Controllable release exosome hydrogel and preparation method and application thereof

Abstract

This invention relates to the field of biomedical technology, and more particularly to a controlled-release exosome hydrogel, its preparation method, and its applications. The controlled-release exosome hydrogel prepared using the method provided by this invention has the following significant advantages over existing exosome delivery systems: 1. It enables precise temporal and spatial control; 2. It enhances the bioactivity of exosomes; 3. It adapts to the wound microenvironment; 4. It integrates functions such as exosome therapy, photothermal antibacterial activity, hemostasis, and angiogenesis promotion; 5. It exhibits excellent biocompatibility and high cell survival rate; 6. Animal experiments show that the controlled-release exosome hydrogel provided by this invention helps promote wound healing; in full-thickness skin defects and deep second-degree burn models, it can accelerate epithelialization and collagen deposition, thereby reducing scar formation. Therefore, it holds promise for applications in the preparation of wound repair products.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a controllable release exosome hydrogel, its preparation method, and its application. Background Technology

[0002] Exosomes, as naturally occurring vesicles secreted by cells, possess a variety of biological functions, including regulating immunity, promoting angiogenesis, and facilitating tissue repair. They have shown great promise in many fields, such as wound healing, inflammation regulation, and cancer treatment. However, existing exosome delivery systems often face challenges in vivo, such as excessively rapid clearance, low release efficiency, and difficulty in adapting to the wound microenvironment.

[0003] Currently, common exosome delivery systems mainly include prefabricated hydrogels and nanocarriers. Both of these systems passively and slowly release exosomes, often making it difficult to precisely control the release rate and timing. Furthermore, they cannot achieve uniform coverage on irregular wound surfaces, leading to a lack of therapeutic stability. Traditional hydrogel systems rely primarily on passive release mechanisms, making precise spatiotemporal control difficult in dynamic and variable wound environments. While existing hydrogels and nanocarriers can maintain exosome bioactivity through slow release, this process lacks flexibility and cannot be precisely adjusted according to the specific needs of the wound.

[0004] Photodynamic drug delivery systems are a drug delivery technology that uses light stimulation as an external control signal to precisely regulate the timing and spatial location of drug release. Although photodynamic drug delivery systems have shown great potential in precision medicine, most existing systems still suffer from insufficient control precision, especially in cases of irregular wound surfaces, making it difficult to ensure the accurate release of exosomes.

[0005] In addition, existing photothermal response systems are usually used in conjunction with traditional antibiotics or physical methods. While these two methods can inhibit bacteria to some extent, they are prone to causing bacterial resistance and require continuous intervention from external equipment, which increases the complexity and cost of treatment.

[0006] Therefore, how to construct a hydrogel that enables the controlled release of exosomes is a technical problem that urgently needs to be solved. Summary of the Invention

[0007] This invention aims to at least solve one of the technical problems existing in related technologies. Therefore, the first objective of this invention is to provide a method for preparing a controllable release exosome hydrogel; the second objective is to provide a controllable release exosome hydrogel; and the third objective is to provide an application of the controllable release exosome hydrogel.

[0008] To achieve the first objective, the technical solution adopted by this invention is as follows:

[0009] A method for preparing a controllable release exosome hydrogel includes the following steps:

[0010] S100, NorHA was obtained by modifying sodium hyaluronate with norbornene anhydride.

[0011] NorHA is a nobornene-functionalized hyaluronic acid.

[0012] S200, using p(AEO4MA- co -NIPAAM)-SH for choline phosphate After modification, TRCP is obtained;

[0013] Among them, p(AEO4MA- co The structural formula of -NIPAAM)-SH is shown below:

[0014] ;

[0015] The structure of TRCP is shown below:

[0016] ;

[0017] In the structural formula, x, y, and z are positive integers, and TRCP is a temperature-responsive polymer modified with choline phosphate.

[0018] p(AEO4MA- co -NIPAAM) is a copolymer of N-isopropylacrylamide (NIPAAM) and 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl methacrylate (AEO4MA);

[0019] S300, AuNPs@DA nanoparticles were prepared using gold nanoparticles and dopamine;

[0020] S400, prepare photoinitiator aqueous solution I and AuNPs@DA nanoparticle dispersion respectively;

[0021] S500. Dissolve NorHA in the photoinitiator aqueous solution I to obtain NorHA-containing photoinitiator aqueous solution II. Add TRCP, AuNPs@DA nanoparticle dispersion and cell exosome suspension to the NorHA-containing photoinitiator aqueous solution II. Prepare a controllable release exosome hydrogel using the photocrosslinking in situ gelation method.

[0022] Hyaluronic acid (HA) molecules are composed of glucuronic acid and N-acetylglucosamine, containing numerous active sites such as hydroxyl (-OH) and carboxyl (-COOH) groups. These sites can covalently couple with the functional groups of alkenes, grafting alkenes onto the HA chain. Under the action of photo / thermal initiators, the double bonds of the alkene-grafted hyaluronic acid undergo free radical polymerization to form a three-dimensional network hydrogel. Furthermore, the alkenes can undergo thiol-alkene click reactions with mercapto-containing substances, thereby achieving rapid cross-linking of the hydrogel.

[0023] TRCP, as one of the core materials of hydrogels, contains thiol groups (-SH) at its ends, enabling it to react with norbornene-functionalized hyaluronic acid to achieve rapid cross-linking. Furthermore, TRCP is a choline phosphate-modified temperature-responsive polymer. Below its lower critical dissolution temperature (LCST), the polymer is in an extended state, capable of loading exosomes. When the temperature rises above its LCST, the polymer contracts, promoting the release of exosomes.

[0024] AuNPs@DA nanoparticles are gold nanoparticles coated with dopamine (DA). These nanoparticles can absorb light energy and convert it into heat energy under near-infrared light irradiation, thereby promoting the contraction of hydrogels and the release of exosomes.

[0025] Preferably, the structural formula of NorHA is as follows:

[0026] ;

[0027] Where n and m are both positive integers, and the sum of n and m is 800.

