Preparation method and application of catalytic hydrogen production dECM gel

Abstract

The invention relates to the technical field of gel for catalytic hydrogen production, and discloses a preparation method and application of dECM gel for catalytic hydrogen production, and the preparation method comprises the following steps: S1, dispersing magnetic nanoparticles in a catalyst precursor solution, adding an amino silane coupling agent, and stirring; adding acid, and carrying out hydrolysis reaction to obtain hydrogel; s2, carrying out drying and heat treatment on the hydrogel; carrying out surface modification on the obtained solid by adopting polyethyleneimine to obtain magnetic particles; s3, suspending the carboxyl-containing dECM in an MES buffer solution, then adding EDC and NHS, and reacting at room temperature to obtain an NHS ester intermediate; and S4, adding magnetic particles into the NHS ester intermediate, and carrying out amidation reaction to obtain the catalytic hydrogen production dECM gel. The prepared decellularized cartilage matrix hydrogel with the characteristic of catalytic hydrogen production can release hydrogen through a catalytic reaction, cell proliferation and differentiation are facilitated, and tissue regeneration is promoted.

Description

Technical Field

[0001] The present invention relates to the technical field of catalytic hydrogen production gel, and in particular to a preparation method and application of catalytic hydrogen production dECM gel. Background Art

[0002] Hydrogen is considered an effective antioxidant that can scavenge free radicals in the body, reduce oxidative stress, and help reduce cell damage and tissue inflammation by selectively neutralizing harmful reactive oxygen species (ROS) molecules. In the treatment of arthritis or cartilage damage, hydrogen can stimulate the proliferation and differentiation of osteoblasts and chondrocytes, accelerating the repair of cartilage tissue. In addition, hydrogen has an inhibitory effect on the inflammatory response in cartilage, helping to slow the degeneration of cartilage. In the treatment of fractures or bone defects, hydrogen can promote bone regeneration and healing by reducing inflammation, promoting angiogenesis, and stimulating the function of osteoblasts. In anti-fibrotic treatment, the inhibitory effect of hydrogen on fibrosis can alleviate or reverse the fibrotic process by regulating fibrosis-related signaling pathways and reducing excessive fibroblast proliferation and collagen deposition.

[0003] Catalytic hydrogen-producing gels have the characteristic of continuous hydrogen production. Therefore, they can play a positive role in arthritis, cartilage damage, bone repair, and fibrosis-related diseases. Hydrogen can be continuously released at the wound site, promoting cell proliferation, repairing damage, and accelerating tissue regeneration, which helps in the long-term repair process. For example, the invention patent with publication number CN117695956A discloses a hydrogel for sustained hydrogen release, its preparation method, and application. The hydrogel comprises the following components in percentage by mass: 0.5-0.7% sodium alginate, 2.0-2.6% polyvinyl alcohol, 0.7-1.0% sodium tetraborate, 0.5-4.0% auxiliary agent, 0.04-0.26% hydrogen production catalyst, and the remainder is deionized water; the hydrogen production catalyst is a heterojunction catalyst with photocatalytic hydrogen production activity, namely NH2-MIL-125@ZIS. However, the hydrogel is only used as a medical dressing and is not used to promote long-term repair processes such as cell proliferation and tissue regeneration. As a result, the binding properties of the hydrogen-producing catalyst in the hydrogel with the hydrogel carrier and the binding properties between the hydrogel and the tissue are difficult to meet long-term use requirements. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides a preparation method and application of catalytic hydrogen production dECM gel. Combining the technology of magnetism and catalytic hydrogen production, through the introduction of hydrogen production catalysts and magnetic nanomaterials and cross-linking treatment of hydrogels, a decellularized cartilage matrix (dECM) gel with catalytic hydrogen production properties is prepared. It can not only release hydrogen through catalytic reactions, which helps cell proliferation and differentiation and promotes tissue regeneration, but also has certain positioning and control advantages under the action of an external magnetic field. The magnetic field can optimize the distribution of the gel in the lesion area and help improve the reaction efficiency between the catalyst and the environment.

[0005] The purpose of the present invention is achieved through the following technical solutions: In a first aspect, the present invention provides a method for preparing a dECM gel for catalytic hydrogen production, comprising the following steps: S1. Dispersing magnetic nanoparticles in a catalyst precursor solution to obtain a mixed solution; adding an aminosilane coupling agent, adjusting the pH to 5-5.5, and performing a hydrolysis reaction to obtain a hydrogel; S2, drying and heat-treating the hydrogel; and then surface-modifying the obtained solid with polyethyleneimine to obtain magnetic particles; S3, suspending the carboxyl-containing dECM in MES buffer, then adding EDC and NHS, and reacting at room temperature to obtain NHS ester intermediate; S4. Add magnetic particles to the NHS ester intermediate to carry out an amidation reaction to obtain a catalytic hydrogen production dECM gel.

