A nano-enzyme-supported biomimetic hydrogel, its preparation method and application

Abstract

This invention discloses a nanozyme-loaded biomimetic hydrogel, its preparation method, and its applications, belonging to the field of biocomposite materials technology. The preparation method includes the following steps: preparing Mn@Co@MOF powder, Mn@Co3O4 powder, and Pt nanoparticles; preparing Mn@Co3O4@Pt nanozymes; preparing a Mn@Co3O4@Pt nanozyme-PVA mixed solution; preparing an Alg-PBA hydrogel precursor; and obtaining the nanozyme-loaded biomimetic hydrogel. The nanozyme-loaded biomimetic hydrogel disclosed in this invention effectively regulates the bone inflammation microenvironment caused by diabetes, significantly reducing inflammatory responses and promoting bone tissue repair and regeneration. The nanozymes loaded in the hydrogel possess highly efficient catalytic performance, mimicking the catalytic function of enzymes and improving treatment efficiency. Simultaneously, the good biocompatibility of the nanozymes ensures the safety and effectiveness of the hydrogel in biomedical applications.

Description

Technical Field

[0001] This invention belongs to the field of biocomposite materials technology, specifically relating to a nanoenzyme-loaded biomimetic hydrogel, its preparation method, and its application. Background Technology

[0002] With the development of biomaterials science and nanotechnology, functionalized porous materials have shown great application potential in the biomedical field. In particular, metal-organic frameworks (MOFs) and their derivatives have become a research hotspot due to their unique porous structure, high specific surface area, and tunable chemical composition. MOFs not only have wide applications in catalysis, gas storage, and separation, but their derived nanozymes have also attracted much attention due to their enzyme-mimicking catalytic activity.

[0003] In the biomedical field, nanozymes are widely studied for their high catalytic efficiency and good biocompatibility, and are used in disease treatment and biosensing. Especially for diabetic bone defects, traditional treatments face many challenges due to the inflammatory response and decreased bone tissue repair capacity caused by the hyperglycemic environment. Therefore, developing novel materials that can simultaneously regulate the inflammatory microenvironment and promote bone tissue repair is particularly important. Summary of the Invention

[0004] The purpose of this invention is to provide a nanozyme-loaded biomimetic hydrogel, its preparation method and application, which has a regulatory effect on the bone inflammation microenvironment caused by diabetes and promotes osteogenic formation.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0006] A method for preparing a nanozyme-supported biomimetic hydrogel includes the following steps:

[0007] S1. Disperse MnCl2·4H2O, Co(NO3)2·6H2O and 2-methylimidazole in anhydrous methanol, mix the solutions quickly, transfer the mixed solutions to a reaction vessel, heat, centrifuge, collect the precipitate, wash repeatedly with anhydrous methanol, and dry to obtain Mn@Co@MOF powder.

[0008] S2. Place the Mn@Co@MOF nanocomposite material obtained in step S1 in a tube furnace, heat it, calcine it, and pyrolyze it to obtain Mn@Co3O4 powder.

[0009] S3. Dissolve chloroplatinic acid in deionized water, add polyvinylpyrrolidone, and stir thoroughly to obtain Pt nanoparticles;

[0010] S4. Disperse the Mn@Co3O4 powder obtained in step S2 in RO water, add Pt nanoparticles, stir at 1500-2000 rpm for 18 h, centrifuge to collect the precipitate, and obtain Mn@Co3O4@Pt nanozyme.

[0011] S5. Disperse the Mn@Co3O4@Pt nanozyme obtained in step S4 in RO water to obtain a nanozyme suspension, add PVA, stir evenly to obtain a mixed solution;

[0012] S6. Dissolve sodium alginate, EDC·HCl and 3-aminophenylboronic acid in deionized water, stir, place in a 3500kd dialysis bag and dialyze with RO water for 7 days, freeze dry to obtain Alg-PBA hydrogel precursor.

[0013] S7. Dissolve the Alg-PBA hydrogel precursor obtained in step S6 in RO water, mix it with the mixed solution obtained in step S5 in equal volume, stir evenly, add 0.1M NaOH aqueous solution, stir, soak in 1% CaCl2 aqueous solution, and load nanozyme biomimetic hydrogel.

[0014] Preferably, in step S1, the mixed solution is transferred to a reaction vessel and heated at 25°C for 12 hours.

