Preparation method of multifunctional nano-enzyme and application of multifunctional nano-enzyme in diabetes treatment medicine

Abstract

The invention relates to a preparation method of a multifunctional nano-enzyme and application of the multifunctional nano-enzyme in diabetes treatment drugs, and belongs to the technical field of medical preparations, the preparation method comprises the following steps: step 01, dissolving cerium acetate and a sodium hydroxide solution in deionized water according to a molar ratio of 2: 1; step 02, adding the mixed solution into a hydrothermal reaction kettle; step 03, carrying out centrifugal filtration on the reaction product of the precipitation obtained in the step 02; step 04, drying the solid product, so as to obtain CeO nano-particles; step 05, dissolving the CeO nano particles and polyvinylpyrrolidone in methanol; step 06, centrifuging the mixture; step 07, centrifugally washing the mixed solution to obtain a CeO-coated ZIF-8 composite material; step 08, dissolving the CeO (at) ZIF-8 and chloroauric acid (HAuCl) in methanol; step 09, dropwise adding a sodium borohydride aqueous solution into the mixed solution; and step 10, obtaining a final product CeO2 (at) ZIF-8 (at) Au. The CeO2 (at) ZIF-8 (at) Au prepared by the invention has remarkable advantages in repairing diabetic bone defects and reducing or relieving chronic inflammation.

Description

Technical Field

[0001] This invention relates to the field of biomedical engineering, and in particular to a method for preparing a multifunctional nanozyme and its application in a drug for treating diabetes. Background Technology

[0002] The occurrence of diabetes and its related complications (such as bone loss due to fractures) is closely related to hyperglycemia, oxidative stress, and chronic inflammation. For example, the published literature Catanzaro, O. (2021). Diabetic oxidative stress and bone loss complications. Endocrinology and Disorders, 5(1), 01–04. It argues that persistent hyperglycemia is the main feature of diabetes, which triggers a series of biochemical reactions leading to increased oxidative stress. Hyperglycemia can promote an increase in oxidative stress through the following pathways: First, the polyol pathway: In a hyperglycemic state, glucose is metabolized through the polyol pathway, leading to the accumulation of sorbitol and the consumption of NADPH. NADPH is necessary to maintain the reduction of glutathione (GSH), and glutathione is an important intracellular antioxidant. Its depletion will exacerbate oxidative stress. Secondly, as described in the literature Wang, N., & Zhang, C. (2024). Oxidative Stress: A Culprit in the Progression of Diabetic Kidney Disease. Antioxidants, 13(4), 455., the hexosamine pathway is activated by hyperglycemia, leading to the production of UDP-GlcNAc, which alters protein function and promotes oxidative stress. Thirdly, the aforementioned literature also elucidates advanced glycation end products (AGEs): hyperglycemia promotes the formation of AGEs, which bind to RAGE receptors on the cell surface, activating downstream signaling pathways including NADPH oxidase, thereby increasing the production of reactive oxygen species (ROS). Fourthly, the aforementioned literature also elucidates protein kinase C (PKC): hyperglycemia also activates the protein kinase C (PKC) pathway, leading to the production of reactive oxygen species (ROS), further exacerbating oxidative stress. In summary, high blood sugar leads to an imbalance of antioxidant enzymes, with a decrease in superoxide dismutase (SOD) and glutathione (GSH), and an increase in reactive oxygen species (ROS) and oxidized glutathione (GSSG), ultimately resulting in oxidative stress.

[0003] Furthermore, oxidative stress can activate inflammatory pathways, and inflammation, in turn, can exacerbate oxidative stress (see Jha, JC, Ho, F., Dan, C., & Jandelet-Dahm, K. (2018). A causal link between oxidative stress and inflammation in cardiovascular and renal complications of diabetes. Clinical Science, 132(16), 1811–1836.). Reactive oxygen species (ROS) can activate transcription factors, such as nuclear factor-κB (NF-κB), leading to increased expression of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6). These cytokines further promote the inflammatory response and lead to the production of more ROS, thus creating a vicious cycle.

[0004] As illustrated in Sheu, A., White, CP, & Center, JR (2024). Bone metabolism in diabetes: a clinician's guide to understanding the bone–glucose interplay. Diabetologia, 67(8), 1493–1506., hyperglycemia directly impairs osteoblast maturation, promotes osteoblast aging, and affects osteoclast survival. Advanced glycation end products (AGEs) impair osteoblast survival and function, disrupt bone mineralization, and affect protein and collagen cross-linking. Reduced bone turnover leads to the gradual accumulation of old bone, thereby weakening bone's load-bearing capacity.

