0015] Preferably, the gelatin-tannic acid solution is composed of a 5-15 wt% gelatin solution and a 10-30 mg / mL tannic acid solution in a volume ratio of 5-10:1.

[0016] Preferably, the method for preparing zinc-modified montmorillonite in step S4 is as follows: Montmorillonite was ultrasonically dispersed in deionized water, zinc salt was added, the mixture was stirred and reacted, and then centrifuged, washed, and freeze-dried to obtain zinc-modified montmorillonite.

[0017] Preferably, the specific preparation method of zinc-modified montmorillonite in step S4 is as follows: Montmorillonite was added to deionized water and ultrasonically dispersed at 200-300W for 30-50 min at room temperature to obtain a 1-5 wt% montmorillonite suspension. Zinc salt (zinc salt to montmorillonite mass ratio of 1:5-10) was then added and stirred at 200-400 rpm for 4-6 h at room temperature. After centrifugation at 3000-5000 rpm for 10-20 min, the precipitate was collected and washed 3-5 times with deionized water. The washed precipitate was pre-frozen at -80℃ for 2-4 h and then freeze-dried at -50℃ to -60℃ and 10-20 Pa for 24-48 h to obtain zinc-modified montmorillonite.

[0018] Preferably, the zinc salt is selected from one or more of zinc chloride, zinc nitrate, and zinc sulfate.

[0019] Preferably, the method for preparing quercetin-chitosan particles in step S4 is as follows: Chitosan was dissolved in acetic acid solution, and the pH was adjusted to obtain a chitosan solution. Quercetin was dissolved in anhydrous ethanol to obtain a quercetin solution. The quercetin solution was added dropwise to the chitosan solution, and the mixture was stirred to obtain a reaction solution. The reaction solution was added dropwise to sodium tripolyphosphate solution, and the mixture was stirred continuously. After centrifugation, the precipitate was collected, washed, and freeze-dried to obtain quercetin-chitosan particles.

[0020] Preferably, the specific preparation method of the quercetin-chitosan particles in step S4 is as follows: Chitosan was dissolved in 0.5-2 wt% acetic acid solution, and the pH was adjusted to 4.0-5.0 with 0.1 M NaOH to obtain a chitosan solution of 1-3 mg / mL. Quercetin was dissolved in 70% anhydrous ethanol to obtain a quercetin solution of 1-2 mg / mL. The quercetin solution was added dropwise to the chitosan solution at a volume ratio of 1:3-5, with the addition time controlled at 20-30 min. The mixture was then stirred at 200-400 rpm for 40-60 min at room temperature to obtain the reaction solution. The reaction solution was then prepared by adding the quercetin solution dropwise at a volume ratio of 1-2: 1. Add the reaction solution dropwise to a 0.5-2 mg / mL sodium tripolyphosphate solution, controlling the dropwise addition time to 40-60 min. At room temperature, continue stirring at 200-400 rpm for 30-60 min, then centrifuge at 8000-12000 rpm for 15-30 min, collect the precipitate, wash it 3-5 times with deionized water, pre-freeze the washed precipitate at -80℃ for 2-4 h, and then freeze-dry it at -50℃ to -60℃ and 10-20 Pa for 24-48 h to obtain quercetin-chitosan particles.

[0021] Preferably, in step S4, the mass ratio of the exosome suspension rich in nampt, zinc-modified montmorillonite, quercetin-chitosan particles, and mixed solution A is 1:3-5:2-4:10-20; the centrifugation speed is 2000-4000 rpm, and the centrifugation time is 2-5 min.

[0022] Preferably, in step S5, the mold material is polytetrafluoroethylene, and the room temperature curing time is 30-90 seconds.

[0023] Secondly, the present invention provides a nampt-rich wound repair exosome hydrogel prepared by the method described above.

[0024] In summary, the present invention has the following beneficial effects: 1. Significantly enhances exosome functional activity and yield This invention employs a dual induction strategy using hypoxia conditions and enrichment culture medium. Nicotinamide (a namptylamine substrate) and deferoxamine (a hypoxia mimic) are added to DMEM / F12 medium to synergistically activate the NAD⁺ metabolic pathway in stem cells. This not only significantly increases exosome production but also enriches namptylamine within the exosomes. These pre-functionalized exosomes exhibit enhanced biological activity in promoting cell proliferation and angiogenesis, solving the problem of limited content of active ingredients in natural exosomes.

[0025] 2. Construct a multifunctional and collaborative intelligent repair microenvironment This invention integrates a hydrogel with dual functional modules: zinc-modified montmorillonite and quercetin-chitosan particles. The quercetin-chitosan particles continuously release quercetin, which can scavenge reactive oxygen species, inhibit inflammatory factors, and alleviate oxidative stress. The zinc-modified montmorillonite utilizes its adsorption properties to trap exosomes, while zinc ions exert broad-spectrum antibacterial effects and promote epithelial cell migration. The synergistic effect of these two components provides dual protection against bacteria and oxidation, preventing exosomes from being consumed by bacteria or reactive oxygen species.