[0015] Preferably, in step S1, the centrifugation parameters are 5000-7000 rpm and the centrifugation time is 15-20 min.

[0016] Preferably, in step S1, after repeated washing with anhydrous methanol, the solid product is dried at 20-25°C for 24 hours.

[0017] Preferably, in step S2, Mn@Co@MOF powder is placed in a tube furnace and heated to 500°C at a rate of 40°C / min in a nitrogen atmosphere, and calcined for 3 hours to obtain Mn@Co3O4 powder through pyrolysis.

[0018] Preferably, in step S6, the stirring process parameters are: stirring rate of 800-1000 rpm / min and stirring time of 18-24 h.

[0019] Preferably, in step S6, the process parameters of the freeze-drying process are: pre-freezing at -40℃ for 180 min, vacuum freeze-drying at -80℃ for 48 h, and vacuum degree of 9 Pa.

[0020] Preferably, in step S7, the soaking time in the 1% CaCl2 aqueous solution is 30 seconds.

[0021] The present invention also provides a method for preparing a nanozyme-loaded biomimetic hydrogel using the method described above.

[0022] The present invention also provides an application of the nanozyme-loaded biomimetic hydrogel prepared by the above preparation method, or the nanozyme-loaded biomimetic hydrogel, in the preparation of products for relieving and / or treating diabetic bone defects.

[0023] Compared with the prior art, the present invention has the following advantages and technical effects:

[0024] This invention discloses a nanozyme-loaded biomimetic hydrogel and its preparation method. This nanozyme-loaded biomimetic hydrogel effectively regulates the inflammatory microenvironment of bone inflammation caused by diabetes, significantly reducing inflammatory responses and promoting bone tissue repair and regeneration. The nanozymes loaded in the hydrogel possess highly efficient catalytic properties, mimicking the catalytic function of enzymes and improving treatment efficiency. Simultaneously, the excellent biocompatibility of the nanozymes ensures the safety and effectiveness of the hydrogel in biomedical applications.

[0025] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0026] Figure 1 Statistical graphs showing the effect of Mn@Co@MOF provided in Examples 1-4 on the activity of nanozymes superoxide dismutase (SOD);

[0027] Figure 2 A statistical graph showing the GOx-like catalytic activity of the Mn@Co3O4 nanozyme provided in Example 5 and the Mn@Co3O4@Pt nanozyme provided in Example 6;

[0028] Figure 3 A statistical chart of the SOD-like catalytic activity of the Mn@Co3O4 nanozyme provided in Example 5 and the Co3O4 nanozyme provided in Example 7;

[0029] Figure 4 The CAT-like catalytic activity of the Mn@Co3O4 nanozyme provided in Example 5 and the Co3O4@C nanozyme provided in Example 8;

[0030] Figure 5 The release curve of Aln functional molecules of the Mn@Co3O4@Pt nanozyme provided in Example 6;

[0031] Figure 6 Mg for the Mn@Co3O4@Pt nanozyme provided in Example 6 2+ Functional molecule release curves;

[0032] Figure 7 Rheological-frequency scans of the composite hydrogels provided in Examples 9 and 10;

[0033] Figure 8Rheological-time scans of the composite hydrogels provided in Examples 9 and 10;

[0034] Figure 9 A comparison chart of the compressive modulus of the composite hydrogel PAA provided in Examples 9 and 10;

[0035] Figure 10 A statistical chart illustrating the characterization of M1 macrophage iL-1 cells by the PAM composite hydrogel provided in Example 9;

[0036] Figure 11 Statistical graph of the characterization of the PAM composite hydrogel provided in Example 9 on the pro-inflammatory marker iNOS of M1 macrophages.