[0028] Preferably, the TRCP structure is as follows:

[0029] .

[0030] Preferably, in step S200, p(AEO4MA- co In the -NIPAAM)-SH structure, x, y, and z are 9, 81, and 3, respectively. The synthesis includes the following steps:

[0031] S210. Using tetraethylene glycol as a raw material, the temperature-responsive monomer AEO4MA was synthesized, with the following structural formula: ;

[0032] S220 utilizes the temperature-responsive monomer AEO4MA, N-isopropylacrylamide (NIPAAM), and chain transfer agent. A reversible addition-fragmentation chain transfer polymerization reaction was carried out in the presence of azobisisobutyronitrile (AEO4MA-) to synthesize the intermediate p(AEO4MA- co -NIPAAM)-CS3, the structure is shown below:

[0033] ;

[0034] S230. The terminal groups are removed by the aminolysis reaction of the intermediate to obtain p(AEO4MA- co -NIPAAM)-SH, the structure is as follows:

[0035] .

[0036] Preferably, the lower critical dissolution temperature of TRCP is 41℃~43℃.

[0037] Preferably, in step S400, the photoinitiator is selected from lithium phenyl-2,4,6-trimethylbenzoyl phosphinate (LAP).

[0038] And / or, in step S500, the cell exosomes are selected from human umbilical cord mesenchymal stem cell exosomes.

[0039] Preferably, in step S300, the particle size distribution range of AuNPs@DA nanoparticles is 65nm to 72nm.

[0040] Preferably, in step S500, the mass-volume concentration of TRCP in the controllably released exosome hydrogel is 2% to 10%.

[0041] And / or the mass-volume concentration of the AuNPs@DA nanoparticle dispersion is 0.25% to 1.25%;

[0042] And / or the concentration of the exosome suspension is 1×10 8 ~1×10 12 The particle size distribution ranges from 70 nm to 100 nm, with a particle size of 1 / mL.

[0043] Preferably, in step S500, the light used in the photocrosslinking in-situ gelation method is selected from blue light, and the irradiation time is 30-90s;

[0044] The wavelength range of the blue light is 380nm to 455nm.

[0045] To achieve the second objective, the technical solution adopted by this invention is as follows:

[0046] A controllable release exosome hydrogel is prepared using the above-described method for preparing controllable release exosome hydrogels.

[0047] To achieve the third objective, the technical solution adopted by this invention is as follows:

[0048] An application of a controllable release exosome hydrogel, using the above-mentioned controllable release exosome hydrogel to prepare antibacterial products and / or wound repair products.

[0049] The above-described one or more technical solutions in the embodiments of the present invention have at least one of the following technical effects:

[0050] The controlled-release exosome hydrogel prepared using the method provided by this invention has the following significant advantages over existing exosome delivery systems:

[0051] 1. Precise timing and air conditioning control: By using near-infrared light irradiation, the hydrogel can achieve precise release of exosomes at the wound site, avoiding the problems of excessively fast or slow exosome release in traditional delivery systems.

[0052] II. Enhancing the bioactivity of exosomes: Since exosomes are loaded in thermosensitive polymers and released only after being stimulated by near-infrared light at the wound site, this controlled release method can significantly improve the stability and bioactivity of exosomes and enhance the wound healing effect.

[0053] 3. Adaptation to the wound microenvironment: The in-situ gelation properties of hydrogels enable them to adapt well to irregular wound shapes, ensuring full coverage of the wound surface and improving healing efficiency.

[0054] IV. Multifunctional Synergy: This invention integrates functions such as exosome therapy, photothermal antibacterial properties, hemostasis, and angiogenesis promotion, and therefore is expected to be used to prepare antibacterial products.

[0055] V. Excellent biocompatibility: high cell survival rate, no toxic damage to major organs.

[0056] VI. Animal experiments showed that the controllable release exosome hydrogel provided by this invention helps promote wound healing. In full-thickness skin defects and deep second-degree burn models, it can accelerate epithelialization and collagen deposition, thereby reducing scar formation. Therefore, it is expected that the exosome hydrogel provided by this invention can be used to prepare wound repair products.

[0057] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0058] Figure 1 This is the NorHA characterization spectrum provided in Embodiment 1 of the present invention.

[0059] Figure 2It is the CP provided in Embodiment 1 of the present invention. 31 P nuclear magnetic resonance spectroscopy (P nuclear magnetic resonance spectroscopy) 31 P NMR).

[0060] Figure 3 The p(AEO4MA-) provided in Embodiment 1 of this invention co -NIPAAM)-CS3 1 H NMR spectrum.

[0061] Figure 4 The p(AEO4MA-) provided in Embodiment 1 of this invention co Gel permeation chromatography (GPC) curve of NIPAAM-CS3.

[0062] Figure 5 The p(AEO4MA-) provided in Embodiment 1 of this invention co Characterization of -NIPAAM)-SH.

[0063] Figure 6 This is the choline phosphate-modified temperature-responsive polymer (TRCP) provided in Example 1 of this invention. 1 H NMR spectrum.

[0064] Figure 7 This is the choline phosphate-modified temperature-responsive polymer (TRCP) provided in Example 1 of this invention. 31 P NMR spectrum.

[0065] Figure 8 This describes the morphology and particle size characterization of AuNPs@DA provided in Embodiment 1 of the present invention.

[0066] Figure 9 This describes the chemical composition and spectral structure characterization of AuNPs@DA provided in Example 1 of this invention.

[0067] Figure 10 This is the photothermal performance characterization of AuNPs@DA provided in Embodiment 1 of the present invention.

[0068] Figure 11 This is the Fourier transform infrared (FT-IR) spectrum of the controllable release exosome hydrogel (NTA-Gel) provided in Embodiment 1 of the present invention.

[0069] Figure 12 This is a scanning electron microscope (SEM) image of the NTA / EVs-Gel provided in Embodiment 1 of the present invention.

[0070] Figure 13 This describes the swelling performance of the NTA / EVs-Gel provided in Example 1 of this invention.