[0005] As illustrated in the literatures Li J, Han F, Ma J, Wang H, Pan J, Yang G, et al. Targeting Endogenous Hydrogen Peroxide at Bone Defects Promotes Bone Repair. Advanced Functional Materials 2022; 32(10):2111208. and Ivanova P, Dzięgielewski K, Drozd M, Skorupska S, Grabowska-Jadach I, Pietrzak M. Nanoparticles of chosennoble metals asreactive oxygen species scavengers. Nanotechnology 2021; 32(5):055704., nanozymes exhibit superior catalytic efficiency, lower cost, and excellent stability under harsh conditions compared to natural enzymes, attracting increasing attention in the field of biomaterials. Integrating nanozymes into tissue-engineered biomaterials has opened up new horizons for their preparation and practical application. However, despite some progress in current research, the functional upper limit of nanozymes remains low in studies related to bone repair in diabetes. Furthermore, existing research focuses too much on improving the diabetic microenvironment by eliminating ROS, neglecting to address the AGEs problem caused by hyperglycemia. The accumulation of AGEs can further exacerbate oxidative stress and inflammatory responses, thereby affecting the effectiveness of bone repair therapy.

[0006] Current treatment options largely rely on supplementing with antioxidants such as vitamin E, vitamin C, and N-acetylcysteine ​​(NAC) to help reduce oxidative stress. The use of nonsteroidal anti-inflammatory drugs (NSAIDs) and glucocorticoids can help alleviate inflammatory responses. However, the pathological microenvironment of diabetic bone defects (hyperglycemia, inflammation, oxidative stress) is detrimental to autologous bone survival and regeneration. How to eliminate reactive oxygen species, suppress inflammation, and reshape the pathological microenvironment of diabetes to promote bone defect repair and regeneration remains a major challenge in the field of bone tissue engineering. In the oral and maxillofacial region, this challenge manifests as impaired periodontal tissue regeneration, implant osseointegration failure, and increased complexity in craniofacial bone defect repair. Although various bone repair biomaterials exist on the market, their effectiveness in diabetic patients often falls short of expectations, especially in the unique environment of the oral cavity where repair materials must withstand cyclic occlusal loads. This underscores the urgent need to develop targeted craniofacial bone repair materials and treatment options for diabetic patients. Summary of the Invention

[0007] This invention provides a method for preparing a multifunctional nanozyme and its application in diabetes treatment drugs, in order to overcome the shortcomings of the prior art. It is a multifunctional material that can regulate cellular glucose metabolism, remove excess reactive oxygen species (ROS), and inhibit inflammation, thereby improving the pathological microenvironment of diabetes and promoting tissue repair.

[0008] In order to achieve the objectives of this invention, the following technologies are proposed: One aspect provides a method for preparing multifunctional nanozymes, including the following steps: Step 01: Dissolve cerium acetate (Ce(CH3COO)3·xH2O) and sodium hydroxide solution (NaOH) in deionized water at a molar ratio of 2:1; Step 02: Add the mixture obtained in Step 01 to the hydrothermal reactor and hydrothermally react at 180°C for 24 hours; Step 03: The precipitated reaction product obtained in Step 02 is centrifuged and filtered, and then washed with deionized water; Step 04: The solid product washed in step 03 is dried in a vacuum drying oven at 60°C to obtain CeO2 nanoparticles. Step 05: The CeO2 nanoparticles obtained in step 04 are dissolved in methanol and mixed at 25°C for 24 hours. Step 06: Centrifuge the mixture obtained in Step 05, and then mix it with zinc nitrate hexahydrate (Zn(NO3)2·6H2O) and 2-methylimidazole (2-MIM) in methanol solvent, and react at room temperature for 30 min. Step 07: After centrifugation and washing of the mixture obtained in step 06, CeO2@ZIF-8 composite material is obtained; Step 08: Dissolve CeO2@ZIF-8 and chloroauric acid (HAuCl4) obtained in step 07 in methanol; Step 09: Add sodium borohydride aqueous solution dropwise to the mixed solution obtained in step 08, and stir the mixture at room temperature for 1 hour. Step 10: After centrifuging and washing the mixture obtained in step 09, the final product CeO2@ZIF-8@Au is obtained.