[0037] Figure 12 A statistical chart illustrating the characterization of CD206 M2 macrophages by the PAM composite hydrogel provided in Example 9;

[0038] Figure 13 The statistical graph shows the characterization of the PAM composite hydrogel provided in Example 9 on the pro-inflammatory marker iL-10 of M2 macrophages;

[0039] Figure 14 The immunofluorescence results of the PAM composite hydrogel provided in Example 9 against the pro-inflammatory marker iNOS of M1 macrophages are shown below. Figure 14 In the figure, 'a' represents the immunofluorescence result on day 3. Figure 14 b in the figure represents the immunofluorescence result on day 5;

[0040] Figure 15 The immunofluorescence results of the PAM composite hydrogel provided in Example 9 on CD206 M2 macrophages are shown below. Figure 15 In the figure, 'a' represents the immunofluorescence result on day 3. Figure 15 b in the figure represents the immunofluorescence result on day 5;

[0041] Figure 16 A statistical chart showing the expression of osteogenic differentiation-related marker ALP by the PAM composite hydrogel provided in Example 9;

[0042] Figure 17 A statistical chart showing the expression of OPN, a marker of osteogenic differentiation, by the PAM composite hydrogel provided in Example 9;

[0043] Figure 18 A statistical chart showing the expression of Runx-2, a biomarker related to osteogenic differentiation, by the PAM composite hydrogel provided in Example 9;

[0044] Figure 19 A statistical chart showing the expression of osteogenic differentiation-related marker OCN by the PAM composite hydrogel provided in Example 9;

[0045] Figure 20 The immunofluorescence results of the PAM composite hydrogel provided in Example 9 for osteogenic differentiation-related markers are shown below. Figure 20 In the figure, 'a' represents the immunofluorescence result on day 7. Figure 20 b in the figure represents the immunofluorescence result on day 14;

[0046] Figure 21 The results of the PAM composite hydrogel provided in Example 9 on the osteogenic differentiation-related markers ALP / ARS, wherein, Figure 21 In this context, 'a' represents the ALP result. Figure 21 In this context, 'b' represents the ARS result.

[0047] Figure 22 Micro-CT image of PAM composite hydrogel provided in Example 9;

[0048] Figure 23 A statistical chart of bone volume fraction of PAM composite hydrogel provided in Example 9;

[0049] Figure 24 Statistical chart of trabecular separation of PAM composite hydrogel provided in Example 9;

[0050] Figure 25 The statistical chart of the number of trabeculae in PAM composite hydrogel provided in Example 9. Detailed Implementation

[0051] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0052] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0053] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels.

[0054] Example 1 This example provides a Mn@Co@MOF composite material

[0055] 1 mmol MnCl2·4H2O and 1 mmol Co(NO3)2·6H2O were dispersed in 4 mL of anhydrous methanol, and 49.9 mmol 2-methylimidazole was dispersed in 26 mL of anhydrous methanol. The solutions were quickly mixed and transferred to a reaction vessel. The mixture was heated at 25 °C for 12 h, centrifuged at 5000 rpm for 20 min, and the precipitate was collected. The precipitate was washed repeatedly with anhydrous methanol and dried at 25 °C for 24 h to obtain Mn@Co@MOF, i.e., M 10 C.

[0056] The preparation method of Example 2 is the same as that of Example 1, except that in step S1, the amount of MnCl2·4H2O added is 0 mmol, resulting in Mn@Co@MOF, i.e., MOC.

[0057] Example 3 was prepared using the same method as Example 1, except that in step S1, the amount of MnCl2·4H2O added was 0.2 mmol, resulting in Mn@Co@MOF, i.e., M2C.

[0058] The preparation method of Example 4 is the same as that of Example 1, except that in step S1, the amount of MnCl2·4H2O added is 0.5 mmol, and Mn@Co@MOF, i.e., M5C, is obtained.

[0059] Example 5 This example provides a Mn@Co3O4 nanozyme

[0060] S1. Disperse 0.5 mmol MnCl2·4H2O and 1 mmol Co(NO3)2·6H2O in 4 mL of anhydrous methanol, and disperse 49.9 mmol 2-methylimidazole in 26 mL of anhydrous methanol. Quickly mix the solutions, transfer the mixture to a reaction vessel, heat at 25 °C, react for 12 h, centrifuge at 5000 rpm for 20 min, collect the precipitate, wash repeatedly with anhydrous methanol, and dry at 25 °C for 24 h to obtain Mn@Co@MOF, i.e., M5C.

[0061] S2. Place the M5C obtained in step S1 in a tube furnace, heat it to 500℃ at a rate of 40℃ / min in a nitrogen atmosphere, calcine for 3h, and obtain Mn@Co3O4 nanozyme through pyrolysis.