[0071] Figure 14 These are the degradation curves of NTA / EVs-Gel with different mass-volume concentrations provided in Example 1 of this invention.

[0072] Figure 15 This describes the mechanical properties of the NTA / EVs-Gel provided in Embodiment 1 of the present invention.

[0073] Figure 16 This invention examines the antibacterial activity and bactericidal effect of the different substances provided in Example 1.

[0074] Figure 17 This is a cumulative release rate curve of different hydrogels provided in Test Example 2 of the present invention.

[0075] Figure 18 This is a statistical graph showing the quantitative analysis results of HUVECs cell viability using the CCK-8 assay provided in Example 3 of this invention.

[0076] Figure 19 This is a diagram showing the detection results of the HUVECs cell migration ability provided in Example 3 of this invention.

[0077] Figure 20 This is a Transwell experimental result diagram showing the effect of different substances on the migration of HUVECs provided in Example 3 of this invention.

[0078] Figure 21 The images provided in Example 3 of this invention are bright-field images of the tubular structures of HUVECs after different material treatments.

[0079] Figure 22 This is an example of the wound healing status of mice in different treatment groups provided in Example 4 of this invention.

[0080] Figure 23 These are pathological sections of mouse wound tissues from different substance groups provided in Example 4 of this invention.

[0081] Figure 24 This is an example of the wound healing status of rats in different treatment groups provided in Example 4 of this invention.

[0082] Figure 25 These are pathological sections of rat wound tissues from different substance groups provided in Example 4 of this invention. Detailed Implementation

[0083] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. The following embodiments are used to illustrate this invention, but cannot be used to limit the scope of this invention.

[0084] In the following embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available, unless otherwise specified, and are carried out in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions.

[0085] Example 1

[0086] The process for preparing a controlled-release exosome hydrogel is as follows:

[0087] I. Preparation of norbornene-functionalized hyaluronic acid (NorHA).

[0088] Sodium hyaluronate 4.03 g of powder was added to a 500 mL three-necked round-bottom flask, followed by 100 mL of water. The mixture was magnetically stirred at 500 rpm for 2 h at 24 °C–26 °C until the sodium hyaluronate was completely dissolved, forming a homogeneous and transparent sodium hyaluronate aqueous solution. Subsequently, under continuous stirring, 80 mL of anhydrous N,N-dimethylformamide (DMF) was slowly added dropwise to the flask through a constant-pressure dropping funnel (dropping rate of 1 mL / min) to obtain a sodium hyaluronate-DMF mixed system.

[0089] Take another 250mL beaker and add norbornene eddy anhydride. 1.64 g of powder and 20 mL of anhydrous DMF were ultrasonically treated (300 W) for 10 min to completely dissolve norbornenic anhydride and obtain a clear norbornenic anhydride DMF solution. This solution was slowly added dropwise (0.5 mL / min) to the above sodium hyaluronate / DMF mixture through a constant pressure dropping funnel. After the addition was complete, the mixture was stirred for 30 min to ensure that the reaction system was fully mixed. The pH of the reaction system was adjusted to 9.9–10.1 using a 5 mol / L NaOH aqueous solution. After adjustment, the three-necked flask was transferred to a 4°C constant-temperature water bath and stirred at 500 rpm for 24 hours. The reaction solution was then transferred to a 1000 mL beaker, and a mixture of ethanol and N,N-dimethylformamide (volume ratio 1:1) was slowly added at a rate of 2 mL / min at room temperature until a large amount of white flocculent precipitate appeared in the solution. Stirring was continued for 30 min to allow complete precipitation. Subsequently, the precipitate was collected by vacuum filtration (using a Buchner funnel with filter paper pore size of 1–3 μm) and washed three times with a 50 mL mixture of ethanol and N,N-dimethylformamide (volume ratio 1:1). The washed precipitate was dissolved in 100 mL of water and transferred to a pre-treated dialysis bag (molecular weight cutoff of 3500 Da) after boiling for 30 min to remove impurities for dialysis purification. The dialysis purification process consisted of two stages: In the first stage, dialysis was performed for 3 days at 4°C using a 0.9% (w / v) NaCl aqueous solution as the dialysis medium, with the dialysis solution changed 3 times daily to remove sodium ions and small molecule impurities. In the second stage, dialysis was performed for 2 days using ultrapure water, with the dialysis solution changed 4 times daily to ensure complete removal of residual solvent and salts. After dialysis, the solution in the dialysis bag was transferred to a lyophilization bottle and placed in a freeze dryer. The solution was then freeze-dried at -50°C under a vacuum of 0.1 mbar for 72 hours to obtain white flocculent NorHA. Solid powder, its characterization is as follows Figure 1 As shown; where Figure A represents HA and NorHA. 1 H nuclear magnetic resonance (H nuclear magnetic resonance) 1 Figure B shows the 1H NMR spectrum, and Figure C shows the Fourier transform infrared (FT-IR) spectrum of HA and NorHA.

[0090] II. Preparation of choline phosphate-modified temperature-responsive polymers (TRCPs).

[0091] First, the synthesis of choline phosphate (CP). Due to the high sensitivity of this substance to humidity, the preparation of CP is carried out under anhydrous conditions.

[0092] Drying of glassware: Schlenk flasks, constant pressure dropping funnels, magnetic stir bar, airless funnels and other glassware were placed in a 120℃ forced-air drying oven for pre-drying overnight (drying time ≥12h). After removal, they were immediately transferred to an inert gas operating table, and the internal dry environment of the glassware was maintained by continuously introducing dry argon gas (purity ≥99.999%) until the glassware cooled to room temperature.

[0093] Acetonitrile solvent was dehydrated by distillation using CaH2 (mass ratio 1:10): acetonitrile and CaH2 were added to a 500 mL round-bottom flask, stirred for 30 min, and then heated to 81 °C for distillation. The fraction collected at 77–78 °C was used as the reaction solvent. Before use, it was distilled again to ensure that the water content was ≤0.05%.