[0006] The catalytic hydrogen-producing dECM gel in this invention can not only be directionally controlled under the influence of an external magnetic field but also generate hydrogen through a catalytic reaction. The magnetic field helps stabilize the gel within the lesion area, improves the efficiency of electron transfer between the catalyst and the carrier, and optimizes local hydrogen production. Furthermore, the decellularized cartilage matrix (dECM) hydrogel matrix is ​​stable and biocompatible, making the dECM gel more suitable for bone tissue repair, promoting tissue regeneration and repairing damage.

[0007] To imbue the dECM gel with catalytic hydrogen production properties, magnetic nanoparticles are introduced into the dECM matrix. As carriers for catalytic hydrogen production, magnetic nanoparticles also possess magnetic responsiveness. After the hydrogen production catalyst is loaded onto the surface of the magnetic nanoparticles, an aminosilane coupling agent is added to improve the overall efficiency of the hydrogen production reaction and the durability of the catalyst. First, the aminosilane coupling agent not only enhances the binding between the catalyst and the magnetic nanoparticles but also undergoes a hydrolysis reaction to form a gel. This gelation reaction further enhances the binding between the catalyst and the magnetic nanoparticles and forms a uniform gel network. Second, the hydrogel is dried to remove the solvent, and the catalyst is reduced through heat treatment. After heat treatment, the silane coupling agent forms a silicon oxide layer composed of silicon dioxide, providing a uniform and chemically stable surface. This helps securely load the reduced metal nanoparticles onto the surface of the magnetic nanoparticles, minimizing catalyst shedding or aggregation. Furthermore, the heat treatment helps expose catalytically active sites, facilitating the adsorption and reaction of water molecules. The silicon oxide layer also improves the electron transport environment, further enhancing reaction efficiency and accelerating hydrogen production.

[0008] The solid obtained after heat treatment has a porous structure, which can improve the permeability and cross-linking between it and the dECM gel network and provide more reaction sites. In addition, surface modification by polyethyleneimine can improve hydrophilicity, dispersibility and compatibility with dECM gel or biological tissue. Moreover, the amino groups in polyethyleneimine can undergo covalent cross-linking reaction (amidation reaction) with the carboxyl groups in dECM to form stable covalent bonds. The magnetic particles can be firmly fixed in the dECM gel network, and finally a catalytic hydrogen production dECM gel is obtained.

[0009] Preferably, in S1, the catalyst precursor is chloroplatinate H2PtCl6, chlororuthenate RuCl3 or nickel nitrate Ni(NO3)2; the concentration of the catalyst precursor solution is 0.1-0.5 mol / L, the solvent is a mixed solution of water and ethanol, and the volume ratio of water to ethanol is 1:0.5-2.

[0010] Preferably, in S1, magnetic nanoparticles are dispersed in the catalyst precursor solution at a ratio of 1-5 mg / mL; the magnetic nanoparticles are iron-based nanoparticles (such as Fe3O4, Fe3O4@SiO2, etc.) or nickel-based nanoparticles (NiFe2O4).

[0011] Preferably, in S1, the aminosilane coupling agent is γ-aminopropyltriethoxysilane or γ-aminopropyltrimethoxysilane; the amount of the aminosilane coupling agent added is 0.5-5% of the volume of the mixed solution; the temperature of the hydrolysis reaction is 50-60° C., and the time is 0.5-2 h.

[0012] The pH of the solution is adjusted to 5-5.5 by adding acid, which can be hydrochloric acid.

[0013] Preferably, in S2, the heat treatment is carried out in a hydrogen atmosphere at 400-600° C. for 2-4 hours.

[0014] The heat treatment can ensure that the catalyst precursor is fully reduced to a metal catalyst and that the formed nano-metal particles are evenly distributed and firmly attached to the surface of the magnetic nano-particles.

[0015] Preferably, in S2, the surface modification comprises the following steps: dispersing the obtained solid in water to control the concentration to 0.5-2 mg / mL, and ultrasonically treating for 5-10 min to obtain a uniform dispersion; adding polyethyleneimine solution dropwise to the dispersion while stirring, and after the addition is completed, reacting at 25-37°C for 1-2 h, and then centrifuging, washing, and drying.

[0016] Preferably, the concentration of the polyethyleneimine solution is 1 mg / mL, the average molecular weight of polyethyleneimine is 10,000-25,000 Da, and the solvent is water; the amount of polyethyleneimine added is 1-10% of the mass of the obtained solid.

[0017] Preferably, in S3, the preparation of the carboxyl-containing dECM comprises the following steps: activating the dECM with p-nitrophenyl chloroformate (CAS No. 7693-46-1), and adding a carboxyl-containing compound to the activated dECM for reaction.

[0018] Preferably, activating dECM using p-nitrophenyl chloroformate comprises the following steps: adding p-nitrophenyl chloroformate to dichloromethane to obtain a p-nitrophenyl chloroformate solution with a concentration of 10-20 mM; mixing dECM and the p-nitrophenyl chloroformate solution in a ratio of 10 mg:1 mL, and reacting in an ice bath for 0.5-2 h.

[0019] Preferably, the reaction of the activated dECM with the carboxyl-containing compound comprises the following steps: adding a solution of a carboxyl-containing compound having a concentration of 45-55 mM to the activated dECM, the ratio of the added amount of dECM to the solution of the carboxyl-containing compound being 10 mg:1 mL, and reacting at 37° C. for 3-6 hours, wherein the carboxyl-containing compound comprises dimethylaminopropionic acid (DMAPA).

