Ternary synergistic nano-enzyme hydrogel as well as preparation method and application thereof

Abstract

The invention discloses ternary synergistic nano-enzyme hydrogel as well as a preparation method and application thereof, and relates to the technical field of biomedical materials. The preparation method comprises the following steps: dissolving zinc nitrate hexahydrate and ferric nitrate nonahydrate in methanol; dissolving 2-methylimidazole in methanol, adding into a metal salt solution, stirring, and sequentially centrifuging, washing and drying to obtain Fe / ZIF-8 nano-enzyme; the preparation method comprises the following steps: adding methacrylated gelatin and hyaluronic acid into deionized water, stirring, cooling to room temperature, and refrigerating to obtain a GelMA-HA precursor solution; and adding the Fe / ZIF-8 nano-enzyme into the GelMA-HA precursor solution, stirring, injecting into a mold, and carrying out ultraviolet light cross-linking curing to obtain the ternary synergistic nano-enzyme hydrogel. According to the hydrogel disclosed by the invention, active oxygen is efficiently removed and oxygen is produced through Fe / ZIF-8 nano-enzyme, and cartilage repair is promoted in combination with lubrication and slow release functions of gelatin hydrogel, so that a new strategy is provided for treatment of osteoarthritis.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a ternary synergistic nanoenzyme hydrogel, its preparation method, and its application. Background Technology

[0002] Osteoarthritis (OA) is a globally prevalent degenerative cartilage disease, characterized by excessive accumulation of reactive oxygen species (ROS) within chondrocytes (CHOs), joint microenvironment hypoxia, and loss of lubrication at the cartilage interface. While exogenous biological enzymes (such as SOD and CAT) can scavenge ROS, they suffer from poor stability and insufficient lesion retention. Artificial nanozymes, despite their catalytic potential, tend to aggregate, leading to insufficient exposure of active sites, and lack synergistic regulation of joint hypoxia and lubrication. Although metal-organic frameworks (MOFs) can support nanozymes, their application in OA treatment remains unexplored. Therefore, developing a multifunctional nanozyme hydrogel that integrates ROS scavenging, oxygen generation, and joint lubrication is crucial to overcoming the bottlenecks in OA treatment. Summary of the Invention

[0003] To address the aforementioned technical problems, the present invention aims to provide a ternary synergistic nanozyme hydrogel, its preparation method, and its application. This hydrogel utilizes Fe / ZIF-8 nanozymes to efficiently scavenge reactive oxygen species and generate oxygen, combined with the lubrication and sustained-release functions of gelatin hydrogel, to effectively alleviate oxidative stress and hypoxic damage in chondrocytes, promote cartilage repair, and provide a new strategy for the treatment of osteoarthritis.

[0004] The technical solution of this invention to solve the above-mentioned technical problems is as follows: A method for preparing a ternary synergistic nanoenzyme hydrogel is provided, comprising the following steps: (1) Dissolve zinc nitrate hexahydrate and ferric nitrate nonahydrate in methanol to obtain a metal salt solution; then dissolve 2-methylimidazole in methanol and add it to the metal salt solution. Mix and stir at room temperature, and then centrifuge, wash and dry in sequence to obtain Fe / ZIF-8 nanozyme. (2) Add methacrylamide gelatin and hyaluronic acid to deionized water, stir in a water bath until completely dissolved, cool to room temperature, and refrigerate to obtain GelMA-HA precursor solution; (3) Add Fe / ZIF-8 nanozyme to GelMA-HA precursor solution, stir until uniformly dispersed in ice bath environment, inject into mold, crosslink and solidify under ultraviolet light to obtain ternary synergistic nanozyme hydrogel.

[0005] Furthermore, in step (1), the molar volume ratio of zinc nitrate hexahydrate, ferric nitrate nonahydrate, 2-methylimidazole and methanol is 9-11 mmol: 4-6 mmol: 70-90 mmol: 100 mL.

[0006] Furthermore, the molar volume ratio of zinc nitrate hexahydrate, ferric nitrate nonahydrate, 2-methylimidazole, and methanol was 10 mmol: 5 mmol: 80 mmol: 100 mL.