[0094] Raw material dehydration: Add CaH2 (mass ratio 1:5) to N,N-dimethyl-2-propyn-1-amine and stir magnetically for 72 h in an ice-water bath at 0 °C. Then connect a vacuum distillation apparatus (vacuum degree ≤10 Pa) and collect the fraction at 45-47 °C. Dry argon gas is continuously introduced during the distillation process to avoid contact between the raw material and air.

[0095] The synthesis of CP is as follows: 2-propanol (7.21 g), dehydrated N,N-dimethyl-2-propyn-1-amine (18.87 g), and dehydrated acetonitrile (50 mL) are added sequentially to a 200 mL Schlenk flask; the Schlenk flask is placed in a -55 °C low-temperature bath (ethanol-dry ice system), and dry argon gas is introduced to replace the air in the system 3 times (each replacement time is 10 min), to obtain a mixed system of N,N-dimethyl-2-propyn-1-amine.

[0096] 7.12 g of 2-chloro-2-oxo-1,3,2-dioxaphosphane was dissolved in 10 mL of anhydrous acetonitrile, and the solution was then transferred to a constant-pressure dropping funnel. Under continuous stirring (300 rpm) and constant temperature (-55 °C), the solution was slowly added dropwise (0.5 mL / min) to a Schlenk flask. During the addition, the system was kept stable by a slight positive pressure of argon (0.02 MPa) to prevent solvent solidification at low temperature from interrupting the addition. After the addition was complete, the reaction was maintained at -55 °C with stirring for 8 h. Then, the temperature was increased (heating rate ≤ 5 °C / h) to approximately 25 °C and maintained for 12–16 h. The temperature was then decreased to -20 °C and maintained for 2 h to allow the byproducts to fully precipitate. Under nitrogen protection, the reaction solution was filtered, and the filtrate was collected and transferred to a Schlenk flask. The Schlenk flask containing the filtrate was placed in an oil bath at 65–70°C and stirred (200 rpm) under an argon atmosphere for 4 days. After cooling to room temperature, anhydrous tetrahydrofuran (100 mL) was slowly added to the system, and the mixture was stirred for 30 min to precipitate the product. After standing for 1 h, the product was collected by filtration. The precipitate was washed repeatedly with anhydrous tetrahydrofuran (50 mL each time) until the supernatant was clear and colorless. The washed precipitate was then dried in a vacuum drying oven (40°C, 0.01 MPa) for 8 h to obtain a yellow waxy solid CP.

[0097] Dissolve CP (5 mg) in deuterated methanol (0.5 mL) and measure the hydrogen nuclear magnetic resonance using a 400 MHz nuclear magnetic resonance spectrometer. 1 H NMR spectrum and 31 Phosphorus nuclear magnetic resonance (PNMR) 31 P NMR spectrum;

[0098] in, 1 The H NMR characterization data are shown below:

[0099] 1 ¹H NMR (400 MHz, methanol-d⁴): δ 4.47 (s, 2H), 4.30 (s, 2H), 3.82–3.74 (m, 2H), 3.63–3.57 (m, 1H), 3.33 (s, 1H), 3.31 (s, 6H), 1.30 (d, J = 6.1 Hz, 6H);

[0100] 31 Phosphorus nuclear magnetic resonance (PNMR) 31 P NMR (spectroscopy) spectrum, such as Figure 2 As shown.

[0101] Dissolve 1 mg of CP in 1 mL of methanol to prepare a 1 mg / mL CP methanol solution. Analyze the solution using an electrospray ionization mass spectrometer (ESI-MS) in positive ion mode. The results are shown below.

[0102] Among them, ESI-MS [M+H] + :C 10 H 20 The calculated value of NO4P is 250.1203, and the measured value is 250.1208.

[0103] Second, the synthesis of 2-(2-(2-(2-)azidoethoxy)ethoxy)ethyl methacrylate (AEO4MA) The process is as follows:

[0104] Tetraethylene glycol (1.0 mol), 4-toluenesulfonyl chloride (0.2 mol), and anhydrous tetrahydrofuran (50 mL) were placed in a dry flask equipped with a dropping funnel containing triethylamine (0.25 mol). The triethylamine was slowly added dropwise at 4 °C for 4 h, and the reaction was stirred overnight at room temperature. The mixture was then filtered, and the filtrate was collected. The filtrate was extracted with dichloromethane, and the solvent was evaporated to obtain the intermediate. DMF (50 mL) and sodium azide (0.25 mol) were added to the intermediate, and the mixture was stirred at 80 °C for 18 h. After cooling to room temperature, the mixture was filtered, and the filtrate was collected again. The filtrate was extracted with dichloromethane and dried over MgSO4 to obtain the crude product. The crude product was purified by flash chromatography using an ethyl acetate / hexane (1:1 v / v) mixture to obtain AEO4. In the presence of triethylamine (0.3 mol) and anhydrous tetrahydrofuran (50 mL), AEO4 was reacted with methacryloyl chloride (0.12 mol). The methacryloyl chloride was added dropwise at 4 °C for 4 h. After stirring the reaction solution overnight at room temperature, the mixture was filtered, and the filtrate was collected. The filtrate was then extracted with dichloromethane and dried with MgSO4 to obtain the crude product. The crude product was purified by rapid chromatography with ethyl acetate / hexane (volume ratio 1:4) to obtain AEO4MA.

[0105] That 1 The H-NMR characterization data are shown below:

[0106] 1 H NMR (400MHz, CDCl3): δ 6.13, 5.57 (–C=C–, s, 1H), 4.31–4.29 (–CH2OOC–, m, 2H), 3.76–3.73 (–OCH2CH2OOC–, m, 2H), 3.6 8–3.67 (–OCH2CH2–, d, J=6.7Hz, 10H), 3.40–3.37 (–CH2N3, t, J=5.0Hz, 2H), 1.94 (–CH3, s, 3H).

[0107] Its mass spectrometry characterization data are shown below:

[0108] ESI-MS[M+NH4]+ :C 12 H 21 The calculated value of N3O5 is 305.1819, and the measured value is 305.1821.