[0026] 3. Form a triple-synergistic repair closed loop In this invention, zinc-modified montmorillonite, quercetin-chitosan particles, and namptyl-rich exosomes form a close synergistic relationship: zinc-modified montmorillonite can inhibit wound infection and reduce exosome consumption; quercetin-chitosan particles can scavenge reactive oxygen species and protect exosome activity; and namptyl-rich exosomes can efficiently exert NAD⁺ metabolic regulation in a low-infection, low-oxidation microenvironment, promoting macrophage M2 polarization. The synergistic effect of these three components constructs a complete therapeutic loop of infection inhibition, oxidative stress scavenging, and active repair.

[0027] 4. Achieving efficient exosome loading and long-lasting sustained release This invention utilizes sodium alginate oxidized with carboxymethyl chitosan via a Schiff base reaction to construct a hydrogel framework, and introduces gelatin-tannic acid to enhance the network structure. This system effectively protects exosomes from enzymatic degradation. More importantly, zinc-modified montmorillonite can electrostatically adsorb and immobilize exosomes within the gel network, avoiding burst release effects and achieving long-term, controllable release of exosomes, thus prolonging the drug's duration of action.

[0028] 5. The preparation process is simple and mild, which is conducive to maintaining the activity of exosomes. This invention utilizes the Schiff base reaction to achieve spontaneous crosslinking at room temperature, eliminating the need for external crosslinking agents, ultraviolet light, or heating as triggering conditions. This avoids the damage to exosomes caused by traditional photocrosslinking or thermal crosslinking, thus preserving their biological activity to the greatest extent. Simultaneously, this exosome hydrogel exhibits excellent injectability and self-healing properties, adapting to wounds of different shapes, making it convenient to use and showing promising clinical application prospects. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention.

[0030] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0031] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0032] Among them, exosome-free fetal bovine serum was purchased from Beijing Bolede Biotechnology Co., Ltd., catalog number: EXO-FBS-50A-1; DMEM / F12 culture medium was purchased from Wuhan Saios Biotechnology Co., Ltd., product number: MED-1006; Carboxymethyl chitosan was purchased from Zhejiang Jinke Pharmaceutical Co., Ltd. Montmorillonite was purchased from Tuoyi New Materials (Guangzhou) Co., Ltd. Chitosan was purchased from Aladdin, catalog number: C1516215, with a degree of deacetylation ≥90% and endotoxin ≤0.05 EU / mg; Quercetin was purchased from Nanjing Dausif Biotechnology Co., Ltd., CAS: 117-39-5; The nampt protein was purchased from Shanghai Newp Biotechnology Co., Ltd.

[0033] Example 1 A method for preparing a nampt-rich exosome hydrogel for wound repair includes the following steps: S1, P3 generation human umbilical cord mesenchymal stem cells were administered at a rate of 1×10⁻⁶. 4 Cells were seeded at a density of cells / cm² into DMEM / F12 medium containing 10% exosome-free fetal bovine serum and cultured to 80% confluence. The medium was then replaced with enrichment medium (composed of DMEM / F12 medium, 1 mM nicotinamide, and 50 μM deferoxamine) and induced to culture at 37°C and 1% oxygen for 36 h. The cell supernatant was collected, centrifuged at 300 × g for 10 min at 4°C, filtered through a 0.45 μm filter membrane, and concentrated by 100 kDa ultrafiltration to 1 / 10 of the original volume of the supernatant to obtain an exosome suspension rich in nampt. S2. Dissolve sodium oxidized alginate in PBS buffer to obtain a 1 wt% sodium oxidized alginate solution; dissolve carboxymethyl chitosan in PBS buffer to obtain a 2 wt% carboxymethyl chitosan solution. S3. Mix sodium alginate solution, carboxymethyl chitosan solution and gelatin-tannic acid solution (composed of 5wt% gelatin solution and 10mg / mL tannic acid solution in a volume ratio of 5:1) evenly according to a volume ratio of 1:1:0.5. Adjust the pH to 7.2 with 0.1M NaOH solution to obtain mixed solution A. S4. Add the exosome suspension, zinc-modified montmorillonite, and quercetin-chitosan particles to mixed solution A at a mass ratio of 1:3:2:10 and mix well. Centrifuge at 2000 rpm for 2 min at room temperature to remove air bubbles and obtain the prepolymer solution. S5. Inject the prepolymer liquid into a polytetrafluoroethylene mold and allow it to stand at room temperature for 30 seconds to cure, thereby obtaining a wound repair exosome hydrogel rich in nampt.

