A ZP@GGA / OCMC composite hydrogel as well as a preparation method and application thereof

Abstract

The application provides a ZP@GGA / OCMC composite hydrogel and a preparation method and application thereof, and belongs to the technical field of hydrogels.The preparation method comprises the following steps: performing a gelation reaction on a gallic acid grafted gelatin solution, an oxidized carboxymethyl cellulose solution and ZIF-8 / 67@PDA nanoparticles to obtain the ZP@GGA / OCMC composite hydrogel.In the application, the Schiff base reaction between GGA and OCMC endows the hydrogel with a rapid in-situ gelation capability and a dynamic reversible network structure, the PDA coating layer significantly improves the hydrophilicity and dispersibility of the ZIF-8 / 67 nanoparticles, and the Zn 2+ and Co 2+ The time-sequential release of two functional ions is realized; the composite hydrogel not only has the properties of injectability, tissue adhesion and degradation behavior matching the bone healing period, but also has multiple functions such as time-sequential ion release, oxidation resistance and bone formation promotion.

Description

Technical Field

[0001] This invention relates to the field of hydrogel technology, and in particular to a ZP@GGA / OCMC composite hydrogel, its preparation method, and its application. Background Technology

[0002] Osteomyelitis is typically caused by infection with pathogens such as methicillin-resistant Staphylococcus aureus (MRSA), severely impacting the healing of bone defects. Metal-organic frameworks (MOFs) have shown great potential in achieving both antibacterial and osteogenic effects. However, the burst release of metal ions and inherent hydrophobicity of single-metal MOFs significantly limit their application in vivo. Furthermore, severe inflammation caused by wound infection can significantly disrupt the infection microenvironment, hindering the bone repair process.

[0003] Therefore, developing a hydrogel system that not only improves the hydrophilicity of MOFs to achieve time-controlled release of metal ions but also possesses injectability and adhesiveness is of profound significance for the clinical treatment of osteomyelitis. Summary of the Invention

[0004] The purpose of this invention is to provide a ZP@GGA / OCMC composite hydrogel, its preparation method, and its application. This invention overcomes the shortcomings of traditional MOF materials, such as strong hydrophobicity and single function. The composite hydrogel combines time-sequential ion release and injectability to meet the dual needs of infection control and bone repair in the treatment of osteomyelitis.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing ZP@GGA / OCMC composite hydrogel, comprising the following steps: 1) Zn source, Co source and organic ligand were reacted in methanol to obtain ZIF-8 / 67 nanoparticles; 2) Disperse ZIF-8 / 67 nanoparticles in Tris-HCl buffer, add dopamine hydrochloride, stir and sonicate to obtain ZIF-8 / 67@PDA nanoparticles; 3) Gallic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide and mixed solvent are mixed, and the resulting mixture and gelatin solution are subjected to grafting reaction to obtain gallic acid grafted gelatin. 4) Carboxymethyl cellulose, dispersing solvent and NaIO4 are mixed and subjected to oxidation reaction. A quencher is added to terminate the reaction to obtain oxidized carboxymethyl cellulose; 5) Gallic acid-grafted gelatin and oxidized carboxymethyl cellulose were mixed with PBS buffer to obtain gallic acid-grafted gelatin solution and oxidized carboxymethyl cellulose solution, respectively. The gallic acid-grafted gelatin solution, oxidized carboxymethyl cellulose solution and ZIF-8 / 67@PDA nanoparticles were subjected to a gelation reaction to obtain ZP@GGA / OCMC composite hydrogel.

[0006] Preferably, in step 1), the Zn source comprises zinc nitrate and / or zinc acetate; the Co source comprises cobalt nitrate hexahydrate and / or cobalt acetate; the organic ligand is dimethylimidazole; the molar ratio of the Zn source to the Co source is 0.5~1.5:0.5~1.5, and the molar ratio of the total number of Zn and Co sources to the organic ligand is 1:1.5~4; the reaction time is 22~26 h.

[0007] Preferably, the pH of the Tris-HCl buffer solution in step 2) is 8.0~9.0; and the mass ratio of dopamine hydrochloride to ZIF-8 / 67 nanoparticles is 0.5~2:1.

[0008] Preferably, the solvent for the gelatin solution in step 3) is water, PBS buffer, or physiological saline, and the mixed solvent is a mixture of N,N-dimethylformamide and water, with a volume ratio of N,N-dimethylformamide to water of 1~3:1~3; the mass ratio of gelatin to gallic acid is 1:0.5~0.8, and the mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, and gallic acid is 1.0~1.2:0.8~1.0:1.

[0009] Preferably, the temperature of the grafting reaction in step 3) is 35~45℃, and the grafting reaction time is 18~36h.

[0010] Preferably, the quenching agent in step 4) is ethylene glycol, the dispersing solvent is water or physiological saline, the mass ratio of carboxymethyl cellulose to NaIO4 is 1:0.4~0.8, and the oxidation reaction time is 18~36h.