[0109] Third, synthesize p(AEO4MA- co -NIPAAM)-CS3 The process is as follows:

[0110] Under an argon atmosphere, the chain transfer agent (0.01 mmol), azobisisobutyronitrile (AIBN) (0.0017 mmol), AEO4MA (300 mg), N-isopropylacrylamide (NIPAAM) (1.2 g), and 1,4-dioxane (3 mL) were sequentially added to a reaction flask. The reaction mixture was degassed by a three-stage freeze-vacuum-thaw cycle. After stirring in a 65°C oil bath for 24 h, the reaction mixture was dissolved in dichloromethane, precipitated with diethyl ether, and the precipitate was collected and dried to obtain p(AEO4MA- co -NIPAAM)-CS3, its nuclear magnetic resonance (NMR) spectrum and gel permeation chromatography (GPC) curve are as follows: Figure 3 and Figure 4 As shown.

[0111] from Figure 4 We can conclude that: p(AEO4MA- co -NIPAAM)-CS3 number-average molecular weight (M n The molecular weight is 12811, the weight-average molecular weight (Mw) is 19699, and the dispersion is 1.538.

[0112] Fourth, synthesize p(AEO4MA- co -NIPAAM)-SH The process is as follows:

[0113] p(AEO4MA- co -NIPAAM)-CS3 (110 mg) was dissolved in 10 mL of freshly distilled anhydrous tetrahydrofuran and transferred to a 25 mL Schlenk flask. The solution was degassed by three freeze-vacuum-thaw cycles. Then, under nitrogen protection, tris(2-carboxyethyl)phosphonic acid hydrochloride (0.035 mmol) and n-hexylamine (0.28 mmol) were added, and residual oxygen was removed by three freeze-vacuum-thaw cycles. After stirring at room temperature in the dark for 16 h, the solution was added dropwise to 10 times the excess of cold diethyl ether to obtain a white precipitate. The precipitation process was repeated twice to remove unreacted amines and byproducts. The crude product was dialyzed (molecular weight cutoff 10 kDa), treated with deionized water for 48 h, and then lyophilized to obtain the purified thiol-terminated polymer p(AEO4MA- co -NIPAAM)-SH, with a yield of 89%, and its characterization is as follows: Figure 5 As shown;

[0114] In Figure A, p(AEO4MA- co -NIPAAM)-CS3 and p(AEO4MA- co -NIPAAM)-SH 1 The H NMR spectrum, Figure B shows p(AEO4MA-) co -NIPAAM)-CS3 and p(AEO4MA- co UV-Vis absorption spectrum of NIPAAM-SH.

[0115] Fifth, the temperature-responsive polymer TRCP modified with choline phosphate was synthesized, and its structural formula is shown below:

[0116] Its synthesis process is as follows:

[0117] p(AEO4MA- co -NIPAAM)-SH(100mg), 100 mg of copper sulfate pentahydrate (II) (1.25 mg), 2.5 mg of sodium ascorbate, and 5 mL of a methanol-water (4:1, v / v) mixture were added to a flask. After reacting at room temperature for 24 h, the reaction mixture was dialyzed against deionized water for 72 h using a dialysis membrane with a molecular weight cutoff of 1 kDa (with the dialysate changed 4–6 times). The purified product, a temperature-responsive choline phosphate-modified polymer (TRCP), was obtained by lyophilization. 1 H NMR spectrum (400MHz), such as Figure 6 As shown; its 31 p NMR spectrum (400MHz), such as Figure 7 As shown.

[0118] III. Preparation of AuNPs@DA nanoparticles.

[0119] The preparation of gold nanoparticles (AuNPs) was carried out as follows: 50 mL of 2.2 mmol / L sodium citrate aqueous solution was added to a round-bottom flask, heated to boiling, and stirred for 15 min. Then, 1 mL of 25 mmol / L HAuCl4 aqueous solution was added and stirred for about 10 min. The temperature was then lowered to 90 °C, and 1 mL of 60 mmol / L sodium citrate aqueous solution and 1 mL of 25 mmol / L gold perchlorate aqueous solution were added. The mixture was stirred for 30 min. This process was repeated until the average diameter of the AuNPs reached about 10 nm.

[0120] The AuNPs@DA nanoparticles were prepared as follows: The AuNPs solution obtained above was centrifuged at 9000 rpm for 10 min. The precipitated AuNPs were added to dopamine solution (1 mg / mL, Tris-HCl buffer, pH 8.5). After stirring at room temperature for 1.5 h, the AuNPs@DA solution was centrifuged with deionized water and resuspended for 1 min. The resulting AuNPs@DA solution was quantitatively diluted to a concentration of 2.5 mg / mL.

[0121] Morphology and particle size characterization, such as Figure 8 Figure A shows a TEM image of AuNPs@DA with a scale bar of 100 μm; Figure B shows a histogram of the hydrodynamic diameter distribution of AuNPs@DA; and Figure C shows a histogram of the zeta potential of AuNPs and AuNPs@DA.

[0122] Chemical composition and spectral structure characterization, such as Figure 9 As shown in the figure; Figure A is the XPS full spectrum of AuNPs@DA; Figure B is the high-resolution XPS spectrum of AuNPs@DA; Figure C is the UV-Vis absorption spectrum of HAuCl4, AuNPs, and AuNPs@DA.

[0123] Photothermal performance characterization, such as Figure 10 As shown; Figure A shows the laser wavelength at 808nm, with different powers (0.5W / cm²). 2 1.0W / cm 2 2.0W / cm 2 Figure A shows the photothermal image of AuNPs@DA at a sample concentration of 100 μg / mL over time. The color bar on the right side of the infrared image represents the temperature (°C). Figure B shows the photothermal heating curves of AuNPs@DA at different power levels. Figure C shows the photothermal temperature rise curve of AuNPs@DA under cyclic near-infrared irradiation (10 min on, 10 min off, repeated five times). Figure D shows the heating-cooling curve of AuNPs@DA under 808 nm laser irradiation. Figure E shows the linear fitting results of the cooling stage, where θ is the difference between the temperature of AuNPs@DA and the ambient temperature, Ln is the natural logarithm, and R0 is the linear coefficient. 2 The coefficient of determination.