[0034] The specific preparation method of sodium alginate oxide is as follows: With a molar ratio of sodium alginate to sodium periodate of 1:0.2, a 0.05 mol / L sodium periodate solution was added dropwise to a 1 wt% sodium alginate solution over a period of 1 hour. The mixture was stirred at 100 rpm for 4 hours at room temperature. Then, ethylene glycol was added at a molar ratio of 1:1 to sodium periodate, and the mixture was stirred for another 20 minutes to terminate the reaction. The reaction solution was transferred to an 8 kDa dialysis bag and dialyzed with 100 times the volume of deionized water at room temperature for 36 hours, with the deionized water being replaced every 6 hours. The dialysate was pre-frozen at -80°C for 1 hour and then freeze-dried at -50°C and 10 Pa for 24 hours to obtain oxidized sodium alginate.

[0035] The specific preparation method of zinc-modified montmorillonite is as follows: Montmorillonite was added to deionized water and ultrasonically dispersed at 200W for 30 min at room temperature to obtain a 1 wt% montmorillonite suspension. Zinc chloride (zinc chloride to montmorillonite mass ratio of 1:5) was then added, and the mixture was stirred at 200 rpm for 4 h at room temperature. After centrifugation at 3000 rpm for 10 min, the precipitate was collected and washed three times with deionized water. The washed precipitate was pre-frozen at -80℃ for 2 h and then freeze-dried at -50℃ and 10 Pa for 24 h to obtain zinc-modified montmorillonite.

[0036] The specific preparation method of quercetin-chitosan particles is as follows: Chitosan was dissolved in 0.5 wt% acetic acid solution, and the pH was adjusted to 4.0 with 0.1 M NaOH to obtain a 1 mg / mL chitosan solution. Quercetin was dissolved in 70% anhydrous ethanol to obtain a 1 mg / mL quercetin solution. The quercetin solution was added dropwise to the chitosan solution at a volume ratio of 1:3, with the addition time controlled at 20 min. The mixture was stirred at 200 rpm for 40 min at room temperature to obtain a reaction solution. The reaction solution was added dropwise to 0.5 mg / mL sodium tripolyphosphate solution at a volume ratio of 1:1, with the addition time controlled at 40 min. The mixture was stirred at 200 rpm for 30 min at room temperature, and then centrifuged at 8000 rpm for 15 min. The precipitate was collected, washed three times with deionized water, pre-frozen at -80℃ for 2 h, and then freeze-dried at -50℃ and 10 Pa for 24 h to obtain quercetin-chitosan particles.

[0037] Example 2 A method for preparing a nampt-rich exosome hydrogel for wound repair includes the following steps: S1, P4 generation human umbilical cord mesenchymal stem cells were used at 3×10 4 Cells were seeded at a density of cells / cm² into DMEM / F12 medium containing 10% exosome-free fetal bovine serum and cultured to 85% confluence. The medium was then replaced with enrichment medium (composed of DMEM / F12 medium, 3mM nicotinamide, and 100μM deferoxamine) and induced to grow at 37°C and 3% oxygen for 54 h. The cell supernatant was collected, centrifuged at 400×g for 12 min at 4°C, filtered through a 0.45μm filter membrane, and concentrated by 100kDa ultrafiltration to 1 / 10 of the original volume of the supernatant to obtain an exosome suspension rich in nampt. S2. Dissolve sodium oxidized alginate in PBS buffer to obtain a 3 wt% sodium oxidized alginate solution; dissolve carboxymethyl chitosan in PBS buffer to obtain a 3 wt% carboxymethyl chitosan solution. S3. Mix sodium alginate solution, carboxymethyl chitosan solution and gelatin-tannic acid solution (composed of 10wt% gelatin solution and 20mg / mL tannic acid solution in a volume ratio of 7.5:1:0.8) evenly, and adjust the pH to 7.4 with 0.1M NaOH solution to obtain mixed solution A; S4. Add the exosome suspension, zinc-modified montmorillonite, and quercetin-chitosan particles to mixed solution A at a mass ratio of 1:4:3:15 and mix well. Centrifuge at 3000 rpm for 3 min at room temperature to remove air bubbles and obtain the prepolymer solution. S5. Inject the prepolymer into a polytetrafluoroethylene mold and allow it to cure at room temperature for 60 seconds to obtain a wound repair exosome hydrogel rich in nampt.

[0038] The specific preparation method of sodium alginate oxide is as follows: With a sodium alginate to sodium periodate molar ratio of 1:0.3, a 0.1 mol / L sodium periodate solution was added dropwise to a 2 wt% sodium alginate solution over a period of 1.5 h. The reaction was carried out at room temperature and 150 rpm for 6 h with stirring. Then, ethylene glycol was added with a ethylene glycol to sodium periodate molar ratio of 1:1.5, and stirring was continued for 30 min to terminate the reaction. The reaction solution was transferred to a 10 kDa dialysis bag and dialyzed with 150 times the volume of the reaction solution of deionized water at room temperature for 42 h, with the deionized water being replaced every 7 h. The dialysate was pre-frozen at -80℃ for 1.5 h and then freeze-dried at -55℃ and 15 Pa for 36 h to obtain oxidized sodium alginate.