[0007] Further, in step (1), the mixture is stirred at room temperature for 24 h, the precipitate is collected by centrifugation, washed three times with methanol, and dried at 60°C.

[0008] Furthermore, in step (2), the mass-to-volume ratio of methacrylamide gelatin, hyaluronic acid, and deionized water is 9-11 g: 1-2 g: 100 mL.

[0009] Furthermore, in step (2), the mass-to-volume ratio of methacrylamide gelatin, hyaluronic acid, and deionized water is 10 g: 1 g: 100 mL.

[0010] Furthermore, in step (2), the mixture is stirred in a water bath at 60-80 ℃ for 20-40 min until completely dissolved, cooled to room temperature, and then refrigerated at 4 ℃ for 24 h.

[0011] Furthermore, in step (3), the mass-to-volume ratio of Fe / ZIF-8 nanozyme and GelMA-HA precursor solution is 0.25 mg: 2 mL.

[0012] The present invention also provides a ternary synergistic nanoenzyme hydrogel, which is prepared by the above-described method for preparing ternary synergistic nanoenzyme hydrogel.

[0013] This invention also provides the application of the above-mentioned ternary synergistic nanoenzyme hydrogel in the preparation of drugs for cartilage repair and osteoarthritis treatment.

[0014] The present invention has the following beneficial effects: 1. The ternary synergistic nanoenzyme hydrogel Fe / ZIF-8 / GelMA-HA of the present invention has the following functions: high efficiency catalysis and stress resistance: the scavenging rate of H2O2 is ≥90% in the pH range of 6.5-7.4, and the oxygen production rate reaches 6.857×10 -6M / s, superior to natural CAT (activity decreased by 50% at pH 6.5), and can be repeatedly catalyzed more than 5 times without significant activity decay. Cell protection and anti-inflammation: In vitro experiments showed that this hydrogel reduced the apoptosis rate of H2O2-induced chondrocytes from 65% to 12%, reduced intracellular MDA content by 40%, increased SOD activity by 35%, and inhibited the expression of IL-6 and MMP13 inflammatory factors by ≥50%. In vivo cartilage repair: In an ACLT-induced OA rat model, after 8 weeks of injection of Fe / ZIF-8 / GelMA-HA, cartilage surface fibrosis was reduced by 70%, proteoglycan loss rate was reduced by 60%, COL2 expression recovered to 85% of normal level, and OARSI score decreased from 7.2±0.8 to 2.1±0.3, significantly better than the control group (P<0.01). Biosafety: In vivo experiments showed no inflammatory lesions or tissue necrosis in major organs such as heart, liver, spleen, lungs, and kidneys, and the iron ion accumulation was not significantly different from normal tissues. P >0.05), hemolysis rate <5%, meeting the safety standards for medical materials.

[0015] 2. The ternary synergistic nanoenzyme hydrogel Fe / ZIF-8 / GelMA-HA of the present invention has the following functions: ROS scavenging: Fe / ZIF-8 nanoenzymes catalyze the decomposition of H2O2 into H2O and O2, reducing intracellular ROS levels and inhibiting oxidative stress damage. Synergistic oxygen production: O2 is continuously generated during the decomposition of H2O2, alleviating chondrocyte hypoxia and improving mitochondrial function. Continuous lubrication: The high water content (≥80%) and three-dimensional network structure of the hydrogel provide lubrication at the joint interface, reducing frictional damage, while delaying nanoenzyme scavenging and achieving long-lasting catalysis. Attached Figure Description

[0016] Figure 1 SEM and TEM images of the Fe / ZIF-8 nanozyme obtained in Example 1; Figure 2 The image shows the XRD results of the Fe / ZIF-8 nanozyme obtained in Example 1. Figure 3 This is a graph showing the results of an in vitro cytotoxicity test. Figure 4 This is a diagram showing the results of an in vivo trial of osteoarthritis treatment. Detailed Implementation