[0020] The carboxyl-containing compound is directly added to the activated dECM obtained after the ice bath reaction to react, ensuring that the activated hydroxyl groups remain active in the solution, thereby reacting more fully with DMAPA.

[0021] In order to introduce carboxyl groups (-COOH) on the dECM surface, p-nitrophenyl chloroformate is needed to first activate the hydroxyl (-OH) groups on the dECM. The activated dECM is reacted with a compound containing a carboxyl (-COOH) functional group. The dECM can effectively introduce carboxyl groups, providing functional groups for subsequent cross-linking reactions. The amino groups modified on the surface of the magnetic particles and the carboxyl groups introduced into the dECM are cross-linked using EDC (1-ethyl-3-(3-dimethylaminopropyl)aminomethylpyridine) and NHS (N-hydroxysuccinimide). After the carboxyl groups are activated, an amidation reaction occurs with the amino groups, and cross-linking forms stable amino-carboxyl covalent bonds. The magnetic particles are firmly fixed in the dECM gel network.

[0022] Preferably, in S3, the carboxyl-containing dECM is suspended in MES buffer, and the concentration of dECM is 1-10 mg / mL; the pH of the MES buffer is 5-6; EDC is added so that the final concentration of EDC in the solution is 0.2-0.3 M; NHS is added so that the final concentration of NHS in the solution is 0.1-0.2 M; and the reaction time at room temperature is 15-30 min.

[0023] Preferably, in S4, the amount of the magnetic particles added is 1-5% relative to the dry weight of the dECM; and the amidation reaction is carried out at 20-37° C. for 2-4 hours.

[0024] In a second aspect, the present invention also provides a use of the catalytic hydrogen-producing dECM gel prepared by the above preparation method in the preparation of bone tissue repair materials.

[0025] Compared with the prior art, the present invention has the following beneficial effects: (1) The hydrogen production catalyst is loaded on the surface of magnetic nanoparticles. The magnetic nanoparticles serve as carriers for catalytic hydrogen production and have magnetic response properties. The resulting catalytic hydrogen production dECM gel can not only produce hydrogen through catalytic reactions but can also be directionally controlled under the action of an external magnetic field. (2) The aminosilane coupling agent can not only enhance the binding between the catalyst and the magnetic nanoparticles, but also undergo a hydrolysis reaction to form a gel. The gelation reaction further enhances the binding between the catalyst and the magnetic nanoparticles and forms a uniform gel network. Afterwards, the catalyst is reduced by heat treatment. The silane coupling agent forms a silicon oxide layer composed of silicon dioxide after heat treatment, which reduces the shedding or agglomeration of the catalyst, further improves the reaction efficiency, and accelerates the generation of hydrogen. (3) Surface modification by polyethyleneimine can improve hydrophilicity, dispersibility, and compatibility with dECM gel or biological tissues; moreover, the amino groups in polyethyleneimine can undergo covalent cross-linking reaction (amidation reaction) with the carboxyl groups in dECM to form stable covalent bonds, and the magnetic particles can be firmly fixed in the dECM gel network, ultimately obtaining catalytic hydrogen production dECM gel. DETAILED DESCRIPTION

[0026] The technical solutions of the present invention are described below with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0027] The preparation method of the catalytic hydrogen production dECM gel of the present invention comprises the following steps: S1. Add the catalyst precursor to a mixed solution of water and ethanol (the volume ratio of water to ethanol is 1:0.5-2) to form a catalyst precursor solution with a concentration of 0.1-0.5 mol / L; disperse the magnetic nanoparticles in the catalyst precursor solution at a ratio of 1-5 mg / mL to obtain a mixed solution; add an aminosilane coupling agent with a volume fraction of 0.5-5% to the mixed solution and stir, then add hydrochloric acid to adjust the pH of the solution to 5-5.5, heat to 50-60°C for hydrolysis reaction, and the reaction time is 0.5-2h to obtain a hydrogel.

[0028] S2. Dry the hydrogel (freeze-dry or hot air dry) to remove the solvent, and then heat-treat it in a hydrogen atmosphere at 400-600°C for 2-4 hours; the obtained solid is surface-modified with polyethyleneimine, and the obtained solid is dispersed in water at a concentration of 0.5-2 mg / mL, and ultrasonically treated for 5-10 minutes to obtain a uniform dispersion; add a 1 mg / mL aqueous solution of polyethyleneimine (average molecular weight of 10,000-25,000 Da) to the dispersion while stirring, and the amount of polyethyleneimine added is 1-10% of the mass of the obtained solid. After the addition is completed, react at 25-37°C for 1-2 hours, and then centrifuge, wash, and dry to obtain magnetic particles.