[0039] The specific preparation method of zinc-modified montmorillonite is as follows: Montmorillonite was added to deionized water and ultrasonically dispersed at 250W for 40 min at room temperature to obtain a 3wt% montmorillonite suspension. Zinc chloride (zinc chloride to montmorillonite mass ratio of 1:5-10) was then added, and the mixture was stirred at 300 rpm for 5 h at room temperature. After centrifugation at 4000 rpm for 15 min, the precipitate was collected and washed four times with deionized water. The washed precipitate was pre-frozen at -80℃ for 3 h and then freeze-dried at -55℃ and 15 Pa for 36 h to obtain zinc-modified montmorillonite.

[0040] The specific preparation method of quercetin-chitosan particles is as follows: Chitosan was dissolved in 1 wt% acetic acid solution, and the pH was adjusted to 4.5 with 0.1 M NaOH to obtain a 2 mg / mL chitosan solution. Quercetin was dissolved in 70% anhydrous ethanol to obtain a 1.5 mg / mL quercetin solution. The quercetin solution was added dropwise to the chitosan solution at a volume ratio of 1:4, with the addition time controlled at 25 min. The mixture was stirred at 300 rpm for 50 min at room temperature to obtain a reaction solution. The reaction solution was added dropwise to a 1 mg / mL sodium tripolyphosphate solution at a volume ratio of 1.5:1, with the addition time controlled at 50 min. The mixture was stirred at 300 rpm for 40 min at room temperature, and then centrifuged at 10000 rpm for 25 min. The precipitate was collected, washed four times with deionized water, pre-frozen at -80℃ for 3 h, and then freeze-dried at -55℃ and 15 Pa for 36 h to obtain quercetin-chitosan particles.

[0041] Example 3 A method for preparing a nampt-rich exosome hydrogel for wound repair includes the following steps: S1, P5 generation human umbilical cord mesenchymal stem cells were injected at a rate of 5 × 10⁻⁶. 4Cells were seeded at a density of cells / cm² into DMEM / F12 medium containing 10% exosome-free fetal bovine serum and cultured to 90% confluence. The medium was then replaced with enrichment medium (composed of DMEM / F12 medium, 5 mM nicotinamide, and 200 μM deferoxamine) and induced to grow at 37°C and 5% oxygen for 72 h. The cell supernatant was collected, centrifuged at 500 × g for 15 min at 4°C, filtered through a 0.45 μm filter membrane, and concentrated by 100 kDa ultrafiltration to 1 / 10 of the original volume of the supernatant to obtain an exosome suspension rich in nampt. S2. Dissolve sodium oxidized alginate in PBS buffer to obtain a 5 wt% sodium oxidized alginate solution; dissolve carboxymethyl chitosan in PBS buffer to obtain a 5 wt% carboxymethyl chitosan solution. S3. Mix sodium alginate solution, carboxymethyl chitosan solution and gelatin-tannic acid solution (composed of 15wt% gelatin solution and 30mg / mL tannic acid solution in a volume ratio of 10:1) in a volume ratio of 2:2:1. Adjust the pH to 7.5 with 0.1M NaOH solution to obtain mixed solution A. S4. Add the exosome suspension, zinc-modified montmorillonite, and quercetin-chitosan particles to mixed solution A at a mass ratio of 1:5:4:20 and mix well. Centrifuge at 4000 rpm for 5 min at room temperature to remove air bubbles and obtain the prepolymer solution. S5. Inject the prepolymer liquid into a polytetrafluoroethylene mold and allow it to cure at room temperature for 90 seconds to obtain a wound repair exosome hydrogel rich in nampt.

[0042] The specific preparation method of sodium alginate oxide is as follows: With a sodium alginate to sodium periodate molar ratio of 1:0.5, a 0.2 mol / L sodium periodate solution was added dropwise to a 3 wt% sodium alginate solution over a period of 2 hours. The reaction was carried out at room temperature and 200 rpm for 8 hours. Then, ethylene glycol was added at a molar ratio of 1:2 to sodium periodate, and the reaction was terminated by stirring for another 40 minutes. The reaction solution was transferred to a 14 kDa dialysis bag and dialyzed with 200 times the volume of the reaction solution of deionized water at room temperature for 48 hours, with the deionized water being replaced every 8 hours. The dialysate was pre-frozen at -80℃ for 2 hours and then freeze-dried at -60℃ and 20 Pa for 48 hours to obtain oxidized sodium alginate.

[0043] The specific preparation method of zinc-modified montmorillonite is as follows: Montmorillonite was added to deionized water and ultrasonically dispersed at 300W for 50 min at room temperature to obtain a 5wt% montmorillonite suspension. Zinc chloride (zinc chloride to montmorillonite mass ratio of 1:10) was then added and stirred at 400 rpm for 6 h at room temperature. After centrifugation at 5000 rpm for 20 min, the precipitate was collected and washed 5 times with deionized water. The washed precipitate was pre-frozen at -80℃ for 4 h and then freeze-dried at -60℃ and 20 Pa for 48 h to obtain zinc-modified montmorillonite.