[0011] Preferably, in step 5), the concentration of the gallic acid-grafted gelatin solution is 15-25 wt%, the concentration of the oxidized carboxymethyl cellulose solution is 3-6 wt%, and the volume ratio of the gallic acid-grafted gelatin solution to the oxidized carboxymethyl cellulose solution is 0.5-1.5:0.5-1.5; the concentration of the ZIF-8 / 67@PDA nanoparticles in the ZP@GGA / OCMC composite hydrogel is 0.3-2 mg / mL.

[0012] Preferably, the temperature of the gelation reaction in step 5) is 35~40℃, and the gelation reaction time is 0.5~2h.

[0013] The present invention also provides a method for preparing the ZP@GGA / OCMC composite hydrogel.

[0014] The present invention also provides the application of the ZP@GGA / OCMC composite hydrogel in the preparation of materials for the treatment of chronic osteomyelitis and / or materials for the repair of infected bone defects.

[0015] The beneficial effects of this invention are: 1) This invention provides a method for preparing a ZP@GGA / OCMC composite hydrogel, comprising: preparation of a zinc-cobalt bimetallic MOF; PDA coating modification of the MOF material; gallic acid modification of gelatin; OCMC preparation; and obtaining a ZP@GGA / OCMC injectable hydrogel by dynamic covalent crosslinking of the above components with Schiff bases. In the preparation method of this invention, the Schiff base reaction between GGA and OCMC endows the hydrogel with rapid in-situ gelation ability and dynamic reversible network structure. The PDA coating layer significantly improves the hydrophilicity and dispersibility of ZIF-8 / 67 nanoparticles, and the Zn on the surface of ZP nanoparticles (ZP NPs) is also improved. 2+ and Co 2+ The time-sequential release of two functional ions—Co—was achieved. 2+ Early rapid release to disrupt bacterial biofilms and remodel the immune microenvironment, Zn 2+ Sustained and long-lasting release promotes osteogenic differentiation. The ZP@GGA / OCMC composite hydrogel prepared in this invention not only possesses injectability, tissue adhesion, and degradation behavior matching the bone healing cycle, but also has multiple functions such as timed ion release, anti-oxidation, and osteopromoting effects. It can be used for minimally invasive treatment of chronic osteomyelitis and infected bone defects, and has positive significance for the development of biofunctional bone repair materials.

[0016] 2) Zn in ZIF-8 / 67 bimetallic MOF 2+ / Co 2+ ZIF-8 / 67 has shown potential applications in antibacterial and osteogenic fields; however, its inherent hydrophobic properties make it difficult to disperse uniformly in aqueous systems, and the ion release behavior of metal ions in physiological environments is difficult to control, limiting its application in injectable hydrogel systems. This invention significantly improves the hydrophilicity and dispersibility of ZIF-8 / 67 nanoparticles by in-situ coating them with a polydopamine layer, while simultaneously achieving Co… 2+ Early release, Zn 2+ The product exhibits a continuous, time-sequential ion release behavior. Furthermore, by combining the modified nanoparticles with a gel matrix, the resulting ZP@GGA / OCMC composite hydrogel possesses excellent injectability and can be delivered to the lesion site via a minimally invasive approach for in-situ shaping. Attached Figure Description

[0017] Figure 1 The images show the XRD patterns of the ZP@GGA / OCMC composite hydrogel of Example 1, the Gel / OCMC hydrogel of Comparative Example 1, and the GGA / OCMC hydrogel of Comparative Example 2. Figure 2 FTIR images of ZP@GGA / OCMC composite hydrogel of Example 1, Gel / OCMC hydrogel of Comparative Example 1, and GGA / OCMC hydrogel of Comparative Example 2; Figure 3 The in vitro degradation curves of ZP@GGA / OCMC composite hydrogel of Example 1, Gel / OCMC hydrogel of Comparative Example 1, and GGA / OCMC hydrogel of Comparative Example 2 are shown. Figure 4 The in vitro antioxidant properties of ZP@GGA / OCMC composite hydrogel of Example 1, Gel / OCMC hydrogel of Comparative Example 1 and GGA / OCMC hydrogel of Comparative Example 2 are compared. Among them, (a) is a bar chart of DDPH free radical scavenging ability and (b) is a bar chart of ABTS free radical scavenging ability. Figure 5 The image shows a macroscopic view of the ZP@GGA / OCMC composite hydrogel of Example 1, where (a) is a schematic diagram of injectability and (b) is a schematic diagram of adhesion. Figure 6 The metal ion release curve of the ZP@GGA / OCMC composite hydrogel in Example 1 is shown. Figure 7 The images show 250 μm SEM images (a~c) of the ZP@GGA / OCMC composite hydrogel of Example 1, the Gel / OCMC hydrogel of Comparative Example 1, and the GGA / OCMC hydrogel of Comparative Example 2, and the EDS image (d) of the composite hydrogel prepared in Example 1. Figure 8 The images shown are 100nm SEM and EDS images of the metal nanoparticles in Example 1, where (a) is the SEM image of ZIF-8 / 67, (b) is the SEM image of ZIF-8 / 67@PDA, and (c) is the EDS image of Zn, Co, C, N and O in ZIF-8 / 67. Figure 9 Zeta plots of ZIF-8 / 67 nanoparticles and ZIF-8 / 67@PDA nanoparticles from Example 1; Figure 10 The static water angle contact diagrams of ZIF-8 / 67 nanoparticles and ZIF-8 / 67@PDA nanoparticles in Example 1 are shown. Detailed Implementation