[0029] S3. Add p-nitrophenyl chloroformate to dichloromethane to obtain a p-nitrophenyl chloroformate solution with a concentration of 10-20 mM; mix dECM and p-nitrophenyl chloroformate solution in a ratio of 10 mg:1 mL, and react in an ice bath for 0.5-2 h; after the reaction is completed, directly add a solution of a carboxyl-containing compound with a concentration of 45-55 mM to the activated dECM, and the ratio of the added amount of dECM to the solution of the carboxyl-containing compound is 10 mg:1 mL. The mixture is reacted at 37°C for 3-6 h and dried to obtain a carboxyl-containing dECM; the carboxyl-containing dECM is then suspended in MES buffer (pH 5-6) at a concentration of 1-10 mg / mL, and EDC and NHS are added so that the final concentration of EDC in the solution is 0.2-0.3 M and the final concentration of NHS is 0.1-0.2 M. The mixture is reacted at room temperature for 15-30 min to obtain an NHS ester intermediate.

[0030] S4. Add magnetic particles to the NHS ester intermediate in an amount of 1-5% relative to the dry weight of the dECM. Perform the amidation reaction at 20-37°C for 2-4 hours. After the reaction is complete, wash the gel multiple times with PBS or deionized water to remove unreacted cross-linker and byproducts to obtain a catalytic hydrogen production dECM gel.

[0031] In a specific embodiment of the present invention, in S1, the catalyst precursor is HPtCl, RuCl, or Ni(NO). The magnetic nanoparticles are iron-based nanoparticles (e.g., FeO, FeO@SiO, etc.) or nickel-based nanoparticles (NiFeO). The aminosilane coupling agent is γ-aminopropyltriethoxysilane or γ-aminopropyltrimethoxysilane.

[0032] In a specific embodiment of the present invention, in S3, dECM is decellularized cartilage matrix extracted from cartilage tissue, cell components in the cartilage tissue are removed by ultrasonication using a cell reagent, and the cartilage tissue is thoroughly washed with deionized water to remove residual chemical reagents, and then freeze-dried into a powder.

[0033] Example 1 S1. Add the catalyst precursor H2PtCl6 to a mixed solution of water and ethanol (the volume ratio of water to ethanol is 1:1) to form a catalyst precursor solution with a concentration of 0.2 mol / L; disperse the magnetic nanoparticles Fe3O4 in the catalyst precursor solution at a ratio of 3 mg / mL to obtain a mixed solution; add 2% volume fraction of γ-aminopropyltriethoxysilane to the mixed solution and stir, then add 0.1M hydrochloric acid to adjust the pH of the solution to 5-5.5, heat to 60°C for hydrolysis reaction, and the reaction time is 2 hours to obtain a hydrogel.

[0034] S2. Freeze-dry the hydrogel to remove the solvent, and then heat-treat it at 500°C at a heating rate of 5°C / min under a hydrogen atmosphere for 3 hours; disperse the obtained solid in water, control the concentration to 1 mg / mL, and ultrasonically treat it for 10 minutes to obtain a uniform dispersion; add a 1 mg / mL aqueous solution of polyethyleneimine (average molecular weight of 10,000 Da) dropwise to the dispersion while stirring, in an amount of polyethyleneimine that is 4% of the mass of the obtained solid. After the addition is completed, react at 25°C for 2 hours, and then centrifuge, wash, and dry to obtain magnetic particles.

[0035] S3. Add p-nitrophenyl chloroformate to dichloromethane to obtain a p-nitrophenyl chloroformate solution with a concentration of 15 mM; mix dECM and p-nitrophenyl chloroformate solution at a ratio of 10 mg:1 mL, and react in an ice bath for 1 hour; after the reaction is completed, directly add a 50 mM aqueous solution of dimethylaminopropionic acid to the activated dECM, and the ratio of the added amount of dECM to the aqueous solution of dimethylaminopropionic acid is 10 mg:1 mL. React at 37°C for 5 hours and obtain carboxyl-containing dECM after drying; then suspend the carboxyl-containing dECM in MES buffer (pH 5-6) at a concentration of 4 mg / mL, add EDC and NHS so that the final concentration of EDC in the solution is 0.2 M and the final concentration of NHS is 0.1 M, and react at room temperature for 20 minutes to obtain an NHS ester intermediate.

[0036] S4. Add magnetic particles to the NHS ester intermediate at an amount of 2.5% relative to the dry weight of the dECM. Perform an amidation reaction at 25°C for 3 h. After the reaction is complete, wash the gel multiple times with PBS or deionized water to remove unreacted cross-linker and byproducts, thereby obtaining a catalytic hydrogen production dECM gel.

[0037] Example 2 S1. Add the catalyst precursor H2PtCl6 to a mixed solution of water and ethanol (the volume ratio of water to ethanol is 1:1) to form a catalyst precursor solution with a concentration of 0.3 mol / L; disperse the magnetic nanoparticles Fe3O4 in the catalyst precursor solution at a ratio of 4 mg / mL to obtain a mixed solution; add 2% volume fraction of γ-aminopropyltriethoxysilane to the mixed solution and stir, then add 0.1M hydrochloric acid to adjust the pH of the solution to 5-5.5, heat to 60°C for hydrolysis reaction, and the reaction time is 2 hours to obtain a hydrogel.