[0062] Example 6 This example provides a Mn@Co3O4@Pt nanozyme

[0063] S1. Disperse 0.5 mmol MnCl2·4H2O and 1 mmol Co(NO3)2·6H2O in 4 mL of anhydrous methanol, and disperse 49.9 mmol 2-methylimidazole in 26 mL of anhydrous methanol. Quickly mix the solutions, transfer the mixture to a reaction vessel, heat at 25 °C, react for 12 h, centrifuge at 5000 rpm for 20 min, collect the precipitate, wash repeatedly with anhydrous methanol, and dry at 25 °C for 24 h to obtain Mn@Co@MOF, i.e., M5C.

[0064] S2. Place the M5C obtained in step S1 in a tube furnace, heat it to 500℃ at a rate of 40℃ / min in a nitrogen atmosphere, calcine for 3h, and pyrolyze to obtain Mn@Co3O4 powder.

[0065] S3. Dissolve chloroplatinic acid in deionized water, add polyvinylpyrrolidone, and stir thoroughly to obtain Pt nanoparticles;

[0066] S4. Disperse the Mn@Co3O4 powder obtained in step S2 in RO water, add Pt nanoparticles, stir at 2000 rpm for 18 h, centrifuge to collect the precipitate, and obtain Mn@Co3O4@Pt nanozyme, i.e. MCPt A.

[0067] Example 7 This example provides a Co3O4@C nanozyme

[0068] 1 mmol Co(NO3)2·6H2O was dispersed in 4 mL of anhydrous methanol, and 49.9 mmol 2-methylimidazole was dispersed in 26 mL of anhydrous methanol. The solutions were quickly mixed and transferred to a reaction vessel. The mixture was heated at 25 °C for 12 h, centrifuged at 5000 rpm for 20 min, and the precipitate was collected. The precipitate was washed repeatedly with anhydrous methanol and dried at 25 °C for 24 h to obtain Co3O4. The product was then placed in a tube furnace and heated to 500 °C at a rate of 40 °C / min under nitrogen atmosphere. The mixture was calcined for 3 h to obtain Co3O4@C powder.

[0069] Example 8 This example provides a Co3O4

[0070] 1 mmol Co(NO3)2·6H2O was dispersed in 4 mL of anhydrous methanol, and 49.9 mmol 2-methylimidazole was dispersed in 26 mL of anhydrous methanol. The solutions were quickly mixed and transferred to a reaction vessel. The mixture was heated at 25 °C and reacted for 12 h. After centrifugation at 5000 rpm for 20 min, the precipitate was collected, washed repeatedly with anhydrous methanol, and dried at 25 °C for 24 h to obtain Co3O4.

[0071] Example 9 This example provides a biomimetic hydrogel loaded with nanozymes.

[0072] S1. Disperse 0.5 mmol MnCl2·4H2O and 1 mmol Co(NO3)2·6H2O in 4 mL of anhydrous methanol, and disperse 49.9 mmol 2-methylimidazole in 26 mL of anhydrous methanol. Quickly mix the solutions, transfer the mixture to a reaction vessel, heat at 25 °C, react for 12 h, centrifuge at 5000 rpm for 20 min, collect the precipitate, wash repeatedly with anhydrous methanol, and dry at 25 °C for 24 h to obtain Mn@Co@MOF, i.e., M5C.

[0073] S2. Place the M5C obtained in step S1 in a tube furnace, heat it to 500℃ at a rate of 40℃ / min in a nitrogen atmosphere, calcine for 3h, and pyrolyze to obtain Mn@Co3O4 powder.

[0074] S3. Dissolve chloroplatinic acid in deionized water, add polyvinylpyrrolidone, and stir thoroughly to obtain Pt nanoparticles;

[0075] S4. Disperse the Mn@Co3O4 powder obtained in step S2 in RO water, add Pt nanoparticles, stir at 2000 rpm for 18 h, centrifuge to collect the precipitate, and obtain Mn@Co3O4@Pt nanozyme.