[0009] Furthermore, during centrifugal filtration in step 03, the centrifugal filtration conditions are 10,000 rpm / min for 10 minutes.

[0010] Furthermore, in step 05, when CeO2 nanoparticles are mixed and dissolved with polyvinylpyrrolidone (PVP), the mass ratio of CeO2 nanoparticles to polyvinylpyrrolidone (PVP) is (0.03-0.1):1.

[0011] Furthermore, in step 05, the molecular weight of polyvinylpyrrolidone (PVP) is 4000-10000 g / mol.

[0012] Furthermore, in step 06, the molar ratio of zinc nitrate hexahydrate (Zn(NO3)2·6H2O) to 2-methylimidazole (2-MIM) is 1:1.

[0013] Furthermore, in step 08, the concentration of chloroauric acid (HAuCl4) is 0.05-0.20M.

[0014] Furthermore, in step 09, the concentration of the sodium borohydride aqueous solution is 0.1M.

[0015] On the other hand, a multifunctional nanozyme is used in the preparation of drugs for treating diabetes. CeO2@ZIF-8@Au, prepared using a multifunctional nanozyme preparation method, is used in the preparation of drugs for repairing bone defects in diabetes.

[0016] The advantages of the above technical solution are: The CeO2@ZIF-8@Au prepared by this invention has significant advantages in repairing bone defects in diabetic patients and reducing or alleviating the occurrence of chronic inflammation. Attached Figure Description

[0017] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will now be described in further detail with reference to the accompanying drawings.

[0018] Figure 1 The synthesis flowchart of CeO2@ZIF-8@Au is shown.

[0019] Figure 2 The microstructure morphology of CeO2 in the CeO2@ZIF-8@Au nanoparticles prepared in Example 1 is shown.

[0020] Figure 3 The microstructure morphology of CeO2@ZIF-8 in the CeO2@ZIF-8@Au nanoparticles prepared in Example 1 is shown.

[0021] Figure 4 The microstructure morphology of the CeO2@ZIF-8@Au nanoparticles prepared in Example 1 is shown.

[0022] Figure 5 The XRD pattern of CeO2@ZIF-8@Au prepared in Example 1 is shown.

[0023] Figure 6The results of the in vitro biological evaluation of the cell activity of CeO2@ZIF-8@Au nanoparticles prepared in Example 1 are shown in the figure.

[0024] Figure 7 The results of the in vitro biological evaluation of the antioxidant activity of CeO2@ZIF-8@Au nanoparticles prepared in Example 1 are shown in the figure.

[0025] Figure 8 The graph shows the effect of CeO2@ZIF-8@Au nanoparticles prepared in Example 1 on glucose levels. Detailed Implementation

[0026] like Figure 1 As shown, a method for preparing a multifunctional nanozyme first uses CeO2 nanoparticles with redox activity as the core, and encapsulates them with zeolite imidazole ester framework material (ZIF-8) to construct a pH-responsive carrier (in the acidic microenvironment of diabetes, pH < 6, the alkaline ZIF-8 will hydrolyze and release CeO2 coated on the core layer under acidic conditions). Finally, gold nanoparticles (Au NPs) are modified on the surface to enhance electron transfer efficiency.

[0027] The principle behind the CeO2@ZIF-8@Au nanoparticles prepared by the above method in the treatment of bone defects caused by diabetes is as follows: Under a hyperglycemic environment, gold nanoparticles (Au NPs) exhibit glucose oxidase-like activity, which can effectively consume local glucose, thereby reducing the damage caused by glucose to osteoblasts. The CeO2 nanoparticles, through Ce³⁺ / Ce 4 The reversible degradation process activates superoxide dismutase and catalase-like activities, continuously scavenging reactive oxygen species (ROS), thereby alleviating chronic inflammatory complications. This, in turn, further reduces ROS production by mitigating chronic inflammation. The zeolite imidazole ester framework material (ZIF-8) degrades in an acidic inflammatory microenvironment, enabling controlled release of therapeutic drugs, thus offering significant advantages in treating diabetes involving bone defects and chronic inflammation.