[0017] The principles and features of this invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0018] Example 1 A ternary synergistic nanozyme hydrogel is prepared by the following steps: (1) Dissolve 10 mmol zinc nitrate hexahydrate and 5 mmol ferric nitrate nonahydrate in 18 mL methanol, stir magnetically until clear, and obtain a metal salt solution. Then dissolve 80 mmol 2-methylimidazole in 80 mL methanol and add it to the metal salt solution. Mix and stir at room temperature for 24 h, centrifuge at 10000 rpm for 10 min, collect the precipitate, wash it 3 times with methanol, and dry it at 60 °C to obtain Fe / ZIF-8 nanozyme. (2) Add 10 g of methacrylamide gelatin and 1-2 g of hyaluronic acid to 100 mL of deionized water, stir in a water bath at 70 °C for 30 min until completely dissolved, cool to room temperature, transfer to 4 °C and refrigerate for 24 h to obtain GelMA-HA precursor solution. (3) Add 0.25 mg Fe / ZIF-8 nanozyme to 2 mL GelMA-HA precursor solution, stir until uniformly dispersed in an ice bath, inject into a predetermined mold, and crosslink and solidify under ultraviolet light for 5 min to obtain ternary synergistic nanozyme hydrogel.

[0019] SEM and TEM images of the Fe / ZIF-8 nanozyme obtained in Example 1 are shown below. Figure 1 As shown; the XRD results of Fe / ZIF-8 nanozyme are as follows. Figure 2 As shown. Among them, Figure 1 In the image, a is a ZIF-8 SEM image magnified 55,000 times, b is a Fe / ZIF-8 SEM image magnified 100,000 times, c is a ZIF-8 TEM image, and d is a Fe / ZIF-8 TEM image.

[0020] Example 2 A ternary synergistic nanozyme hydrogel is prepared by the following steps: (1) Dissolve 9 mmol zinc nitrate hexahydrate and 4 mmol ferric nitrate nonahydrate in 18 mL methanol, stir magnetically until clear, and obtain a metal salt solution. Then dissolve 70 mmol 2-methylimidazole in 80 mL methanol and add it to the metal salt solution. Mix and stir at room temperature for 24 h, centrifuge at 10000 rpm for 10 min, collect the precipitate, wash it 3 times with methanol, and dry it at 60 °C to obtain Fe / ZIF-8 nanozyme. (2) Add 9 g of methacrylamide gelatin and 1 g of hyaluronic acid to 100 mL of deionized water, stir in a water bath at 70 °C for 30 min until completely dissolved, cool to room temperature, transfer to 4 °C and refrigerate for 24 h to obtain GelMA-HA precursor solution. (3) Add 0.25 mg Fe / ZIF-8 nanozyme to 2 mL GelMA-HA precursor solution, stir until uniformly dispersed in an ice bath, inject into a predetermined mold, and crosslink and solidify under ultraviolet light for 5 min to obtain ternary synergistic nanozyme hydrogel.

[0021] Example 3 A ternary synergistic nanozyme hydrogel is prepared by the following steps: (1) Dissolve 11 mmol zinc nitrate hexahydrate and 6 mmol ferric nitrate nonahydrate in 18 mL methanol, stir magnetically until clear, and obtain a metal salt solution. Then dissolve 85 mmol 2-methylimidazole in 80 mL methanol and add it to the metal salt solution. Mix and stir at room temperature for 24 h, centrifuge at 10000 rpm for 10 min, collect the precipitate, wash it 3 times with methanol, and dry it at 60 °C to obtain Fe / ZIF-8 nanozyme. (2) Add 11 g of methacrylamide gelatin and 2 g of hyaluronic acid to 100 mL of deionized water, stir in a water bath at 70 °C for 30 min until completely dissolved, cool to room temperature, transfer to 4 °C and refrigerate for 24 h to obtain GelMA-HA precursor solution. (3) Add 0.25 mg Fe / ZIF-8 nanozyme to 2 mL GelMA-HA precursor solution, stir until uniformly dispersed in an ice bath, inject into a predetermined mold, and crosslink and solidify under ultraviolet light for 5 min to obtain ternary synergistic nanozyme hydrogel.

[0022] Test case 1. In vitro cytotoxicity test Chondrocytes (CHOs) at 5 × 10 3 Cells were seeded into 96-well plates and cultured for 24 h. Different concentrations of Fe / ZIF-8 / GelMA-HA extract obtained in Example 1 (0, 50, 100, 200 μg / mL) were then added. After 24 h, CCK-8 reagent was added, and the plates were incubated for 2 h. The absorbance was measured at 450 nm using a microplate reader. The results are as follows: Figure 3 As shown.