[0124] IV. Preparation of controllable release exosome hydrogels.

[0125] First, the photoinitiator LAP was dissolved in deionized water to prepare a solution with a w / v ratio of 0.25%. An AuNPs@DA aqueous solution with a concentration of 1.2 mg / mL was prepared using ultrasound-assisted dispersion. NorHA was dissolved in the LAP solution at a predetermined concentration, and TRCP and AuNPs@DA dispersions were added sequentially. Then, a quantitative amount of human umbilical cord mesenchymal stem cell exosomes (hUCMSC-EVs) suspension was mixed in, and the mixture was vortexed at room temperature to obtain a homogeneous precursor solution. Finally, a power density of 25 mW / cm³ was used to prepare the solution. 2 Irradiation with 405nm blue light for 60s triggers a photocrosslinking reaction, achieving in-situ gelation and forming a hydrogel loaded with exosomes, denoted as NTA / EVs-Gel hydrogel.

[0126] Taking the preparation of 500 μL hydrogel as an example: using 0.25% LAP (500 μL) stock solution as a base, NorHA (10 mg, corresponding to a final concentration of 2%) was added and stirred to dissolve. Then, TRCP (2.5 mg) and AuNPs@DA (3.75 mg, corresponding to a final concentration of 7.5 mg / mL) were added and vortexed to mix. Finally, human umbilical cord mesenchymal stem cell exosomes (hUCMSC-EVs) (50 μg, corresponding to a final concentration of 100 μg / mL) were mixed in. The mixture was continuously vortexed at room temperature until the solution was homogeneous, resulting in a precursor solution (500 μL). This solution was then placed under 405 nm blue light (light intensity 25 mW / cm²). 2 Irradiation for 60 seconds initiates a photocrosslinking reaction to achieve in-situ gelation, ultimately forming a controllable release exosome hydrogel loaded with hUCMSC-EVs, denoted as: 2%NorHA-Gel;

[0127] The amount of NorHA added in the above-mentioned preparation of hydrogels was replaced with (20 mg, corresponding to 4% final concentration), (30 mg, corresponding to 6% final concentration), (40 mg, corresponding to 8% final concentration) and (100 mg, corresponding to 10% final concentration) respectively to prepare NTA / EVs-Gel hydrogels containing different mass volume concentrations of NorHA, which were denoted as 4%NorHA-Gel, 6%NorHA-Gel, 8%NorHA-Gel and 10%NorHA-Gel respectively.

[0128] V. Characterization of NTA / EVs-Gel Gel.

[0129] Fourier transform infrared spectroscopy (FT-IR) was used to characterize the chemical structures and functional group interactions of NorHA, TRCP, AuNPs@DA, and NTA-Gel to verify the success of the gel crosslinking reaction. Scanning electron microscopy (SEM) was used to observe the microporous structure of the gel and the dispersion state of hUCMSC-EVs in NTA / EVs-Gel. The swelling performance of the gel in PBS buffer at 37℃ and pH=7.4 was determined by gravimetric analysis. In vitro degradation experiments were conducted to investigate the degradation behavior of hydrogels at different mass / volume concentrations in PBS buffer and to analyze their degradation kinetics. The storage modulus (G') and loss modulus (G'') of the gel were measured using a rheometer to evaluate its mechanical stability and viscoelasticity under different strains and angular frequencies. Details are as follows:

[0130] (I) Characterization of chemical structure and functional groups.

[0131] Test method: Fourier transform infrared spectroscopy (FT-IR);

[0132] Test subjects: NorHA, TRCP, AuNPs@DA, and NTA-Gel;

[0133] Characterization objective: To verify the chemical structure of each component and the success of the gel crosslinking reaction;

[0134] Characterization results: such as Figure 11 As shown.

[0135] (ii) Microscopic morphological characterization.

[0136] Test method: Scanning electron microscopy (SEM);

[0137] Test subject: NTA / EVs-Gel lyophilized sample;

[0138] Characterization objective: To observe the microstructure, pore size distribution, and dispersion state of hUCMSC-EVs in the matrix;

[0139] Characterization results: such as Figure 12 As shown;

[0140] Figure A shows the micron-scale three-dimensional porous network morphology of the NTA / EVs-Gel freeze-dried sample; Figure B shows the nanoscale dispersed particles of the NTA / EVs-Gel freeze-dried sample.

[0141] (III) Characterization of swelling properties.

[0142] Test method: Gravimetric method;

[0143] Test conditions: 37 o C. PBS buffer with pH 7.4;

[0144] Characterization objective: To determine the gel swelling rate and swelling kinetics;

[0145] Characterization results: such as Figure 13 As shown;

[0146] Figure A shows the relationship between the swelling rate and time for hydrogels with different mass volume concentrations of NorHA.

[0147] Figure B shows images of the NTA / EVs-Gel hydrogel before and after swelling equilibrium.

[0148] (iv) Characterization of degradation behavior.

[0149] Test method: In vitro degradation experiment;

[0150] Test conditions: NTA / EVs-Gel of different mass / volume concentrations were incubated in PBS buffer at room temperature;

[0151] Characterization objective: To analyze gel degradation behavior and degradation kinetics;

[0152] Characterization results: such as Figure 14 As shown.

[0153] (V) Characterization of mechanical properties

[0154] Test method: rheometer test;

[0155] Test parameters: energy storage modulus (G'), loss modulus (G'');

[0156] Test conditions: different strains (0.1%~1000%), different angular frequencies (0.1~100Hz);

[0157] Characterization objective: To evaluate the mechanical stability and viscoelasticity of the gel;

[0158] Characterization results: such as Figure 15 As shown;

[0159] Figure A shows the strain amplitude scanning curves of storage modulus (G') and loss modulus (G") (test conditions: 25℃, angular frequency 1Hz, strain range 0.1%~1000%).