[0038] S2. Freeze-dry the hydrogel to remove the solvent, and then heat-treat it at 500°C at a heating rate of 5°C / min under a hydrogen atmosphere for 3 hours; disperse the obtained solid in water, control the concentration to 1 mg / mL, and ultrasonically treat it for 10 minutes to obtain a uniform dispersion; add a 1 mg / mL aqueous solution of polyethyleneimine (average molecular weight of 10,000 Da) dropwise to the dispersion while stirring, in an amount of polyethyleneimine that is 4% of the mass of the obtained solid. After the addition is completed, react at 25°C for 2 hours, and then centrifuge, wash, and dry to obtain magnetic particles.

[0039] S3. Add p-nitrophenyl chloroformate to dichloromethane to obtain a p-nitrophenyl chloroformate solution with a concentration of 15 mM; mix dECM and p-nitrophenyl chloroformate solution in a ratio of 10 mg:1 mL, and react in an ice bath for 1 hour; after the reaction is completed, directly add a 50 mM aqueous solution of dimethylaminopropionic acid to the activated dECM, and the ratio of the added amount of dECM to the aqueous solution of dimethylaminopropionic acid is 10 mg:1 mL. React at 37°C for 5 hours and obtain carboxyl-containing dECM after drying; then suspend the carboxyl-containing dECM in MES buffer (pH 5-6) at a concentration of 5 mg / mL, add EDC and NHS so that the final concentration of EDC in the solution is 0.3 M and the final concentration of NHS is 0.2 M, and react at room temperature for 30 minutes to obtain an NHS ester intermediate.

[0040] S4. Add magnetic particles to the NHS ester intermediate at an amount of 3.5% relative to the dry weight of the dECM. Perform the amidation reaction at 37°C for 3 h. After the reaction is complete, wash the gel multiple times with PBS or deionized water to remove unreacted cross-linker and byproducts, thereby obtaining a catalytic hydrogen production dECM gel.

[0041] Example 3 S1. Add the catalyst precursor H2PtCl6 to a mixed solution of water and ethanol (the volume ratio of water to ethanol is 1:1) to form a catalyst precursor solution with a concentration of 0.4 mol / L; disperse the magnetic nanoparticles Fe3O4 in the catalyst precursor solution at a ratio of 4 mg / mL to obtain a mixed solution; add 3% volume fraction of γ-aminopropyltriethoxysilane to the mixed solution and stir, then add 0.1M hydrochloric acid to adjust the pH of the solution to 5-5.5, heat to 60°C for hydrolysis reaction, and the reaction time is 2 hours to obtain a hydrogel.

[0042] S2. Freeze-dry the hydrogel to remove the solvent, and then heat-treat it at 600°C at a heating rate of 5°C / min under a hydrogen atmosphere for 3 hours; disperse the obtained solid in water, control the concentration to 1 mg / mL, and ultrasonically treat it for 10 minutes to obtain a uniform dispersion; add a 1 mg / mL aqueous solution of polyethyleneimine (average molecular weight of 10,000 Da) dropwise to the dispersion while stirring, in an amount of polyethyleneimine that is 5% of the mass of the obtained solid. After the addition is completed, react at 25°C for 2 hours, and then centrifuge, wash, and dry to obtain magnetic particles.

[0043] S3. Add p-nitrophenyl chloroformate to dichloromethane to obtain a p-nitrophenyl chloroformate solution with a concentration of 15 mM; mix dECM and p-nitrophenyl chloroformate solution in a ratio of 10 mg:1 mL, and react in an ice bath for 1 hour; after the reaction is completed, directly add a 50 mM aqueous solution of dimethylaminopropionic acid to the activated dECM, and the ratio of the added amount of dECM to the aqueous solution of dimethylaminopropionic acid is 10 mg:1 mL. React at 37°C for 5 hours and obtain carboxyl-containing dECM after drying; then suspend the carboxyl-containing dECM in MES buffer (pH 5-6) at a concentration of 5 mg / mL, add EDC and NHS so that the final concentration of EDC in the solution is 0.3 M and the final concentration of NHS is 0.2 M, and react at room temperature for 30 minutes to obtain an NHS ester intermediate.

[0044] S4. Magnetic particles were added to the NHS ester intermediate at a concentration of 4.5% relative to the dry weight of the dECM. The amidation reaction was carried out at 37°C for 4 h. After the reaction was completed, the unreacted cross-linker and by-products were removed by multiple washing with PBS or deionized water to obtain a catalytic hydrogen production dECM gel.

[0045] Comparative Example 1 The difference from Example 1 is that no magnetic nanoparticles are added.

[0046] S1. Add the catalyst precursor H2PtCl6 to a mixed solution of water and ethanol (the volume ratio of water to ethanol is 1:1) to form a catalyst precursor solution with a concentration of 0.2 mol / L; add 2% volume fraction of γ-aminopropyltriethoxysilane to the catalyst precursor solution and stir, then add 0.1M hydrochloric acid to adjust the pH of the solution to 5-5.5, heat to 60°C for hydrolysis reaction, and the reaction time is 2h to obtain a hydrogel.