[0028] To specifically demonstrate the significant advantages of CeO2@ZIF-8@Au in treating bone defects and chronic inflammation in diabetes, the following examples were conducted.

[0029] Example 1 A method for preparing a multifunctional nanozyme involves accurately weighing 10 mg of cerium acetate (Ce(CH3COO)3·xH2O), dissolving it in 20 mL of deionized water, adding 8 mL of 0.1 M NaOH solution, transferring the solution to a hydrothermal reactor, and reacting at 180°C for 24 hours. The reaction solution is then centrifuged at 10,000 rpm for 10 minutes, washed three times with deionized water, and vacuum dried at 60°C to obtain CeO2 nanoparticles.

[0030] 100 mg of the above CeO2 nanoparticles were dissolved together with polyvinylpyrrolidone (PVP, 1000 g / mol, 1 g) in 50 mL of methanol and stirred at 25°C for 24 hours. After centrifugation, zinc nitrate hexahydrate (Zn(NO3)2·6H2O, 300 mg) and 2-methylimidazole (2-MIM, 500 mg) were added to the precipitate, and the mixture was stirred in 50 mL of methanol at room temperature for 30 minutes. After centrifugation, washing, and vacuum drying, the CeO2@ZIF-8 composite material was obtained.

[0031] 100 mg CeO2@ZIF-8 was dispersed in 20 mL of deionized water, and 15 mL of 1 mM HAuCl4 solution was added. The mixture was stirred at room temperature for 2 hours, followed by dropwise addition of 0.1 M sodium borohydride aqueous solution, and the reaction was continued for 1 hour. After centrifugation, washing, and drying, CeO2@ZIF-8@Au nanoparticles were obtained.

[0032] Example 2 Accurately weigh 10 mg of cerium acetate (Ce(CH3COO)3·xH2O), dissolve it in 20 mL of deionized water, add 8 mL of 0.1 M NaOH solution, transfer to a hydrothermal reactor, and react at 180°C for 24 hours. Centrifuge the reaction solution at 10000 rpm for 10 minutes, wash three times with deionized water, and vacuum dry at 60°C to obtain CeO2 nanoparticles.

[0033] 50 mg of the above CeO2 nanoparticles were dissolved together with polyvinylpyrrolidone (PVP, 5000 g / mol, 1 g) in 50 mL of methanol and stirred at 25°C for 24 hours. After centrifugation, zinc nitrate hexahydrate (Zn(NO3)2·6H2O, 300 mg) and 2-methylimidazole (2-MIM, 500 mg) were added to the precipitate, and the mixture was stirred in 50 mL of methanol at room temperature for 30 minutes. After centrifugation, washing, and vacuum drying, the CeO2@ZIF-8 composite material was obtained.

[0034] 100 mg CeO2@ZIF-8 was dispersed in 20 mL of deionized water, and 15 mL of 1 mM HAuCl4 solution was added. The mixture was stirred at room temperature for 2 hours, followed by dropwise addition of 0.1 M sodium borohydride aqueous solution, and the reaction was continued for 1 hour. After centrifugation, washing, and drying, CeO2@ZIF-8@Au nanoparticles were obtained.

[0035] Example 3 Accurately weigh 10 mg of cerium acetate (Ce(CH3COO)3·xH2O), dissolve it in 20 mL of deionized water, add 8 mL of 0.1 M NaOH solution, transfer to a hydrothermal reactor, and react at 180°C for 24 hours. Centrifuge the reaction solution at 10000 rpm for 10 minutes, wash three times with deionized water, and vacuum dry at 60°C to obtain CeO2 nanoparticles.

[0036] 50 mg of the above CeO2 nanoparticles were dissolved together with polyvinylpyrrolidone (PVP, 5000 g / mol, 1 g) in 50 mL of methanol and stirred at 25°C for 24 hours. After centrifugation, zinc nitrate hexahydrate (Zn(NO3)2·6H2O, 300 mg) and 2-methylimidazole (2-MIM, 500 mg) were added to the precipitate, and the mixture was stirred in 50 mL of methanol at room temperature for 30 minutes. After centrifugation, washing, and vacuum drying, the CeO2@ZIF-8 composite material was obtained.

[0037] 100 mg CeO2@ZIF-8 was dispersed in 20 mL of deionized water, and 20 mL of 2 mM HAuCl4 solution was added. The mixture was stirred at room temperature for 2 hours, followed by dropwise addition of 0.1 M sodium borohydride aqueous solution, and the reaction was continued for 1 hour. After centrifugation, washing, and drying, CeO2@ZIF-8@Au nanoparticles were obtained.