[0018] This invention provides a method for preparing ZP@GGA / OCMC composite hydrogel, comprising the following steps: 1) Zn source, Co source and organic ligand were reacted in methanol to obtain ZIF-8 / 67 nanoparticles; 2) Disperse ZIF-8 / 67 nanoparticles in Tris-HCl buffer, add dopamine hydrochloride and stir and sonicate to obtain ZIF-8 / 67@PDA nanoparticles (denoted as ZP NPs); 3) Gallic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS) are mixed with a mixed solvent, and the resulting mixture is subjected to a grafting reaction with a gelatin solution to obtain gallic acid-grafted gelatin (denoted as GGA). 4) Carboxymethyl cellulose (CMC), dispersing solvent and NaIO4 are mixed and subjected to oxidation reaction. A quencher is added to terminate the reaction to obtain oxidized carboxymethyl cellulose (denoted as OCMC). 5) Gallic acid-grafted gelatin and oxidized carboxymethyl cellulose were mixed with PBS buffer to obtain gallic acid-grafted gelatin solution and oxidized carboxymethyl cellulose solution, respectively. The gallic acid-grafted gelatin solution, oxidized carboxymethyl cellulose solution and ZIF-8 / 67@PDA nanoparticles were subjected to a gelation reaction to obtain ZP@GGA / OCMC composite hydrogel.

[0019] In this invention, the Zn source in step 1) preferably comprises zinc nitrate and / or zinc acetate; the Co source preferably comprises cobalt nitrate hexahydrate and / or cobalt acetate; the organic ligand is preferably dimethylimidazole; the molar ratio of the Zn source to the Co source is preferably 0.5~1.5:0.5~1.5, more preferably 0.8~1.2:0.8~1.2, and more preferably 1:1; the molar ratio of the total number of Zn source and Co source to the organic ligand is preferably 1:1.5~4, more preferably 1:2~3.5, and more preferably 1:2.5~3; the reaction time is preferably 22~26 h, more preferably 23~25 h, and more preferably 24 h.

[0020] In this invention, the reaction described in step 1) is a coordination reaction. After the coordination reaction is completed, the product is centrifuged, the purple precipitate is collected, the purple precipitate is washed three times with methanol, and then dried overnight at 55~65℃ to obtain ZIF-8 / 67 nanoparticles.

[0021] In this invention, the pH value of the Tris-HCl buffer solution in step 2) is preferably 8.0~9.0, more preferably 8.2~8.8, and even more preferably 8.5; the mass ratio of dopamine hydrochloride to ZIF-8 / 67 nanoparticles is preferably 0.5~2:1, and even more preferably 1~1.5:1.

[0022] In this invention, the specific process of stirring and ultrasonication in step 2) is as follows: stirring and ultrasonication are repeated, with each ultrasonication being performed 3 times. The time for each stirring and ultrasonication is preferably 4 to 6 minutes, and more preferably 5 minutes. During the stirring and ultrasonication process, dopamine hydrochloride is uniformly adsorbed and reacted on the surface of ZIF-8 / 67 nanoparticles to form a uniform and dense polydopamine coating layer.

[0023] In this invention, the mixture after stirring and ultrasonication (coating reaction) is centrifuged to collect the solid product. The solid product is washed with water and then dried to obtain ZIF-8 / 67@PDA core-shell nanoparticles. The centrifugation speed is preferably 7000~9000 rpm, more preferably 8000 rpm. The centrifugation time is preferably 3~8 min, more preferably 4~7 min, and more preferably 5~6 min. The purpose of washing with water is to remove unreacted dopamine hydrochloride and other impurities. The drying temperature is preferably 55~65℃, more preferably 60℃.

[0024] In this invention, the solvent of the gelatin solution in step 3) is preferably water, PBS buffer, or physiological saline, and the mixed solvent is preferably a mixture of N,N-dimethylformamide (DMF) and water. The volume ratio of N,N-dimethylformamide to water is preferably 1~3:1~3, more preferably 1.5~2.5:1.5~2.5, and more preferably 2:2. The mass ratio of gelatin to gallic acid is preferably 1:0.5~0.8, more preferably 1:0.6~0.7, and more preferably 1:0.65. The mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, and gallic acid is preferably 1.0~1.2:0.8~1.0:1, more preferably 1.05~1.15:0.85~0.95:1, and more preferably 1.1:0.9:1.