[0044] The specific preparation method of quercetin-chitosan particles is as follows: Chitosan was dissolved in a 2 wt% acetic acid solution, and the pH was adjusted to 5.0 with 0.1 M NaOH to obtain a 3 mg / mL chitosan solution. Quercetin was dissolved in 70% anhydrous ethanol to obtain a 2 mg / mL quercetin solution. The quercetin solution was added dropwise to the chitosan solution at a volume ratio of 1:5, with the addition time controlled at 30 min. The mixture was then stirred at 400 rpm for 60 min at room temperature to obtain a reaction solution. The reaction solution was added dropwise to a 2 mg / mL sodium tripolyphosphate solution at a volume ratio of 2:1, with the addition time controlled at 60 min. The mixture was then stirred at 400 rpm for 60 min at room temperature, centrifuged at 12000 rpm for 30 min, and the precipitate was collected. The precipitate was washed 5 times with deionized water, pre-frozen at -80℃ for 4 h, and then freeze-dried at -60℃ and 20 Pa for 48 h to obtain quercetin-chitosan particles.

[0045] Comparative Example 1 This comparative example provides a method for preparing a wound repair exosome hydrogel rich in nampt. The only difference from Example 2 is that nicotinamide and deferoxamine are not used for induction in step S1, and hypoxia is not used. Instead, conventional culture is used based on normal oxygen concentration. The other raw material types, amounts, and preparation process parameters are completely consistent with Example 2.

[0046] Specifically: S1, P4 generation human umbilical cord mesenchymal stem cells were used at 3×10 4 Cells were seeded at a density of cells / cm² into DMEM / F12 medium containing 10% exosome-free fetal bovine serum and cultured to 85% confluence. The medium was then replaced with DMEM / F12 and cultured at 37°C and 21% oxygen for 54 h. The cell supernatant was collected, centrifuged at 400×g for 12 min at 4°C, filtered through a 0.45 μm filter membrane, and concentrated by 100 kDa ultrafiltration to 1 / 10 of the original volume of the supernatant to obtain a normal exosome suspension.

[0047] Comparative Example 2 This comparative example provides a method for preparing a wound repair exosome hydrogel rich in nampt. The only difference from Example 2 is that an equal amount of nampt protein solution (prepared from nampt protein and deionized water) with the same nampt concentration is used to replace the nampt-rich exosome suspension. All other raw material types, amounts, and preparation process parameters are completely consistent with Example 2.

[0048] Comparative Example 3 This comparative example provides a method for preparing a wound repair exosome hydrogel rich in nampt. The only difference from Example 2 is that a physical mixture of zinc chloride and montmorillonite is used to replace zinc-modified montmorillonite. The mass of zinc chloride and montmorillonite is the same as that of zinc chloride and montmorillonite in Example 2. All other raw material types, amounts, and preparation process parameters are completely consistent with those in Example 2.

[0049] Comparative Example 4 This comparative example provides a method for preparing a wound repair exosome hydrogel rich in nampt, which differs from Example 2 only in that: a physical mixture of quercetin and chitosan is used to replace the quercetin-chitosan particles. The mass of quercetin and chitosan is the same as that of quercetin and chitosan in Example 2, and the other raw material types, amounts and preparation process parameters are completely consistent with those in Example 2.

[0050] Comparative Example 5 This comparative example provides a method for preparing a wound repair exosome hydrogel rich in nampt. The only difference from Example 2 is that gelatin-tannic acid solution is not added in step S3. The other raw material types, amounts, and preparation process parameters are completely consistent with Example 2.

[0051] Performance testing I. Rheological property testing 1. Testing Method The nampt-rich exosome hydrogels for wound repair prepared in Examples 1-3 and Comparative Examples 1-5 were cut into circular samples with a diameter of 20 mm and a thickness of 2 mm, with 3 parallel samples per group. A rotational rheometer (model: TA DHR-2) equipped with a 20 mm parallel plate clamp was used. The test temperature was set to 37℃, the clamp gap to 1000 μm, and silicone oil was applied to the edges of the samples to prevent moisture evaporation. The following tests were performed: Basic network stability test: First, dynamic strain scanning was performed, with a strain range of 0.1-100% and a fixed frequency of 1Hz, to determine the linear viscoelastic region of the sample. Then, within the linear region, a fixed strain of 0.5% was selected, and dynamic frequency scanning was performed to record the storage modulus (G′) and loss modulus (G′′) at an angular frequency of 10 rad / s to evaluate the basic network stability of the hydrogel. The ratio of G′ / G′′ was calculated, which reflects the viscoelastic characteristics of the hydrogel. If G′ / G′′>1, it indicates that the material is in a gel state, and the larger the ratio, the stronger the elasticity and the more stable the network.