[0160] Figure B shows the time scan curves of storage modulus (G') and loss modulus (G") (test conditions: 25℃, angular frequency 1Hz, strain 1%).

[0161] In the following test examples, if the treated hydrogel component contains NorHA, its mass-volume concentration is 2%; if it contains AuNPs@DA, its mass-volume concentration is 0.75%; and if it contains exosomes, it is hUCMSC-EVs (concentration 1×10⁻⁶).8 (particles / mL, particle size 70–100 nm).

[0162] Comparative Example 1

[0163] Except for the absence of hUCMSC-EVs suspension, the rest of the process is the same as the 2% NorHA-Gel hydrogel preparation process in Example 1, denoted as NTA-Gel hydrogel.

[0164] Comparative Example 2

[0165] Except for the absence of AuNPs@DA dispersion and hUCMSC-EVs suspension, the rest of the process is the same as the 2% NorHA-Gel hydrogel preparation process in Example 1, denoted as NorHA / TRCP.

[0166] Comparative Example 3

[0167] Except for the absence of TRCP and hUCMSC-EVs suspension, the rest of the process is the same as the 2% NorHA-Gel hydrogel preparation process in Example 1, denoted as NorHA / AuNPs@DA.

[0168] Test Example 1

[0169] Colony counting was performed using the optical density (OD) method and the plate method; viable / dead bacteria staining and SEM observation were used to evaluate its resistance to methicillin-resistant Staphylococcus aureus (MRSA). Methicillin-resistant Staphylococcus aureus , MRSA The antibacterial activity and bactericidal effect of )

[0170] Groups: Control (negative control, treated with PBS buffer), AMP (positive control, treated with ampicillin), Gel 1 (treated with 2% NorHA solution), Gel 2 (treated with NorHA / TRCP), Gel 3 (treated with NorHA / AuNPs@DA), Gel 4 (treated with 2% NorHA solution and NIR irradiation), Gel 5 (treated with NTA / EVs-Gel), Gel 6 (treated with NTA / EVs-Gel and NIR irradiation);

[0171] NIR stands for near-infrared spectrum;

[0172] Test results, such as Figure 16 As shown;

[0173] Figure A shows the results after different sample treatments. MRSAColony growth display diagram; Figure B is the bacterial morphology display diagram after different sample treatments magnified 20,000 times (20.0k×) and 50,000 times (50.0k×) by scanning electron microscope, and the scale bar is 500 nm; Figure C is the display diagram of live bacteria, dead bacteria and fusion fluorescence staining results after different sample treatments; Figure D is the columnar diagram of OD direct quantification at 590 nm after different sample treatments.

[0174] Test Example 2

[0175] Analysis of the on-demand release efficiency and cumulative release rate of exosomes under different NIR irradiation conditions by fluorescence quantification method of exosomes labeled with 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO), and the NIR irradiation power is 2.0 W / cm 2 , cyclic irradiation (10 min on / 10 min off), and the results are as Figure 17 shown.

[0176] Test Example 3

[0177] I. Effects of different treatment substances on cell biological activity.

[0178] Test method: CCK-8 method (detect cell proliferation); scratch test, transwell test (evaluate cell migration); tube formation test (investigate angiogenesis);

[0179] Test cells: HUVECs cells;

[0180] Test purpose: To evaluate the effects of the gel on cell proliferation, migration and angiogenesis;

[0181] Grouping: Control is the PBS buffer treatment group, NTA-Gel treatment group, EVs is the exosome treatment group, and NTA / EVs-Gel treatment group.

[0182] The quantitative analysis results of the viability of HUVECs cells by CCK-8 method are as Figure 18 shown. In the figure, ns represents no statistical significance, * represents 0.01 < P ≤ 0.05, ** represents 0.001 < P ≤ 0.01, and *** represents P ≤ 0.001.

[0183] The detection results of the migration ability of HUVECs cells are as Figure 19 shown; The display diagram of the transwell test results of the effects of different substances on the migration of HUVECs is as Figure 20 shown; The bright-field images of the tubular structure formation of HUVECs after different substance treatments are as Figure 21 shown, and the scale bar is 100 µm.

[0184] Test Example 4

[0185] Through in vivo animal experiments (full-thickness skin defect wound model, deep second-degree burn model), combined with wound healing photography, histological section H&E staining (evaluating epithelialization and tissue repair), and Masson's trichrome staining (analyzing collagen deposition), the in vivo wound healing effect was systematically evaluated as follows:

[0186] I. Construction of a full-thickness skin defect wound model in mice.

[0187] Male Kunming mice aged 5 - 6 weeks and weighing 25 - 35 g were selected and fed adaptively for 1 week before the experiment. All operations were carried out under sterile conditions. After anesthetizing the mice with isoflurane, the back hair was shaved and the remaining hair was thoroughly removed using depilatory cream. The limbs and tail were fixed on a sterile surgical board, and the exposed back skin was disinfected with 75% ethanol. A full-thickness skin defect wound with a diameter of 8 mm was prepared in the center of the back using a sterile biopsy punch. In some models, a suspension of 10 7 CFU / mL was applied to the wound surface to construct an infected full-thickness skin defect wound. The mice were randomly divided into 5 groups, namely the sterile wound dressing group (control group), NTA-Gel + NIR irradiation group, free EVs treatment group, NTA / EVs-Gel group (without NIR irradiation), and NTA / EVs-Gel + NIR irradiation group. According to the grouping, the corresponding treatments were applied locally to the wound surface. MRSA The appearance and scabbing of the wound surface were recorded regularly, and the wound closure rate was calculated. The results are as

[0188] shown; Figure 22 as follows;

[0189] Among them, Figure A is a display diagram of the healing of the full-thickness skin defect wounds in mice of different treatment groups;

[0190] Figure B is a quantitative analysis result diagram of the wound closure rate in mice. ns represents no statistical significance, * represents 0.01 < P ≤ 0.05, ** represents 0.001 < P ≤ 0.01, and *** represents P ≤ 0.001.