[0047] S2. Freeze-dry the hydrogel to remove the solvent, and then heat-treat it at 500°C at a heating rate of 5°C / min under a hydrogen atmosphere for 3 hours; disperse the obtained solid in water, control the concentration to 1 mg / mL, and ultrasonically treat it for 10 minutes to obtain a uniform dispersion; add a 1 mg / mL aqueous solution of polyethyleneimine (average molecular weight of 10,000 Da) dropwise to the dispersion while stirring, in an amount of polyethyleneimine that is 4% of the mass of the obtained solid. After the addition is completed, react at 25°C for 2 hours, and then centrifuge, wash, and dry to obtain magnetic particles.

[0048] S3. Add p-nitrophenyl chloroformate to dichloromethane to obtain a p-nitrophenyl chloroformate solution with a concentration of 15 mM; mix dECM and p-nitrophenyl chloroformate solution at a ratio of 10 mg:1 mL, and react in an ice bath for 1 hour; after the reaction is completed, directly add a 50 mM aqueous solution of dimethylaminopropionic acid to the activated dECM, and the ratio of the added amount of dECM to the aqueous solution of dimethylaminopropionic acid is 10 mg:1 mL. React at 37°C for 5 hours and obtain carboxyl-containing dECM after drying; then suspend the carboxyl-containing dECM in MES buffer (pH 5-6) at a concentration of 4 mg / mL, add EDC and NHS so that the final concentration of EDC in the solution is 0.2 M and the final concentration of NHS is 0.1 M, and react at room temperature for 20 minutes to obtain an NHS ester intermediate.

[0049] S4. Add magnetic particles to the NHS ester intermediate at an amount of 2.5% relative to the dry weight of the dECM. Perform an amidation reaction at 25°C for 3 h. After the reaction is complete, wash the gel multiple times with PBS or deionized water to remove unreacted cross-linker and byproducts, thereby obtaining a catalytic hydrogen production dECM gel.

[0050] Comparative Example 2 The difference from Example 1 is that no aminosilane coupling agent is added.

[0051] S1. Add the catalyst precursor H2PtCl6 to a mixed solution of water and ethanol (the volume ratio of water to ethanol is 1:1) to form a catalyst precursor solution with a concentration of 0.2 mol / L; disperse the magnetic nanoparticles Fe3O4 in the catalyst precursor solution at a ratio of 3 mg / mL to obtain a mixed solution.

[0052] S2. Dry the mixed solution to remove the solvent, and then heat-treat it at 500°C at a heating rate of 5°C / min under a hydrogen atmosphere for 3 hours; disperse the obtained solid in water, control the concentration to 1 mg / mL, and ultrasonically treat it for 10 minutes to obtain a uniform dispersion; add a 1 mg / mL aqueous solution of polyethyleneimine (average molecular weight of 10,000 Da) dropwise to the dispersion while stirring, in an amount of polyethyleneimine that is 4% of the mass of the obtained solid. After the addition is completed, react at 25°C for 2 hours, and then centrifuge, wash, and dry to obtain magnetic particles.

[0053] S3. Add p-nitrophenyl chloroformate to dichloromethane to obtain a p-nitrophenyl chloroformate solution with a concentration of 15 mM; mix dECM and p-nitrophenyl chloroformate solution at a ratio of 10 mg:1 mL, and react in an ice bath for 1 hour; after the reaction is completed, directly add a 50 mM aqueous solution of dimethylaminopropionic acid to the activated dECM, and the ratio of the added amount of dECM to the aqueous solution of dimethylaminopropionic acid is 10 mg:1 mL. React at 37°C for 5 hours and obtain carboxyl-containing dECM after drying; then suspend the carboxyl-containing dECM in MES buffer (pH 5-6) at a concentration of 4 mg / mL, add EDC and NHS so that the final concentration of EDC in the solution is 0.2 M and the final concentration of NHS is 0.1 M, and react at room temperature for 20 minutes to obtain an NHS ester intermediate.

[0054] S4. Add magnetic particles to the NHS ester intermediate at an amount of 2.5% relative to the dry weight of the dECM. Perform an amidation reaction at 25°C for 3 h. After the reaction is complete, wash the gel multiple times with PBS or deionized water to remove unreacted cross-linker and byproducts, thereby obtaining a catalytic hydrogen production dECM gel.

[0055] Comparative Example 3 The difference from Example 1 is that the amount of aminosilane coupling agent added is too much.

[0056] S1. Add the catalyst precursor H2PtCl6 to a mixed solution of water and ethanol (the volume ratio of water to ethanol is 1:1) to form a catalyst precursor solution with a concentration of 0.2 mol / L; disperse the magnetic nanoparticles Fe3O4 in the catalyst precursor solution at a ratio of 3 mg / mL to obtain a mixed solution; add 6% volume fraction of γ-aminopropyltriethoxysilane to the mixed solution and stir, then add 0.1M hydrochloric acid to adjust the pH of the solution to 5-5.5, heat to 60°C for hydrolysis reaction, and the reaction time is 2 hours to obtain a hydrogel.