[0052] Gelation kinetics test: The uncured prepolymer was immediately transferred to the parallel plate fixture of the rheometer. Using time-scan mode, with a fixed strain of 0.5% and a fixed frequency of 1Hz, the changes in storage modulus (G') and loss modulus (G'') over time were monitored at 37℃. The gelation time was defined as the time point corresponding to the intersection of G' and G'' (G'=G'').

[0053] 2. Test Results and Analysis The specific test results are shown in Table 1.

[0054] Table 1. Rheological properties of nampt-rich wound repair exosome hydrogels The test results in Table 1 show that the nampt-rich exosome hydrogels for wound repair prepared in Examples 1-3 exhibit rapid gelation, dense gel networks, excellent elasticity, and structural stability, meeting the mechanical support and morphological maintenance requirements for wound repair. The hydrogel properties of each comparative example showed varying degrees of decline: ordinary exosomes without nampt enrichment induction and free nampt protein replacing nampt-rich exosomes both resulted in reduced mechanical properties; the physical mixture of zinc chloride and montmorillonite, and the mixture of quercetin and chitosan, respectively, replacing zinc-modified montmorillonite and quercetin-chitosan particles, significantly worsened the gelation rate or mechanical stability; without the addition of the gelatin-tannic acid system, the gelation rate was significantly slowed, and network stability decreased significantly. In summary, the components of this invention work synergistically, giving the hydrogel excellent rheological properties, making it more suitable for clinical wound repair applications.

[0055] II. Mechanical Property Testing 1. Testing Method The nampt-rich exosome hydrogels for wound repair prepared in Examples 1-3 and Comparative Examples 1-5 were cut into strips 20 mm long, 5 mm wide, and 2 mm thick, with 5 parallel samples in each group. Using an electronic universal testing machine at a temperature of 37°C and a tensile rate of 10 mm / min, the following two tests were performed: Basic mechanical property testing: Tensile tests were conducted on the wound repair exosome hydrogel samples rich in nampt to measure and record the tensile strength at break (MPa) and elongation at break (%) to evaluate the basic mechanical compatibility of the hydrogel.

[0056] 2. Test Results and Analysis The specific test results are shown in Table 2.

[0057] Table 2. Mechanical property test results of nampt-rich wound repair exosome hydrogels As shown in Table 2, the exosome hydrogels rich in nampt prepared in Examples 1-3 exhibited higher elongation at break and higher tensile strength at break than the comparative examples 1-4, indicating that the hydrogels possess good flexibility and extensibility. Example 2 showed the best performance, meeting the mechanical compatibility requirements for skin wound dressings. Due to the absence of components or the substitution of other substances, the mechanical properties of each comparative example were weaker than those of Examples 1-3: Comparative Example 1 used ordinary exosomes instead of nampt enriched exosomes, resulting in a significant decrease in mechanical properties. Nampt enriched exosomes can enhance the biological function and mechanical properties of the hydrogel; Comparative Example 2 used free nampt protein instead of nampt enriched exosomes, and the uneven distribution led to internal structural defects; Comparative Example 3 used free zinc chloride and montmorillonite instead of zinc-modified montmorillonite, resulting in weak interfacial bonding and easy brittle fracture during stretching; Comparative Example 4 used free quercetin and chitosan instead of quercetin-chitosan particles, and the precipitation of free quercetin crystals increased the brittleness of the hydrogel; Comparative Example 5, lacking gelatin-tannic acid, showed an abnormally high tensile strength at break but the lowest elongation at break, and the hydrogel was hard and brittle, making it unsuitable as a wound dressing.

[0058] III. Antibacterial Performance Test 1. Testing Method The nampt-rich exosome hydrogels for wound repair prepared in Examples 1-3 and Comparative Examples 1-5 were cut into circular samples with a diameter of 4 mm and a thickness of 2 mm. Three parallel samples were prepared for each group. After sterilization by ultraviolet irradiation, they were used for later use. Staphylococcus aureus (S. aureus) was selected. Staphylococcus aureus ATCC 25923), Escherichia coli ( Escherichia coli ATCC25922) and Pseudomonas aeruginosa ( Pseudomonas aeruginosa (ATCC 27853), the three strains were inoculated into the culture medium and cultured at 37°C for 12 h, and then diluted to 2×10. 5 A bacterial suspension was prepared at CFU / mL.

[0059] Sterilized wound repair exosome hydrogel samples rich in nampt were placed in 96-well plates. 200 μL of the above bacterial suspension was added to each well. The plates were incubated at 37°C with constant temperature shaking at 100 rpm for 24 h. The bacterial suspension of each well was serially diluted 10-fold, and 100 μL of each well was spread onto an agar plate. After incubation at 37°C for 24 h, the number of colonies (CFU) was counted. The bacterial suspension without the addition of nampt-rich wound repair exosome hydrogel was used as the blank group. The sterilization rate was calculated as follows: sterilization rate = (CFU of blank group - CFU of sample group) / CFU of blank group × 100%.