[0191] The H&E staining and Masson's trichrome staining conditions of different treatment groups are as Figure 23 shown;

[0192] Among them, Figure A is the H&E staining condition of different treatment groups, and the scale bar is 2 nm; Figure B is the Masson's trichrome staining condition of different treatment groups, and the scale bar is 2 nm.

[0193] II. Construction of a deep second-degree burn model in rats.

[0194] Healthy SD rats (weighing 200-250g) were selected and acclimatized for one week before the experiment. They were housed individually in a clean, well-ventilated environment with free access to food and water. The fur on the rats' backs was carefully trimmed to avoid damaging the subcutaneous tissue. After lightly anesthetizing the rats with ether, they were fixed supine on a sterile surgical board. A hollow plastic cylinder with an inner diameter of 1cm was vertically and firmly placed against the skin on the back. 100ml of ether was injected into the cylinder using a preheated syringe. o Boiling water (5 mL) was continuously applied to the skin for 10 seconds, then the cylinder was quickly inverted to drain the boiling water, creating a standardized circular deep second-degree burn wound with a diameter of 1 cm on the back. The control group rats underwent the same procedure, but room temperature distilled water was injected instead of boiling water. After modeling, pathological sections confirmed that the wounds conformed to the characteristics of deep second-degree burns, namely epidermal necrosis, dermal vascular damage, and extensive inflammatory cell infiltration.

[0195] Regularly record the appearance and scab formation of the wound, calculate the wound healing rate, and the results are as follows: Figure 24 As shown;

[0196] Figure A shows the wound healing status of rats in different treatment groups, and Figure B is a heat map showing the wound healing rate of rats.

[0197] H&E staining and Masson's trichrome staining in different treatment groups, such as Figure 25 As shown;

[0198] Figure A shows the H&E staining results of different treatment groups, with a scale bar of 2 nm; Figure B shows the Masson trichrome staining results of different treatment groups, with a scale bar of 2 nm.

[0199] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a controllable release exosome hydrogel, characterized in that, Includes the following steps: S100, NorHA was obtained by modifying sodium hyaluronate with norbornene anhydride. NorHA is a nobornene-functionalized hyaluronic acid. S200, using p(AEO4MA- co -NIPAAM)-SH for choline phosphate After modification, TRCP is obtained; Among them, p(AEO4MA- co The structural formula of -NIPAAM)-SH is shown below: ； The structure of TRCP is shown below: ； In the structural formula, x, y, and z are positive integers, and TRCP is a temperature-responsive polymer modified with choline phosphate. S300, AuNPs@DA nanoparticles were prepared using gold nanoparticles and dopamine; S400, prepare photoinitiator aqueous solution I and AuNPs@DA nanoparticle dispersion respectively; S500. Dissolve NorHA in the photoinitiator aqueous solution I to obtain NorHA-containing photoinitiator aqueous solution II. Add TRCP, AuNPs@DA nanoparticle dispersion and cell exosome suspension to the NorHA-containing photoinitiator aqueous solution II. Prepare a controllable release exosome hydrogel using the photocrosslinking in situ gelation method.

2. The method for preparing the controllable release exosome hydrogel as described in claim 1, characterized in that, The structural formula of NorHA is shown below: ； Where n and m are both positive integers, and the sum of n and m is 800.

3. The method for preparing the controllable release exosome hydrogel as described in claim 1, characterized in that, In step S200, p(AEO4MA- co In the -NIPAAM)-SH structure, x, y, and z are 9, 81, and 3, respectively. The synthesis includes the following steps: S210. Using tetraethylene glycol as a raw material, the monomer AEO4MA of the temperature-responsive polymer was synthesized, with the following structural formula: ; S220, utilizing the temperature-responsive monomers AEO4MA, NIPAAM, and chain transfer agents. A reversible addition-fragmentation chain transfer polymerization reaction was carried out in the presence of azobisisobutyronitrile (AEO4MA-) to synthesize the intermediate p(AEO4MA- co -NIPAAM)-CS3, the structure is shown below: ; S230. The terminal groups are removed by the aminolysis reaction of the intermediate to obtain p(AEO4MA- co -NIPAAM)-SH, the structure is as follows: 。 4. The method for preparing the controllable release exosome hydrogel as described in claim 1, characterized in that, The lower critical dissolution temperature of TRCP is 41℃~43℃.

5. The method for preparing the controllable release exosome hydrogel as described in claim 1, characterized in that, In step S400, the photoinitiator is selected from lithium phenyl-2,4,6-trimethylbenzoylphosphinate; And / or, in step S500, the cell exosomes are selected from human umbilical cord mesenchymal stem cell exosomes.

6. The method for preparing the controllable release exosome hydrogel as described in claim 1, characterized in that, In step S300, the particle size distribution of AuNPs@DA nanoparticles ranges from 65 nm to 72 nm.

7. The method for preparing the controllable release exosome hydrogel as described in claim 1, characterized in that, In step S500, the mass-volume concentration of TRCP in the controllably released exosome hydrogel is 2%–10%. And / or the mass-volume concentration of the AuNPs@DA nanoparticle dispersion is 0.25% to 1.25%; And / or the concentration of the exosome suspension is 1×10 8 ~1×10 12 The particle size distribution ranges from 70 nm to 100 nm, with a particle size of 1 / mL.

8. The method for preparing the controllable release exosome hydrogel as described in claim 1, characterized in that, In step S500, the light used in the photocrosslinking in-situ gelation method is selected from blue light, and the irradiation time is 30-90s; The wavelength range of the blue light is 380nm to 455nm.

9. A controllable release exosome hydrogel, characterized in that, It was prepared using the method for preparing controllable release exosome hydrogel as described in any one of claims 1 to 8.

10. An application of a controllable release exosome hydrogel, characterized in that, Antibacterial products and / or wound repair products can be prepared using the controllable release exosome hydrogel as described in claim 9.

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