[0076] S5. Disperse 100 mg of Mn@Co3O4@Pt nanozyme obtained in step S4 in 10 mL of RO water to obtain nanozyme suspension, add 1 g of PVA, stir evenly to obtain mixed solution;

[0077] S6. Dissolve 2.0 g sodium alginate, 1.92 g EDC·HCl, and 0.78 g 3-aminophenylboronic acid in 200 mL of deionized water, stir at 1000 rpm / min for 18 h, place in a 3500 kDa dialysis bag and dialyze with RO water for 7 days, pre-freeze at -40℃ for 180 min, freeze-dry under vacuum at -80℃ for 48 h, wherein the vacuum degree is 9 Pa, to obtain Alg-PBA hydrogel precursor;

[0078] S7. Dissolve the Alg-PBA hydrogel precursor obtained in step S6 in RO water to prepare an Alg-PBA aqueous solution with a mass fraction of 10%. Mix it with the mixed solution obtained in step S5 in equal volume, stir evenly, add 0.1M NaOH aqueous solution, stir, and soak in 1% CaCl2 aqueous solution for 30s to obtain PAM composite hydrogel.

[0079] The preparation method of Example 10 is the same as that of Example 9, except that Mn@Co3O4@Pt nanozyme is not added when preparing the mixed solution in step S5, and PA composite hydrogel is obtained.

[0080] The effectiveness of the above embodiments was verified through the following experiments:

[0081] 1. The effect of Mn@Co@MOF provided in Examples 1-4 on the activity of nanozyme superoxide dismutase (SOD) was determined. The activity was measured using a Shanghai enzyme-linked SOD activity assay kit. The results are as follows: Figure 1 .

[0082] Depend on Figure 1 It can be seen that the nanozyme synthesized with an M5C, i.e., MnCl2·4H2O addition ratio of 0.5 mmol has the best SOD activity, and this ratio of nanozyme was selected for subsequent experiments.

[0083] 2. The catalytic activities of the nanozymes provided in Examples 5-7, including glucose oxidase-like (GOx) catalytic activity, SOD-like catalytic activity, and catalase-like (CAT) catalytic activity, were determined using the following experimental protocol:

[0084] The GOx-like catalytic activity of the Mn@Co3O4 nanozymes of Example 5 and the Mn@Co3O4@Pt nanozymes of Example 6 was tested by catalytic oxidation of TMB. A control group was prepared by adding the same buffer solution and glucose, and an equal volume of RO water as the sample. 3 mL of 3 mL HAC-NaAc buffer (0.1 M, pH 4.0), 20 μL of a sample at a certain concentration, and 80 μL of glucose (12.0 mM) were added sequentially. The mixture was incubated at 37 °C for 30 min, followed by the addition of 200 μL of 2 mM TMB. The absorbance was measured at 652 nm, and the UV spectra were scanned using a UV spectrometer. The results are as follows: Figure 2 .

[0085] Depend on Figure 2 It can be seen that the Mn@Co3O4@Pt provided in Example 6 can catalyze the decomposition of glucose into hydrogen peroxide, and then catalyze the oxidation of TMB, exhibiting a characteristic ox-TMB UV absorption peak at 652 nm. No absorption peak was observed in the control group and the Mn@Co3O4 provided in Example 5, verifying the GOX activity of Mn@Co3O4@Pt.

[0086] The SOD-like catalytic activity of Mn@Co3O4 in Example 5 and Co3O4 in Example 7 was tested using an SOD assay kit (MLbio) according to the manufacturer's instructions, with an equal volume of RO water as a control group. The results are as follows: Figure 3 .

[0087] Depend on Figure 3 It can be seen that Mn@Co3O4 can remove superxanthine and the superoxide anion (O3O4) produced by the xanthine oxidase reaction system. 2- ), (O 2- SOD can reduce nitroblue tetrazolium to form blue formazan, which has absorption at 560 nm. 2- This inhibits the formation of formazan; Mn doping enhances the SOD activity of the material, resulting in a decrease in ultraviolet absorption at 560 nm.

[0088] The CAT-like catalytic activity of Mn@Co3O4 in Example 5 and Co3O4@C in Example 8 was tested by measuring O2 concentration hourly using a dissolved oxygen meter for up to 6 hours, with an equal volume of RO water as a control group. Results are as follows: Figure 4 .

[0089] Depend on Figure 4 It can be seen that the amount and rate of oxygen released in Mn@Co3O4 within 6h are the highest. Mn doping improves the CAT activity of the material and can catalyze the decomposition of hydrogen peroxide.