[0060] 2. Test Results and Analysis The specific test results are shown in Table 3.

[0061] Table 3. Antibacterial properties of nampt-rich wound repair exosome hydrogels. As shown in Table 3, the nampt-rich exosome hydrogels for wound repair prepared in Examples 1-3 exhibited excellent broad-spectrum antibacterial activity against three common wound infection bacteria, with significantly higher bactericidal rates than the comparative examples. Example 2 showed the best antibacterial performance, meeting the antibacterial requirements for infected wound dressings. Due to component deficiencies or substitutions, the antibacterial performance of the comparative examples was weaker than that of Examples 1-3, as detailed in the analysis below. Comparative Example 1 used ordinary exosomes instead of nampt-enriched exosomes, and the bactericidal rate was significantly lower than that of Example 2, indicating that nampt-enriched exosomes can indirectly enhance the antibacterial microenvironment of hydrogels through immune regulation, and the antibacterial performance decreased significantly after their absence. Comparative Example 2 used free nampt protein instead of nampt to enrich exosomes. The bactericidal rate was only slightly improved compared with Comparative Example 1, and there was a significant difference compared with Example 2. This indicates that free nampt protein lacks the targeted delivery and multi-factor synergistic effect of exosomes and cannot replace the complete exosome system. Comparative Example 3 used a physical mixture of free zinc chloride and montmorillonite instead of zinc-modified montmorillonite nanosheets, and the bactericidal rate was lower than that of Example 2. This indicates that zinc-modified montmorillonite nanosheets can achieve long-lasting antibacterial effect through the slow release of Zn²⁺, while the Zn²⁺ in the physical mixture is released too quickly, resulting in insufficient antibacterial durability. Comparative Example 4 used a physical mixture of free quercetin and chitosan instead of quercetin-chitosan particles, and the bactericidal rate was lower than that of Example 2. This indicates that quercetin-chitosan particles can achieve long-lasting antibacterial effect through the slow release of quercetin, while quercetin in the physical mixture is prone to crystallization and precipitation, resulting in a decrease in antibacterial effect. Comparative Example 5, which did not contain gelatin-tannic acid solution, had the lowest bactericidal rate, indicating that tannic acid in the gelatin-tannic acid system is the key source of antibacterial activity of the hydrogel, and its absence significantly weakens the antibacterial performance.

[0062] IV. Cytotoxicity Test 1. Testing Method ① Sample preparation: Take the nampt-rich exosome hydrogels for wound repair prepared in Examples 1-3 and Comparative Examples 1-5, cut them into circular samples with a diameter of 4 mm and a thickness of 2 mm, and set up 3 parallel samples in each group. After sterilization by ultraviolet irradiation, they are ready for use.

[0063] ②Preparation of extract: Place the sterilized exosome hydrogel sample rich in namptol into a sterile container, add complete culture medium (containing 10wt% fetal bovine serum, 100U / mL penicillin, and 100μg / mL streptomycin) at a sample surface area to culture medium volume ratio of 3cm² / mL, and extract at 37℃ for 24h. Collect the supernatant as the hydrogel extract and store at 4℃ for later use.

[0064] ③ Cell Culture and Seeding: Human skin fibroblasts (HSF) were used as test cells and cultured in a 37°C, 5% CO2 incubator with complete culture medium, changing the medium every 2 days. Cells in the logarithmic growth phase were seeded into 48-well plates at a density of 20,000 cells per well, with 500 μL of complete culture medium added to each well, and cultured for 12 h to allow the cells to adhere.

[0065] ④ Experimental grouping and treatment: Discard the original culture medium in the wells and treat according to the following groups (each group has 3 replicates): Sample group: 500 μL of hydrogel extraction buffer was added to each well; Negative control group: 500 μL of fresh complete culture medium was added to each well; Blank group: no cells were seeded, and 500 μL of fresh complete culture medium was added to each well (to subtract background absorbance). The culture plates were placed in a 37℃, 5% CO2 incubator and cultured for 72 h.

[0066] ⑤ Cell viability assay (MTT method): After culture, add 20 μL of MTT solution (5 mg / mL) to each well and incubate at 37℃ in a 5% CO2 incubator for 4 h. Discard the supernatant, add 150 μL of DMSO to each well, and shake for 10 min to completely dissolve the formazan crystals. Measure the absorbance (OD value) at 490 nm using a microplate reader.

[0067] ⑥ Result calculation: Calculate the relative cell activity according to the formula: Relative cell activity = (Absorbance of sample group - Absorbance of blank group) / (Absorbance of negative control group - Absorbance of blank group) × 100%.

[0068] 2. Test Results and Analysis The specific test results are shown in Table 4.