[0057] S2. Freeze-dry the hydrogel to remove the solvent, and then heat-treat it at 500°C at a heating rate of 5°C / min under a hydrogen atmosphere for 3 hours; disperse the obtained solid in water, control the concentration to 1 mg / mL, and ultrasonically treat it for 10 minutes to obtain a uniform dispersion; add a 1 mg / mL aqueous solution of polyethyleneimine (average molecular weight of 10,000 Da) dropwise to the dispersion while stirring, in an amount of polyethyleneimine that is 4% of the mass of the obtained solid. After the addition is completed, react at 25°C for 2 hours, and then centrifuge, wash, and dry to obtain magnetic particles.

[0058] S3. Add p-nitrophenyl chloroformate to dichloromethane to obtain a p-nitrophenyl chloroformate solution with a concentration of 15 mM; mix dECM and p-nitrophenyl chloroformate solution at a ratio of 10 mg:1 mL, and react in an ice bath for 1 hour; after the reaction is completed, directly add a 50 mM aqueous solution of dimethylaminopropionic acid to the activated dECM, and the ratio of the added amount of dECM to the aqueous solution of dimethylaminopropionic acid is 10 mg:1 mL. React at 37°C for 5 hours and obtain carboxyl-containing dECM after drying; then suspend the carboxyl-containing dECM in MES buffer (pH 5-6) at a concentration of 4 mg / mL, add EDC and NHS so that the final concentration of EDC in the solution is 0.2 M and the final concentration of NHS is 0.1 M, and react at room temperature for 20 minutes to obtain an NHS ester intermediate.

[0059] S4. Add magnetic particles to the NHS ester intermediate at an amount of 2.5% relative to the dry weight of the dECM. Perform an amidation reaction at 25°C for 3 h. After the reaction is complete, wash the gel multiple times with PBS or deionized water to remove unreacted cross-linker and byproducts, thereby obtaining a catalytic hydrogen production dECM gel.

[0060] Comparative Example 4 The difference from Example 1 is that no heat treatment was performed.

[0061] S1. Add the catalyst precursor H2PtCl6 to a mixed solution of water and ethanol (the volume ratio of water to ethanol is 1:1) to form a catalyst precursor solution with a concentration of 0.2 mol / L; disperse the magnetic nanoparticles Fe3O4 in the catalyst precursor solution at a ratio of 3 mg / mL to obtain a mixed solution; add 2% volume fraction of γ-aminopropyltriethoxysilane to the mixed solution and stir, then add 0.1M hydrochloric acid to adjust the pH of the solution to 5-5.5, heat to 60°C for hydrolysis reaction, and the reaction time is 2 hours to obtain a hydrogel.

[0062] S2. Freeze-dry the hydrogel to remove the solvent; disperse the obtained solid in water, control the concentration to 1 mg / mL, and ultrasonicate for 10 minutes to obtain a uniform dispersion; add a 1 mg / mL aqueous solution of polyethyleneimine (average molecular weight of 10,000 Da) dropwise to the dispersion while stirring, and the amount of polyethyleneimine added is 4% of the mass of the obtained solid. After the addition is completed, react at 25°C for 2 hours, then centrifuge, wash, and dry to obtain magnetic particles.

[0063] S3. Add p-nitrophenyl chloroformate to dichloromethane to obtain a p-nitrophenyl chloroformate solution with a concentration of 15 mM; mix dECM and p-nitrophenyl chloroformate solution at a ratio of 10 mg:1 mL, and react in an ice bath for 1 hour; after the reaction is completed, directly add a 50 mM aqueous solution of dimethylaminopropionic acid to the activated dECM, and the ratio of the added amount of dECM to the aqueous solution of dimethylaminopropionic acid is 10 mg:1 mL. React at 37°C for 5 hours and obtain carboxyl-containing dECM after drying; then suspend the carboxyl-containing dECM in MES buffer (pH 5-6) at a concentration of 4 mg / mL, add EDC and NHS so that the final concentration of EDC in the solution is 0.2 M and the final concentration of NHS is 0.1 M, and react at room temperature for 20 minutes to obtain an NHS ester intermediate.

[0064] S4. Add magnetic particles to the NHS ester intermediate at an amount of 2.5% relative to the dry weight of the dECM. Perform an amidation reaction at 25°C for 3 h. After the reaction is complete, wash the gel multiple times with PBS or deionized water to remove unreacted cross-linker and byproducts, thereby obtaining a catalytic hydrogen production dECM gel.

[0065] Hydrogen production performance test: 100 mg of catalytic hydrogen production dECM gel was placed in a sealed reaction container. PBS buffer was added to the container to immerse the catalytic hydrogen production dECM gel. The container was placed in a 37°C temperature-controlled box to carry out photocatalytic hydrogen production under simulated sunlight conditions.

[0066] Continuous hydrogen production time: the time point when hydrogen production stops increasing.

[0067] Hydrogen production rate: The average hydrogen production rate is calculated based on the continuous hydrogen production time and hydrogen production amount.

[0068] Magnetism: Magnet adsorption was used to observe the magnetic adsorption phenomenon of dECM gel for catalytic hydrogen production.