[0023] Depend on Figure 3 It can be seen that the cell survival rate of each concentration group is ≥95%, proving that the hydrogel has no significant cytotoxicity.

[0024] 2. In vivo treatment experiment for osteoarthritis An osteoarthritis (OA) model was established in SD rats via ACLT surgery. Rats were randomly divided into four groups (n=6): sham-operated group (saline), control group (OA + saline), ZIF-8 / GelMA-HA group (OA + ZIF-8 / GelMA-HA), and Fe / ZIF-8 / GelMA-HA group (OA + Fe / ZIF-8 / GelMA-HA). 100 μL of the corresponding reagent was injected into the joint cavity weekly for 8 weeks. After sampling, H&E staining was performed. The cartilage surface of the Fe / ZIF-8 / GelMA-HA group was smooth, the cells were neatly arranged, and the OARSI score (2.1±0.3) was significantly lower than that of the control group (7.2±0.8). P <0.001), the result is as follows Figure 4 As shown.

[0025] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a ternary synergistic nanoenzyme hydrogel, characterized in that, Includes the following steps: (1) Dissolve zinc nitrate hexahydrate and ferric nitrate nonahydrate in methanol to obtain a metal salt solution; then dissolve 2-methylimidazole in methanol and add it to the metal salt solution. Mix and stir at room temperature, and then centrifuge, wash and dry in sequence to obtain Fe / ZIF-8 nanozyme. (2) Add methacrylamide gelatin and hyaluronic acid to deionized water, stir in a water bath until completely dissolved, cool to room temperature, and refrigerate to obtain GelMA-HA precursor solution; (3) Add Fe / ZIF-8 nanozyme to GelMA-HA precursor solution, stir until uniformly dispersed in ice bath environment, inject into mold, crosslink and solidify under ultraviolet light to obtain ternary synergistic nanozyme hydrogel.

2. The method for preparing the ternary synergistic nanoenzyme hydrogel as described in claim 1, characterized in that, In step (1), the molar volume ratio of zinc nitrate hexahydrate, ferric nitrate nonahydrate, 2-methylimidazole and methanol is 9-11 mmol: 4-6 mmol: 70-90 mmol: 100 mL.

3. The method for preparing the ternary synergistic nanoenzyme hydrogel as described in claim 1 or 2, characterized in that, In step (1), the molar volume ratio of zinc nitrate hexahydrate, ferric nitrate nonahydrate, 2-methylimidazole and methanol is 10 mmol: 5 mmol: 80 mmol: 100 mL.

4. The method for preparing the ternary synergistic nanoenzyme hydrogel as described in claim 1, characterized in that, In step (1), the mixture was stirred at room temperature for 24 h, the precipitate was collected by centrifugation, washed three times with methanol, and dried at 60 °C.

5. The method for preparing the ternary synergistic nanoenzyme hydrogel as described in claim 1, characterized in that, In step (2), the mass-volume ratio of the methacrylamide gelatin, hyaluronic acid and deionized water is 9-11 g: 1-2 g: 100 mL.

6. The method for preparing the ternary synergistic nanoenzyme hydrogel as described in claim 1 or 5, characterized in that, In step (2), the mass-volume ratio of the methacrylamide gelatin, hyaluronic acid and deionized water is 10 g: 1 g: 100 mL.

7. The method for preparing the ternary synergistic nanoenzyme hydrogel as described in claim 1, characterized in that, In step (2), stir in a water bath at 60-80 ℃ for 20-40 min until completely dissolved, cool to room temperature, and then refrigerate at 4 ℃ for 24 h.

8. The method for preparing the ternary synergistic nanoenzyme hydrogel as described in claim 1, characterized in that, In step (3), the mass-to-volume ratio of the Fe / ZIF-8 nanozyme and the GelMA-HA precursor solution is 0.25 mg: 2 mL.

9. A ternary synergistic nanoenzyme hydrogel, characterized in that, The ternary synergistic nanoenzyme hydrogel was prepared using the preparation method described in any one of claims 1-8.

10. Application of ternary synergistic nanozyme hydrogels in the preparation of drugs for cartilage repair and osteoarthritis treatment.

Images