[0025] In this invention, the temperature of the grafting reaction in step 3) is preferably 35~45℃, more preferably 37~42℃, and even more preferably 40℃, and the time of the grafting reaction is preferably 18~36h, more preferably 24~30h, and even more preferably 26~28h.

[0026] In this invention, the mixing time in step 3) is preferably 0.5~1.5h, more preferably 1h. During the mixing process, the activation reaction is carried out in the EDC and NHS condensing agent system. After the grafting reaction is completed, the reaction solution is sequentially dialyzed, pre-frozen, and freeze-dried to obtain gallic acid grafted gelatin (GGA). The molecular weight cutoff for dialysis is preferably 5000~10000Da, more preferably 7000~8000Da. The dialysis time is preferably 3~5 days, more preferably 4 days. The pre-freezing temperature is preferably -25~-15℃, more preferably -20℃. The pre-freezing time is preferably 4~8h, more preferably 5~7 days, more preferably 6 days. The freeze-drying temperature is preferably -85~-75℃, more preferably -80℃. The freeze-drying time is preferably 4~6 days, more preferably 5 days.

[0027] In this invention, the quenching agent in step 4) is preferably ethylene glycol, and the dispersing solvent is preferably water or physiological saline; the mass ratio of carboxymethyl cellulose to NaIO4 is preferably 1:0.4~0.8, more preferably 1:0.5~0.7, and even more preferably 1:0.6; the oxidation reaction time is preferably 18~36h, more preferably 24~30h, and even more preferably 26~28h.

[0028] In this invention, step 4) involves adding a quencher and stirring for 0.5-2 hours to terminate the reaction and quench unreacted NaIO4. Stirring is preferably performed for 1-1.5 hours. The resulting reaction solution is then sequentially dialyzed, pre-frozen, and freeze-dried to obtain oxidized carboxymethyl cellulose. The molecular weight cutoff for dialysis is preferably 5000-10000 Da, more preferably 6000-9000 Da, and even more preferably 7000-8000 Da. The dialysis time is preferably 60-84 hours, more preferably 65-80 hours, and even more preferably 70-72 hours. The pre-freezing temperature is preferably -25 to -15°C, more preferably -20°C. The pre-freezing time is preferably 4-8 hours, more preferably 5-7 days, and even more preferably 6 days. The freeze-drying temperature is preferably -85 to -75°C, more preferably -80°C. The freeze-drying time is preferably 4-6 days, and even more preferably 5 days.

[0029] In this invention, sodium periodate (NaIO4) oxidizes the hydroxyl groups on the CMC molecular chain to aldehyde groups, providing active sites for subsequent Schiff base crosslinking.

[0030] In this invention, the mixing in step 4) is preferably with carboxymethyl cellulose dissolved in a dispersing solvent, and then NaIO4 is added. The concentration of the carboxymethyl cellulose solution is preferably 2-6 wt%, more preferably 3-5 wt%, and even more preferably 4 wt%. The oxidation reaction is preferably carried out at room temperature in the dark. The volume ratio of the quencher to the dispersing solvent is preferably 0.6-1.8:100, more preferably 0.8-1.3:100, and even more preferably 1:100.

[0031] In this invention, the concentration of the gallic acid-grafted gelatin solution in step 5) is preferably 15-25 wt%, more preferably 17-23 wt%, and even more preferably 20 wt%; the concentration of the oxidized carboxymethyl cellulose solution is preferably 3-6 wt%, more preferably 4-5 wt%, and even more preferably 4.5 wt%; the volume ratio of the gallic acid-grafted gelatin solution to the oxidized carboxymethyl cellulose solution is preferably 0.5-1.5:0.5-1.5, more preferably 0.8-1.2:0.8-1.2, and even more preferably 1:1; the concentration of the ZIF-8 / 67@PDA nanoparticles in the ZP@GGA / OCMC composite hydrogel is preferably 0.3-2 mg / mL, more preferably 0.5-1.5 mg / mL, and even more preferably 1 mg / mL.

[0032] In this invention, the temperature of the gelation reaction in step 5) is preferably 35~40℃, more preferably 36~39℃, and even more preferably 37~38℃, and the gelation reaction time is preferably 0.5~2h, and even more preferably 1~1.5h; during the gelation process, GGA and OCMC, and ZP NPs and OCMC undergo Schiff base reaction to obtain ZP@GGA / OCMC composite hydrogel.

[0033] In this invention, the preferred temperature for mixing gallic acid-grafted gelatin with PBS buffer is 35-45°C, and more preferably 40°C.

[0034] The present invention also provides a method for preparing the ZP@GGA / OCMC composite hydrogel.

[0035] The present invention also provides the application of the ZP@GGA / OCMC composite hydrogel in the preparation of materials for the treatment of chronic osteomyelitis and / or materials for the repair of infected bone defects.

[0036] Unless otherwise specified, all raw materials or reagents used in this invention are commercially available products well known to those skilled in the art; and all experimental and detection methods are conventional methods.