[0069] Table 1 The catalytic hydrogen production dECM gel in Examples 1-3 of the present invention uses decellularized cartilage matrix (dECM) as a gel matrix and can continuously release hydrogen, promote tissue regeneration, repair cartilage damage, and repair the surface cartilage of the defective part. As shown in Table 1, magnetic nanoparticles are not added in Comparative Example 1, and the resulting hydrogen production gel has no magnetic characteristics, and when the hydrogen production catalyst is loaded on the surface of the magnetic nanoparticles, the uniform dispersion of the catalyst is better, and has a large catalytic specific surface area for effective reaction. Therefore, the lack of magnetic nanoparticles can affect the dispersibility and stability of the catalyst, may lead to faster deactivation, and the hydrogen production rate will also be affected, and then the hydrogen production amount and continuous hydrogen production time will also be affected. The addition of aminosilane coupling agent in Comparative Example 2 and aminosilane coupling agent in Comparative Example 3 is too much, which will affect the binding between the hydrogen production catalyst and the carrier, and will also affect the dispersibility of the hydrogen production catalyst, and then affect the hydrogen production efficiency. Among them, the excess aminosilane coupling agent may form a thick silicon oxide layer on the surface of the magnetic nanoparticles during the gelation and heat treatment process, which may cover the catalyst active sites, reduce the probability of catalyst contact with water, and may also interfere with the transmission of electrons between the catalyst and the magnetic particles, ultimately reducing the catalytic hydrogen production rate and total amount. In Comparative Example 4, no heat treatment was performed. Since the hydrogen production catalyst was not heat-treated and reduced, it would directly affect the hydrogen production efficiency. At the same time, the magnetic hydrogel was not heat-treated to form a porous structure, which would affect its binding with the dECM gel, and thus also affect the sustained hydrogen production effect.

[0070] The above are only preferred embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the description of the present invention, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for preparing a dECM gel for catalytic hydrogen production, characterized in that: The steps include: S1. Dispersing magnetic nanoparticles in a catalyst precursor solution to obtain a mixed solution; adding an aminosilane coupling agent, adjusting the pH to 5-5.5, and performing a hydrolysis reaction to obtain a hydrogel; S2, drying and heat-treating the hydrogel; and then surface-modifying the obtained solid with polyethyleneimine to obtain magnetic particles; S3, suspending the carboxyl-containing dECM in MES buffer, then adding EDC and NHS, and reacting at room temperature to obtain NHS ester intermediate; S4. Add magnetic particles to the NHS ester intermediate to carry out an amidation reaction to obtain a catalytic hydrogen production dECM gel.

2. The method for preparing the catalytic hydrogen production dECM gel according to claim 1, characterized in that: In S1, the catalyst precursor is H2PtCl6, RuCl3 or Ni(NO3)2; the concentration of the catalyst precursor solution is 0.1-0.5 mol / L.

3. The method for preparing the catalytic hydrogen production dECM gel according to claim 1, characterized in that: In S1, magnetic nanoparticles are dispersed in a catalyst precursor solution at a ratio of 1-5 mg / mL; the magnetic nanoparticles are iron-based nanoparticles or nickel-based nanoparticles.

4. The method for preparing the catalytic hydrogen production dECM gel according to any one of claims 1 to 3, characterized in that: In S1, the amount of the aminosilane coupling agent added is 0.5-5% of the volume of the mixed solution; the temperature of the hydrolysis reaction is 50-60° C., and the time is 0.5-2 h.

5. The method for preparing the catalytic hydrogen production dECM gel according to claim 1, characterized in that: In S2, the heat treatment is carried out in a hydrogen atmosphere at 400-600° C. for 2-4 hours.

6. The method for preparing the catalytic hydrogen production dECM gel according to claim 1 or 5, characterized in that: In S2, the surface modification comprises the following steps: dispersing the obtained solid in water, adding polyethyleneimine solution dropwise while stirring, reacting at 25-37°C for 1-2 hours after the addition is completed, and then centrifuging and drying.

7. The method for preparing the catalytic hydrogen production dECM gel according to claim 1, characterized in that: In S3, the preparation of the carboxyl-containing dECM comprises the following steps: activating the dECM with p-nitrophenyl chloroformate, and reacting the activated dECM with a carboxyl-containing compound.

8. The method for preparing the catalytic hydrogen production dECM gel according to claim 1 or 7, characterized in that: In S3, the carboxyl-containing dECM is suspended in MES buffer at a concentration of 1-10 mg / mL; EDC is added to a final concentration of 0.2-0.3 M; NHS is added to a final concentration of 0.1-0.2 M; and the reaction time at room temperature is 15-30 min.

9. The method for preparing the catalytic hydrogen production dECM gel according to claim 1, characterized in that: In S4, the amount of the magnetic particles added is 1-5% relative to the dry weight of the dECM; and the amidation reaction is carried out at 20-37° C. for 2-4 hours.

10. Use of the catalytic hydrogen-producing dECM gel prepared by the preparation method according to any one of claims 1 to 9 in preparing bone tissue repair materials.

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