[0038] Example 4 Accurately weigh 10 mg of cerium acetate (Ce(CH3COO)3·xH2O), dissolve it in 20 mL of deionized water, add 8 mL of 0.1 M NaOH solution, transfer to a hydrothermal reactor, and react at 180°C for 24 hours. Centrifuge the reaction solution at 10000 rpm for 10 minutes, wash three times with deionized water, and vacuum dry at 60°C to obtain CeO2 nanoparticles.

[0039] 50 mg of the above CeO2 nanoparticles were dissolved together with polyvinylpyrrolidone (PVP, 5000 g / mol, 1 g) in 50 mL of methanol and stirred at 25°C for 24 hours. After centrifugation, zinc nitrate hexahydrate (Zn(NO3)2·6H2O, 300 mg) and 2-methylimidazole (2-MIM, 500 mg) were added to the precipitate, and the mixture was stirred in 50 mL of methanol at room temperature for 30 minutes. After centrifugation, washing, and vacuum drying, the CeO2@ZIF-8 composite material was obtained.

[0040] 100 mg CeO2@ZIF-8 was dispersed in 20 mL of deionized water, and 30 mL of 3 mM HAuCl4 solution was added. The mixture was stirred at room temperature for 2 hours, followed by dropwise addition of 0.1 M sodium borohydride aqueous solution, and the reaction was continued for 1 hour. After centrifugation, washing, and drying, CeO2@ZIF-8@Au nanoparticles were obtained.

[0041] Example 5 Accurately weigh 10 mg of cerium acetate (Ce(CH3COO)3·xH2O), dissolve it in 20 mL of deionized water, add 8 mL of 0.1 M NaOH solution, transfer to a hydrothermal reactor, and react at 180°C for 24 hours. Centrifuge the reaction solution at 10000 rpm for 10 minutes, wash three times with deionized water, and vacuum dry at 60°C to obtain CeO2 nanoparticles.

[0042] 30 mg of the above CeO2 nanoparticles were dissolved together with polyvinylpyrrolidone (PVP, 8000 g / mol, 1 g) in 50 mL of methanol and stirred at 25°C for 24 hours. After centrifugation, zinc nitrate hexahydrate (Zn(NO3)2·6H2O, 300 mg) and 2-methylimidazole (2-MIM, 500 mg) were added to the precipitate, and the mixture was stirred in 50 mL of methanol at room temperature for 30 minutes. After centrifugation, washing, and vacuum drying, the CeO2@ZIF-8 composite material was obtained.

[0043] 100 mg CeO2@ZIF-8 was dispersed in 20 mL of deionized water, and 20 mL of 2 mM HAuCl4 solution was added. The mixture was stirred at room temperature for 2 hours, followed by dropwise addition of 0.1 M sodium borohydride aqueous solution, and the reaction was continued for 1 hour. After centrifugation, washing, and drying, CeO2@ZIF-8@Au nanoparticles were obtained.

[0044] To analyze and test the functionality of the prepared CeO2@ZIF-8@Au nanoparticles, the following methods are provided for analysis and verification: Material characterization The morphology of the CeO2@ZIF-8@Au nanoparticle sample prepared in Example 1 was observed using transmission electron microscopy, such as... Figures 2 to 4As shown, the results indicate that the material has a distinct core-shell structure, with CeO2 nanoparticles (approximately 20 nm) uniformly encapsulated by the ZIF-8 framework, and Au nanoparticles of approximately 5 nm dispersed on the surface.

[0045] like Figure 5 As shown, the presence of CeO2 (PDF#34-0394), ZIF-8 (PDF#62-1030), and Au (PDF#04-0784) crystalline phases of the CeO2@ZIF-8@Au nanoparticles prepared in Example 1 was confirmed again by X-ray diffraction (XRD analysis).