[0037] In this invention, the three-dimensional porous network structure within the composite hydrogel provides crucial spatial channels for cell migration, proliferation, and efficient exchange of nutrients and metabolites. Macropores (pore size > 100 μm) facilitate cell growth and tissue infiltration, while micropores promote cell adhesion and protein adsorption. The synergistic effect of the hierarchical porous structure significantly enhances the osteogenic activity of the scaffold. In the treatment of osteomyelitis, zinc ions not only possess broad-spectrum antibacterial activity but also promote osteogenic function by facilitating osteogenic differentiation and matrix mineralization of bone marrow mesenchymal stem cells. Cobalt ions can effectively disrupt methicillin-resistant Staphylococcus aureus (MRSA) biofilms, reducing their adhesion rate by more than 80%, and reshape the immune microenvironment conducive to bone healing by regulating macrophage polarization towards the anti-inflammatory M2 phenotype. Therefore, constructing a hydrogel system that combines a hierarchical porous structure with the sequential release function of zinc / cobalt ions holds promise for achieving synergistic treatment of infection control, immune regulation, and bone regeneration.

[0038] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0039] In the examples and comparative examples, the pH of the PBS buffer was 7.4.

[0040] Example 1

[0041] 0.4587 g zinc acetate (2.5 mmol) and 0.7277 g cobalt nitrate hexahydrate (2.5 mmol) were dissolved in 40 mL methanol to obtain solution A; 0.8211 g dimethylimidazole (10 mmol) was dissolved in 40 mL methanol to obtain solution B; at room temperature, solution B was slowly added dropwise to solution A at a rate of 0.1 mL / s, and the mixture was stirred continuously for 5 min and then allowed to stand for 24 h to promote crystal growth; after the reaction was completed, the purple precipitate was collected by centrifugation, washed three times with methanol, and dried overnight at 60 °C to obtain ZIF-8 / 67 nanoparticles.

[0042] 100 mg of ZIF-8 / 67 nanoparticles were uniformly dispersed in 50 mL of Tris-HCl buffer (pH=8.5) and sonicated at 40 kHz to ensure thorough dispersion. Then, 100 mg of dopamine hydrochloride was added to the system, and the mixture was stirred for 5 min and sonicated for 5 min (sonication frequency 40 kHz) at room temperature. Each stirring and sonication was performed three times to induce in-situ uniform coating of ZIF-8 / 67 nanoparticles with dopamine. After the reaction was completed, the mixture was centrifuged at 8000 rpm for 5 min, the solid product was collected, and washed repeatedly with deionized water to remove unreacted dopamine and other impurities. Finally, the product was dried in a 60 ℃ oven to obtain ZIF-8 / 67@PDA nanoparticles (ZP NPs).

[0043] At 40℃, 5g of gelatin was dissolved in 150mL of deionized water to obtain a gelatin solution. 3.4g of gallic acid was dissolved in 125mL of a DMF / H2O (DMF to H2O volume ratio 2:3) mixed solvent, and 3.82g of EDC and 3.2g of NHS were added. After stirring at room temperature for 1 hour, the resulting mixture was added to the gelatin solution, and the grafting reaction was carried out at 40℃ for 24 hours. The reaction solution after the grafting reaction was completed was placed in a dialysis bag (molecular weight cutoff 7000 Da) and dialyzed for 4 days. Then, it was pre-frozen at -20℃ for 6 days and freeze-dried at -80℃ for 5 days to obtain gallic acid-grafted gelatin (GGA).

[0044] 4g of CMC was dissolved in 100mL of deionized water, and 2g of NaIO4 was added. The oxidation reaction was carried out at room temperature in the dark for 24h. Then, 1mL of ethylene glycol was added and stirred for 1h to terminate the reaction. The mixture was then transferred to a dialysis bag (molecular weight cutoff 7000Da) and dialyzed for 72h. After dialysis, the mixture was pre-frozen at -20℃ for 6 days and freeze-dried at -80℃ for 5 days to obtain oxidized carboxymethyl cellulose (OCMC).

[0045] At 40°C, 200 mg GGA was dissolved in 1 mL PBS buffer to obtain a 20 wt% GGA solution; at room temperature, 40 mg OCMC was dissolved in 1 mL PBS buffer to obtain a 4 wt% OCMC solution; 1 mg ZP NPs were dispersed in the above GGA solution and mixed with an equal volume of OCMC solution. The mixture was allowed to stand at 37°C for 1 h to carry out the gelation reaction, resulting in a ZP@GGA / OCMC composite hydrogel.

[0046] Example 2

[0047] 0.3788 g zinc nitrate (2 mmol) and 0.5311 g cobalt acetate (3 mmol) were dissolved in 40 mL methanol to obtain solution A; 1.2316 g dimethylimidazole (15 mmol) was dissolved in 40 mL methanol to obtain solution B; at room temperature, solution B was slowly added dropwise to solution A at a rate of 0.1 mL / s, and the mixture was stirred continuously for 5 min and then allowed to stand for 24 h to promote crystal growth; after the reaction was completed, the purple precipitate was collected by centrifugation, washed three times with methanol, and dried overnight at 60 °C to obtain ZIF-8 / 67 nanoparticles.