[0090] 3. Testing the effect of the Mn@Co3O4@Pt nanozyme provided in Example 6 on Aln and Mg 2+ Release curves of functional molecules. The experimental protocol is as follows:

[0091] Add 15 mg Aln, 50 mg MgCl2, and 100 mg Mn@Co3O4@Pt nanozyme to 10 mL of pH 5.0 phosphate buffer. Stir overnight at room temperature, centrifuge at 13000 rpm, wash three times with RO water, centrifuge to collect the precipitate, dry to obtain product powder, thus realizing the reaction of Aln and Mg. 2+ Controllable loading of functional molecules was demonstrated by ICP-MS (inductively coupled plasma mass spectrometry) in a weakly alkaline buffer (PBS, pH 7.4) to detect Aln and Mg. 2+ Release curves of functional molecules. Results are as follows. Figure 5 and Figure 6 .

[0092] Depend on Figure 5 It can be seen that Aln is released rapidly in the first 15 days and then slowly until it is basically completely released by day 24, which verifies the loading of Aln in Mn@Co3O4@Pt and its effective release under normal physiological conditions.

[0093] Depend on Figure 6 It can be seen that Mg 2+ The rapid release followed by a slow release over the first 13 days, until near-complete release by day 24, validated the presence of Mg. 2+ Loading in Mn@Co3O4@Pt and effective release under normal physiological conditions.

[0094] 4. The rheological and mechanical properties of the composite hydrogels provided in Examples 9 and 10 were characterized using the following experimental procedure: a rheometer and a DMA dynamic mechanical analyzer were used to determine the rheological and mechanical properties of the hydrogels, respectively. The results are as follows: Figure 7-9 .

[0095] Depend on Figure 7 It can be seen that the storage modulus of the hydrogel is greater than the loss modulus at different scanning frequencies, and it can be stably gelled.

[0096] Depend on Figure 8 It can be seen that under continuous shear force changes, the storage modulus of the hydrogel is always greater than the loss modulus, and it can be stably gelled.

[0097] Depend on Figure 9 It can be seen that the PAM provided in Example 9 and the PA provided in Example 10 have similar compressive modulus and similar mechanical strength. The PAM is slightly higher than the PA, which may be because the doping of nanomaterials improves the mechanical properties of the composite hydrogel to a certain extent.

[0098] 5. The experimental protocol for regulating the inflammatory microenvironment is as follows:

[0099] A simulated inflammatory environment was created by adding 400 μM H2O2 to the macrophage (RAW264.7) culture environment (DMEM, 10% of FBS, 100 U / mL penicillin, 100 μg / mL streptomycin). Nanozymes were co-incubated with RAW264.7 cells in the H2O2 environment. Cellular and molecular analyses, including qRT-PCR and immunofluorescence (scale bar = 20 μm), were used to characterize M1 macrophages and their pro-inflammatory markers (iL-1, iNOS), and M2 macrophages and their anti-inflammatory markers (CD206, iL-10). The effects of nanozyme-loaded hydrogels on macrophage polarization and their regulatory performance on the inflammatory microenvironment were explored. qRT-PCR results are shown below. Figure 10-13 Immunofluorescence, such as Figure 14-15 .

[0100] Depend on Figure 10-13 It was found that the H2O2 environment can induce the expression of pro-inflammatory factors (IL-1, iNOS) in RAW264.7 and inhibit the expression of anti-inflammatory factors (IL-10, CD206), while the hydrogel loaded with Mn@Co3O4@Pt nanozymes can alleviate this induction and restore it to normal levels. Mn@Co3O4@Pt can promote the expression of anti-inflammatory factors (IL-10, CD206) and inhibit the expression of pro-inflammatory factors (IL-1, iNOS) under inflammatory conditions.

[0101] Depend on Figure 14-15 It was found that the H2O2 environment can induce the expression of pro-inflammatory cytokines (iNOS) and inhibit the expression of anti-inflammatory cytokines (CD206) in RAW264.7, while the hydrogel loaded with Mn@Co3O4@Pt nanozymes can alleviate this induction and restore it to normal levels. MCPtA can promote the expression of anti-inflammatory cytokines (CD206) and inhibit the expression of pro-inflammatory cytokines (iNOS) under inflammatory conditions.