[0069] Table 4. Cytotoxicity test results of nampt-rich wound repair exosome hydrogels As shown in Table 4, the nampt-rich exosome hydrogels for wound repair prepared in Examples 1-3 all exhibited a relative cell activity of over 93% after 72 hours of HSF cell culture. This indicates that the nampt-rich exosome hydrogels for wound repair prepared in this invention have no significant cytotoxicity, excellent biocompatibility, and that the functional components in the hydrogel system work synergistically with release concentrations within the biosafety range. The relative cell activity of each comparative example was also at a high level (>85%), with no significant cytotoxicity, meeting the safety requirements for biomedical materials.

[0070] The above specific embodiments are merely explanations of the present invention and are not intended to limit the present invention. After reading this specification, those skilled in the art can make modifications to these embodiments without contributing any inventive step, but as long as they are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. A method for preparing a wound repair exosome hydrogel rich in nampt, characterized in that, Includes the following steps: S1. Human umbilical cord mesenchymal stem cells were seeded into the basic culture medium and cultured to 80-90% confluence. The medium was then replaced with enrichment medium and induced to culture under hypoxic conditions. The cell supernatant was collected, centrifuged, filtered, and ultrafiltered to obtain an exosome suspension rich in nampt. S2. Dissolve sodium oxidized alginate in PBS buffer to obtain sodium oxidized alginate solution; dissolve carboxymethyl chitosan in PBS buffer to obtain carboxymethyl chitosan solution; S3. Mix sodium alginate oxidized solution, carboxymethyl chitosan solution and gelatin-tannic acid solution evenly, adjust pH to obtain mixed solution A; S4. Add the exosome suspension rich in nampt, zinc-modified montmorillonite and quercetin-chitosan particles to mixed solution A, mix well, centrifuge to remove air bubbles, and obtain the prepolymer solution. S5. Inject the prepolymer into the mold and allow it to solidify at room temperature to obtain a wound repair exosome hydrogel rich in nampt.

2. The method for preparing the nampt-rich wound repair exosome hydrogel according to claim 1, characterized in that, In step S1, the enrichment medium consists of DMEM / F12 medium, nicotinamide, and deferoxamine, with the nicotinamide concentration being 1-5 mM and the deferoxamine concentration being 50-200 μM.

3. The method for preparing the nampt-rich wound repair exosome hydrogel according to claim 1, characterized in that, The method for preparing oxidized sodium alginate in step S2 is as follows: Sodium periodate solution was added dropwise to sodium alginate solution and stirred to react; ethylene glycol was added and stirred to terminate the reaction; after dialysis and freeze-drying, sodium alginate oxide was obtained.

4. The method for preparing the nampt-rich wound repair exosome hydrogel according to claim 1, characterized in that, In step S2, the mass fraction of the oxidized sodium alginate solution is 1-5%; the mass fraction of the carboxymethyl chitosan solution is 2-5%.

5. The method for preparing the nampt-rich wound repair exosome hydrogel according to claim 1, characterized in that, In step S3, the volume ratio of sodium alginate solution, carboxymethyl chitosan solution and gelatin-tannic acid solution is 1-2:1-2:0.5-1; adjust the pH to 7.2-7.

5.

6. The method for preparing the nampt-rich wound repair exosome hydrogel according to claim 1, characterized in that, The preparation method of zinc-modified montmorillonite in step S4 is as follows: Montmorillonite was ultrasonically dispersed in deionized water, zinc salt was added, the mixture was stirred and reacted, and then centrifuged, washed, and freeze-dried to obtain zinc-modified montmorillonite.

7. The method for preparing the nampt-rich wound repair exosome hydrogel according to claim 6, characterized in that, The zinc salt is selected from one or more of zinc chloride, zinc nitrate, and zinc sulfate.

8. The method for preparing the nampt-rich wound repair exosome hydrogel according to claim 1, characterized in that, The method for preparing quercetin-chitosan particles in step S4 is as follows: Chitosan was dissolved in acetic acid solution, and the pH was adjusted to obtain a chitosan solution; quercetin was dissolved in anhydrous ethanol to obtain a quercetin solution. Quercetin solution was added dropwise to chitosan solution, and the reaction was stirred to obtain a reaction solution. The reaction solution was then added dropwise to sodium tripolyphosphate solution, and the mixture was stirred continuously. After centrifugation, the precipitate was collected, washed, and freeze-dried to obtain quercetin-chitosan particles.

9. The method for preparing the nampt-rich wound repair exosome hydrogel according to claim 1, characterized in that, In step S4, the mass ratio of the exosome suspension rich in nampt, zinc-modified montmorillonite, quercetin-chitosan particles, and mixed solution A is 1:3-5:2-4:10-20; the centrifugation speed is 2000-4000 rpm, and the centrifugation time is 2-5 min.

10. A wound repair exosome hydrogel rich in nampt as described in any one of claims 1-9.

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