[0048] 100 mg of ZIF-8 / 67 nanoparticles were uniformly dispersed in 50 mL of Tris-HCl buffer (pH=8.7) and sonicated at 40 kHz to ensure thorough dispersion. Then, 120 mg of dopamine hydrochloride was added to the system, and the mixture was stirred for 5 min and sonicated for 5 min (sonication frequency 40 kHz) at room temperature. The stirring and sonication were performed three times each to induce in-situ uniform coating of ZIF-8 / 67 nanoparticles with dopamine. After the reaction was completed, the mixture was centrifuged at 9000 rpm for 4 min, the solid product was collected, and washed repeatedly with deionized water to remove unreacted dopamine and other impurities. Finally, the product was dried in a 60 ℃ oven to obtain ZIF-8 / 67@PDA nanoparticles (ZP NPs).

[0049] At 40℃, 5g of gelatin was dissolved in 150mL of deionized water to obtain a gelatin solution. 3g of gallic acid was dissolved in 125mL of a DMF / H2O mixed solvent (DMF and H2O volume ratio 1:2), and 3.6g of EDC and 2.7g of NHS were added. After stirring at room temperature for 1 hour, the resulting mixture was added to the gelatin solution, and the grafting reaction was carried out at 45℃ for 20 hours. The reaction solution after the grafting reaction was completed was placed in a dialysis bag (molecular weight cutoff 6000 Da) and dialyzed for 3 days. Then, it was pre-frozen at -25℃ for 5 days and freeze-dried at -80℃ for 5 days to obtain gallic acid-grafted gelatin (GGA).

[0050] 4g of CMC was dissolved in 100mL of deionized water, and 2.4g of NaIO4 was added. The oxidation reaction was carried out at room temperature in the dark for 30h. Then, 1.2mL of ethylene glycol was added and stirred for 1h to terminate the reaction. The mixture was then transferred to a dialysis bag (molecular weight cutoff 8000Da) and dialyzed for 65h. After dialysis, the mixture was pre-frozen at -25℃ for 5 days and freeze-dried at -80℃ for 5 days to obtain oxidized carboxymethyl cellulose (OCMC).

[0051] At 40°C, 180 mg GGA was dissolved in 1 mL PBS buffer to obtain an 18 wt% GGA solution; at room temperature, 50 mg OCMC was dissolved in 1 mL PBS buffer to obtain a 5 wt% OCMC solution; 1 mg ZP NPs were dispersed in the above GGA solution and mixed with an equal volume of OCMC solution. The mixture was allowed to stand at 37°C for 1 h to carry out the gelation reaction, resulting in a ZP@GGA / OCMC composite hydrogel.

[0052] Comparative Example 1

[0053] 200 mg of unmodified gelatin was dissolved in 1 mL of PBS buffer at 40 °C to obtain a 20 wt% gelatin solution.

[0054] 4g of CMC was dissolved in 100mL of deionized water, and 2g of NaIO4 was added. The oxidation reaction was carried out at room temperature in the dark for 24h. Then, 1mL of ethylene glycol was added and stirred for 1h to terminate the reaction. The mixture was then transferred to a dialysis bag (molecular weight cutoff 7000Da) and dialyzed for 72h. After dialysis, the mixture was pre-frozen at -20℃ for 6 days and freeze-dried at -80℃ for 5 days to obtain oxidized carboxymethyl cellulose (OCMC).

[0055] 40 mg of OCMC was dissolved in 1 mL of PBS buffer at room temperature to obtain a 4 wt% OCMC solution; the gelatin solution and OCMC solution were mixed evenly at a volume ratio of 1:1 and the mixture was subjected to a gelation reaction at 37 °C for 1 h to obtain a gel / OCMC hydrogel.

[0056] Comparative Example 2

[0057] At 40℃, 5g of gelatin was dissolved in 150mL of deionized water to obtain a gelatin solution. 3.4g of gallic acid was dissolved in 125mL of a DMF / H2O (DMF to H2O volume ratio 2:3) mixed solvent, and 3.82g of EDC and 3.2g of NHS were added. After stirring at room temperature for 1 hour, the resulting mixture was added to the gelatin solution, and the grafting reaction was carried out at 40℃ for 24 hours. The reaction solution after the grafting reaction was completed was placed in a dialysis bag (molecular weight cutoff 7000 Da) and dialyzed for 4 days. Then, it was pre-frozen at -20℃ for 6 days and freeze-dried at -80℃ for 5 days to obtain gallic acid-grafted gelatin (GGA).

[0058] 4g of CMC was dissolved in 100mL of deionized water, and 2g of NaIO4 was added. The oxidation reaction was carried out at room temperature in the dark for 24h. Then, 1mL of ethylene glycol was added and stirred for 1h to terminate the reaction. The mixture was then transferred to a dialysis bag (molecular weight cutoff 7000Da) and dialyzed for 72h. After dialysis, the mixture was pre-frozen at -20℃ for 6 days and freeze-dried at -80℃ for 5 days to obtain oxidized carboxymethyl cellulose (OCMC).