[0102] 6. The experimental protocol for studying the regulatory properties of the bone immune microenvironment is as follows:

[0103] Bone marrow mesenchymal stem cells (rBMSCs) were cultured in osteogenic medium (α-MEM, 10 mM β-glycerophosphate, 50 μg / mL ascorbic acid, and 10 nM dexamethasone), with 50 mM glucose added to simulate a high-glucose environment. Nanozymes were added and co-cultured with rBMSCs. The expression of osteogenic differentiation-related markers (Runx-2, OCN, OPN, ALP) was analyzed using qRT-PCR, immunofluorescence (scale bar = 20 μm), and ALP / ARS (scale bar = 500 μm) methods. This in vitro study explored the mechanism by which nanozymes promote osteoogenesis through a synergistic bone immune microenvironment regulation strategy. qRT-PCR results are shown below. Figure 16-19 Immunofluorescence results are as follows Figure 20ALP / ARS results are as follows Figure 21 .

[0104] Depend on Figure 16-19 It is known that the H2O2 environment inhibits osteogenic differentiation of rBMSCs and downregulates the gene expression of osteogenic-related factors OPN, OCN, RUNX-2, and ALP. However, the hydrogel loaded with Mn@Co3O4@Pt nanozymes can alleviate this inhibition and restore the expression to normal levels. Furthermore, due to the osteogenic components doped in the material, the expression of related factors can be promoted.

[0105] Depend on Figure 20 It is known that the H2O2 environment inhibits osteogenic differentiation of rBMSCs. Immunofluorescence shows that the H2O2 environment inhibits the protein expression of osteogenic-related factor OPN. However, the hydrogel loaded with Mn@Co3O4@Pt nanozymes can alleviate this inhibition and restore it to normal levels. Furthermore, due to the osteogenic components doped in the material, the expression of OPN is promoted.

[0106] Depend on Figure 21 It is known that the H2O2 environment inhibits osteogenic differentiation of rBMSCs. No obvious alkaline phosphatase and calcium nodule formation were observed in ALP and ARS staining at 7 and 14 days. The H2O2 environment inhibits the maturation and formation of osteoblasts. However, the hydrogel loaded with Mn@Co3O4@Pt nanozymes can alleviate this inhibition and restore the cells to normal levels. Due to the osteogenic components doped in the material, osteoblast maturation is promoted. In ALP and ARS staining, obvious alkaline phosphatase and calcium nodule formation are observed.

[0107] 7. The experimental protocol for evaluating the repair effect of diabetic bone defects is as follows:

[0108] A diabetic SD rat model was established using streptozotocin injection. After anesthesia with sodium pentobarbital, a 5mm diameter circular incision was made anterior-posteriorly in the midline of the skull. Postoperatively, the rats were injected with antibiotics to prevent wound infection. Nanozyme-loaded biomimetic hydrogel was implanted into the skull defect site. Rats were sacrificed at 4, 8, and 12 weeks, and skull tissue was harvested and fixed in 4% paraformaldehyde. Three-dimensional reconstruction was performed using micro-CT (scale bar = 1mm) and accompanying software to measure parameters such as bone volume fraction, trabecular separation, and trabecular number. At 12 weeks post-implantation, the bone volume fraction in the material-filled group increased by 85.3% compared to the non-implanted group and by 35.3% compared to the non-diabetic material-implanted group, further validating the material's regulatory effect on the bone inflammatory microenvironment and its osteogenic promoting effect in vivo. Micro-CT results are shown below. Figure 22 The bone volume fraction results are as follows: Figure 23 The results of trabecular separation are as follows: Figure 24 The results of the trabecular bone count are as follows: Figure 25 .

[0109] Depend on Figure 22 It was found that from 4 to 12 weeks, the healing rate of skull defects in diabetic rats was slow, and the same was true for the PA hydrogel doping group. However, the healing effect of skull defects in rats in the PAM composite hydrogel group doped with MCPtA was ideal. At 8 weeks, the healing status of skull defects was close to that of the control group of non-diabetic rats, and at 12 weeks, it was higher than that of the normal group, with almost complete healing. This is related to the fact that MCPtA improves the diabetic microenvironment and has an osteogenic promoting effect.

[0110] Depend on Figure 23 It was found that at 12 weeks, the bone volume / total volume (BV / TV) (4.98% ± 1.78%) was 1.32 times higher than at 4 weeks, while in the PA group (11.4% ± 0.93%) it was 1.43 times higher than at 4 weeks. The control group showed a normal bone regeneration process, with BV / TV at 12 weeks (17.2% ± 4.05%) being 1.99 times higher than at 4 weeks. However, composite hydrogel implantation significantly weakened the inhibition and promoted bone regeneration, reaching 32.5% ± 4.01%, which was 3.26 times that at 4 weeks, 2.47 times that in the diabetic group, and 1.64 times that in the control group at 12 weeks.