[0059] 200 mg GGA was dissolved in 1 mL PBS buffer at 40 °C to obtain a 20 wt% GGA solution; 40 mg OCMC was dissolved in 1 mL PBS buffer at room temperature to obtain a 4 wt% OCMC solution; the GGA solution and OCMC solution were mixed evenly at a volume ratio of 1:1 and reacted at 37 °C for 1 h to obtain a GGA / OCMC hydrogel.

[0060] Figure 1 The images show the XRD patterns of the ZP@GGA / OCMC composite hydrogel of Example 1, the Gel / OCMC hydrogel of Comparative Example 1, and the GGA / OCMC hydrogel of Comparative Example 2. Figure 1It can be seen that all three groups of materials exhibit broad, diffuse diffraction peaks with amorphous characteristics near 2θ=20°. The peak shape and position of the diffraction curve of ZP@GGA / OCMC are highly consistent with those of Gel / OCMC and GGA / OCMC. No characteristic crystalline diffraction peaks of ZPNPs were observed, indicating that the diffraction signal of ZP NPs was masked by the background of the amorphous gel matrix. This proves that the bimetallic MOF particles are uniformly coated and dispersed in the gel network.

[0061] Figure 2 The images show the FTIR spectra of the ZP@GGA / OCMC composite hydrogel of Example 1, the Gel / OCMC hydrogel of Comparative Example 1, and the GGA / OCMC hydrogel of Comparative Example 2. Figure 2 It can be seen that after loading ZP NPs, the characteristic peaks of the material change significantly: 1590~1690 cm⁻¹ -1 The emergence of new stretching vibrations and the slight decrease and redshift of the characteristic peak intensity of the material indicate that the introduction of ZPNPs enhances the interaction between components.

[0062] Figure 3 The images show the in vitro degradation curves of the ZP@GGA / OCMC composite hydrogel of Example 1, the Gel / OCMC hydrogel of Comparative Example 1, and the GGA / OCMC hydrogel of Comparative Example 2. Figure 3 It can be seen that the ZP@GGA / OCMC composite hydrogel doped with MOF has a significant advantage in structural stability.

[0063] Figure 4 This is a comparison of the in vitro antioxidant properties of the ZP@GGA / OCMC composite hydrogel of Example 1, the Gel / OCMC hydrogel of Comparative Example 1, and the GGA / OCMC hydrogel of Comparative Example 2. (a) is a bar chart showing the DDPH free radical scavenging ability, and (b) is a bar chart showing the ABTS free radical scavenging ability. Figure 4 It can be seen that the ZP@GGA / OCMC composite hydrogel loaded with MOF particles has the highest antioxidant level.

[0064] Figure 5 This is a macroscopic view of the ZP@GGA / OCMC composite hydrogel of Example 1, where (a) is a schematic diagram of injectability and (b) is a schematic diagram of adhesion. Figure 5 It can be seen that the composite hydrogel has good injectability and adhesion.

[0065] Figure 6 The metal ion release curve of the ZP@GGA / OCMC composite hydrogel in Example 1 shows that Co 2+ In the initial stage of release, it rapidly reaches an effective therapeutic concentration and enters a release plateau, while Zn 2+The continuous and stable long-term release characteristics indicate that the present invention has successfully achieved the time-controlled release of two functional metal ions.

[0066] Figure 7 The images show 250 μm SEM images (a-c) of the ZP@GGA / OCMC composite hydrogel of Example 1, the Gel / OCMC hydrogel of Comparative Example 1, and the GGA / OCMC hydrogel of Comparative Example 2, and the EDS image (d) of the composite hydrogel prepared in Example 1. Figure 7 An ordered distribution of pore sizes can be clearly observed in (a~c). Figure 7 The EDS plots of C, N, O, Zn, and Co in d show that Zn and Co elements are evenly distributed in a scattered pattern, indicating that ZP NPs have been uniformly loaded in the three-dimensional gel network and have improved the stability of the material and the controllable drug delivery capability rather than simply physical mixing.

[0067] Figure 8 The images show 100nm SEM and EDS images of the metal nanoparticles from Example 1, where (a) is the SEM image of ZIF-8 / 67, (b) is the SEM image of ZIF-8 / 67@PDA, and (c) is the EDS image of Zn, Co, C, N, and O in ZIF-8 / 67. Figure 8 It can be seen that PDA has successfully coated metal nanoparticles and changed their morphology and elemental composition.

[0068] Figure 9 This is a Zeta plot of the ZIF-8 / 67 nanoparticles and ZIF-8 / 67@PDA nanoparticles from Example 1. (Source: [Insert source here]) Figure 9 It can be seen that after coating PDA, the potential on the surface of nanoparticle materials will change from positive to negative.

[0069] Figure 10 This is a static water contact diagram of ZIF-8 / 67 nanoparticles and ZIF-8 / 67@PDA nanoparticles from Example 1. Figure 10 It can be seen that the hydrophilicity and hydrophobicity of the material surface are significantly changed after PDA is coated, and the material changes from hydrophobic to hydrophilic.

[0070] In this invention, ZP NPs are uniformly dispersed in a GGA solution and then dynamically covalently crosslinked with an OCMC solution via a Schiff base reaction, followed by gelation at room temperature to obtain a ZP@GGA / OCMC composite hydrogel. The composite hydrogel prepared by this invention not only possesses excellent mechanical properties but also exhibits advantages such as anti-inflammatory, antibacterial, osteopromoting, and biocompatibility properties. The resulting hydrogel is suitable for minimally invasive treatment of chronic osteomyelitis and infected bone defects, and has positive significance for the development of biocompatible bone repair materials.

[0071] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a ZP@GGA / OCMC composite hydrogel, characterized in that, It includes the following steps: 1) Zn source, Co source and organic ligand were reacted in methanol to obtain ZIF-8 / 67 nanoparticles; 2) Disperse ZIF-8 / 67 nanoparticles in Tris-HCl buffer, add dopamine hydrochloride, stir and sonicate to obtain ZIF-8 / 67@PDA nanoparticles; 3) Gallic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide and mixed solvent are mixed, and the resulting mixture and gelatin solution are subjected to grafting reaction to obtain gallic acid grafted gelatin. 4) Carboxymethyl cellulose, dispersing solvent and NaIO4 are mixed and subjected to oxidation reaction. A quencher is added to terminate the reaction to obtain oxidized carboxymethyl cellulose; 5) Gallic acid-grafted gelatin and oxidized carboxymethyl cellulose were mixed with PBS buffer to obtain gallic acid-grafted gelatin solution and oxidized carboxymethyl cellulose solution, respectively. The gallic acid-grafted gelatin solution, oxidized carboxymethyl cellulose solution and ZIF-8 / 67@PDA nanoparticles were subjected to a gelation reaction to obtain ZP@GGA / OCMC composite hydrogel.

2. The preparation method of the ZP@GGA / OCMC composite hydrogel according to claim 1, characterized in that, Step 1) The Zn source comprises zinc nitrate and / or zinc acetate; the Co source comprises cobalt nitrate hexahydrate and / or cobalt acetate; the organic ligand is dimethylimidazole; the molar ratio of the Zn source to the Co source is 0.5~1.5:0.5~1.5, and the molar ratio of the total number of Zn source and Co source to the organic ligand is 1:1.5~4; the reaction time is 22~26 h.

3. The method for preparing the ZP@GGA / OCMC composite hydrogel according to claim 1 or 2, characterized in that, Step 2) The pH value of the Tris-HCl buffer solution is 8.0~9.0; the mass ratio of dopamine hydrochloride to ZIF-8 / 67 nanoparticles is 0.5~2:

1.

4. The preparation method of the ZP@GGA / OCMC composite hydrogel according to claim 3, characterized in that, Step 3) The solvent for the gelatin solution is water, PBS buffer, or physiological saline. The mixed solvent is a mixture of N,N-dimethylformamide and water, with a volume ratio of N,N-dimethylformamide to water of 1~3:1~3. The mass ratio of gelatin to gallic acid is 1:0.5~0.8, and the mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, and gallic acid is 1.0~1.2:0.8~1.0:

1.

5. The preparation method of the ZP@GGA / OCMC composite hydrogel according to claim 4, characterized in that, Step 3) The grafting reaction temperature is 35~45℃, and the grafting reaction time is 18~36h.

6. The method for preparing the ZP@GGA / OCMC composite hydrogel according to claim 4 or 5, characterized in that, Step 4) The quenching agent is ethylene glycol, the dispersing solvent is water or physiological saline; the mass ratio of carboxymethyl cellulose to NaIO4 is 1:0.4~0.8, and the oxidation reaction time is 18~36h.

7. The method for preparing the ZP@GGA / OCMC composite hydrogel according to claim 6, characterized in that, Step 5) The concentration of the gallic acid grafted gelatin solution is 15~25wt%, the concentration of the oxidized carboxymethyl cellulose solution is 3~6wt%, and the volume ratio of the gallic acid grafted gelatin solution to the oxidized carboxymethyl cellulose solution is 0.5~1.5:0.5~1.5; the concentration of the ZIF-8 / 67@PDA nanoparticles in the ZP@GGA / OCMC composite hydrogel is 0.3~2mg / mL.

8. The method for preparing the ZP@GGA / OCMC composite hydrogel according to claim 7, characterized in that, Step 5) The gelation reaction temperature is 35~40℃ and the gelation reaction time is 0.5~2h.

9. The ZP@GGA / OCMC composite hydrogel prepared by the method according to any one of claims 1 to 8.

10. The use of the ZP@GGA / OCMC composite hydrogel of claim 9 in the preparation of materials for the treatment of chronic osteomyelitis and / or materials for the repair of infected bone defects.

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