[0111] Depend on Figure 24 It can be seen that at 12 weeks, the trabecular separation (Tb.SP) of the composite hydrogel-doped group was significantly lower than that of the unfilled diabetic group, which verifies the in vivo cranial bone repair effect of composite hydrogel in diabetic rats.

[0112] Depend on Figure 25 It was found that at 12 weeks, the number of trabecular bone (Tb.N) in the composite hydrogel-doped group was significantly higher than that in the unfilled diabetic group, which verified the in vivo cranial bone repair effect of composite hydrogel in diabetic rats.

[0113] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing a nanozyme-supported biomimetic hydrogel, characterized in that, Includes the following steps: S1. Disperse MnCl2·4H2O, Co(NO3)2·6H2O and 2-methylimidazole in anhydrous methanol, mix the solutions quickly, transfer the mixed solutions to a reaction vessel, heat, centrifuge, collect the precipitate, wash repeatedly with anhydrous methanol, and dry to obtain Mn@Co@MOF powder. S2. Place the Mn@Co@MOF nanocomposite material obtained in step S1 in a tube furnace, heat it, calcine it, and pyrolyze it to obtain Mn@Co3O4 powder. S3. Dissolve chloroplatinic acid in deionized water, add polyvinylpyrrolidone, and stir thoroughly to obtain Pt nanoparticles; S4. Disperse the Mn@Co3O4 powder obtained in step S2 in RO water, add the Pt nanoparticles obtained in step S3, stir at 1500~2000 rpm for 18 h, centrifuge to collect the precipitate, and obtain Mn@Co3O4@Pt nanozyme. S5. Disperse the Mn@Co3O4@Pt nanozyme obtained in step S4 in RO water to obtain a nanozyme suspension, add PVA, stir evenly to obtain a mixed solution; S6. Dissolve sodium alginate, EDC·HCl, and 3-aminophenylboronic acid in deionized water, stir, place in a 3500kd dialysis bag, dialyze with RO water for 7 days, freeze dry, and obtain Alg-PBA hydrogel precursor. S7. Dissolve the Alg-PBA hydrogel precursor obtained in step S6 in RO water, mix it with the mixed solution obtained in step S5 in equal volume, stir evenly, add 0.1 M NaOH aqueous solution, stir, soak in 1% CaCl2 aqueous solution, and load nanozyme biomimetic hydrogel.

2. The preparation method according to claim 1, characterized in that, In step S1, the mixed solution is transferred to a reaction vessel and heated at 25°C for 12 h.

3. The preparation method according to claim 1, characterized in that, In step S1, the centrifugation parameters are 5000~7000 rpm and the centrifugation time is 15~20 min.

4. The preparation method according to claim 1, characterized in that, In step S1, after repeated washing with anhydrous methanol, the solid product is dried at 20~25℃ for 24 h.

5. The preparation method according to claim 1, characterized in that, In step S2, Mn@Co@MOF powder is placed in a tube furnace and heated to 500°C at a rate of 40°C / min in a nitrogen atmosphere, and calcined for 3 hours to obtain Mn@Co3O4 powder through pyrolysis.

6. The preparation method according to claim 1, characterized in that, In step S6, the stirring process parameters are: stirring rate of 800~1000 rpm / min and stirring time of 18~24 h.

7. The preparation method according to claim 1, characterized in that, In step S6, the process parameters for the freeze-drying process are: pre-freezing at -40℃ for 180 min, vacuum freeze-drying at -80℃ for 48 h, and vacuum degree of 9 Pa.

8. The preparation method according to claim 1, characterized in that, In step S7, the soaking time in the 1% CaCl2 aqueous solution is 30 s.

9. The nanozyme-loaded biomimetic hydrogel prepared by the preparation method according to any one of claims 1-8.

10. The use of the nanozyme-loaded biomimetic hydrogel prepared by any one of claims 1-8 or the nanozyme-loaded biomimetic hydrogel of claim 9 in the preparation of a drug for treating diabetic bone defects.

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