[0046] Functional testing To verify the application of CeO2@ZIF-8@Au nanoparticles in the treatment of diabetes, the CeO2@ZIF-8@Au nanoparticles prepared in Example 1 were used for the treatment of bone marrow mesenchymal stem cells cultured in high glucose: Firstly, cell viability: In cell viability assays, bone marrow mesenchymal stem cells (BMSCs) were used as a model. Cells were seeded at a density of 5 × 10³ cells / well in 96-well plates and pre-cultured in DMEM medium containing 10% fetal bovine serum (FBS) for 24 hours (37℃, 5% CO2). Then, CeO2@ZIF-8@Au nanocomposite materials at concentrations of 0, 25, 50, and 100 μg / mL were added, and incubation continued for another 24 hours. For CCK-8 assays, 10 μL of CCK-8 reagent was added to each well, and after incubation in the dark for 2 hours, absorbance was measured at 450 nm using a microplate reader. Blank wells (medium and reagent only), negative controls (untreated cells), and positive controls (cells containing 10% DMSO) were included to eliminate background interference. CCK-8 assays showed that at a concentration of 100 μg / mL, cell viability remained above 92%. The results are as follows: Figure 6 As shown, it is clear that after co-culturing BMSCs with CeO2@ZIF-8@Au nanocomposite materials at concentrations of 25, 50, and 100 μg / mL for 24 hours, the cell viability decreased from 95% to 92% with increasing material concentration, but the cell viability of all three groups was greater than 90%. This indicates that the CeO2@ZIF-8@Au nanocomposite material has good biocompatibility and no obvious cytotoxicity.

[0047] Secondly, antioxidant activity (H2O2 scavenging): This experiment used electron spin resonance (ESR) technology to detect the H2O2 scavenging ability of CeO2@ZIF-8@Au nanozyme. 5,5-Dimethyl-1-pyrrolline-N-oxide (DMPO) was used as a spin trapping agent to capture hydroxyl radicals (·OH) generated by the H2O2 reaction, forming a DMPO-OH adduct. Its antioxidant activity was assessed by changes in ESR signal intensity. The specific procedure was as follows: CeO2@ZIF-8@Au nanozyme was dispersed in PBS buffer (pH 7.4), sonicated for 10 min to ensure uniform dispersion, and the concentration was set to 100 μg / mL. 100 μL of PBS buffer was taken, and 10 μL of H2O2 (final concentration 1 mM) and 10 μL of DMPO (final concentration 50 mM) were added. After mixing, the mixture was immediately transferred to a quartz capillary tube for ESR detection. The control group contained only H2O2 / DMPO. The H2O2 scavenging rate was calculated by comparing the changes in the signal intensity of the characteristic peak (1:2:2:1 quartet) of the DMPO-OH adduct. The formula is: Scavenging rate (%) = (1 - I_sample / I_control) × 100%, where I_control is the signal intensity without nanozyme and I_sample is the signal intensity with nanozyme. Figure 7 As shown, the results indicate that with the addition of CeO2@ZIF-8@Au, the DMPO-OH signal intensity gradually decreased, indicating that its ability to scavenge H2O2 was enhanced. The scavenging rate reached 85.3% at 100 μg / mL, which is attributed to the redox activity of CeO2 and the synergistic catalytic effect of Au nanoparticles. This verifies the ROS scavenging potential of this nanozyme in the oxidative stress microenvironment of diabetes.

[0048] Thirdly, the regulatory role of glucose metabolism: This was determined using the glucose oxidase method (GOD-POD method). BMSCs were treated with CeO2@ZIF-8@Au (25, 50, 100 μg / mL) for 24 hours. The cell culture supernatant was collected, centrifuged (3000 rpm, 10 min) to remove cell debris, and then mixed with glucose detection reagents (containing glucose oxidase, peroxidase, and chromogenic substrate) in a specific ratio. The mixture was incubated at 37°C in the dark for 30 minutes. The absorbance was then measured at 505 nm using a microplate reader. Glucose consumption was calculated using a standard curve, and normalization analysis was performed using the untreated group as a baseline. All experiments were repeated three times independently to ensure data reliability. Glucose detection showed that glucose consumption increased 2.3-fold in the treated groups. The results are as follows: Figure 8As shown, glucose consumption increased in a dose-dependent manner with increasing CeO2@ZIF-8@Au concentration, reaching 34 μM / min in the 100 μg / mL treatment group. This phenomenon is mainly attributed to the glucose oxidase-mimicking activity of the Au nanoparticles in the nanocomposite material; the exposed Au atoms on its surface effectively promote the glucose metabolism process in cells by catalyzing the glucose oxidation reaction.

[0049] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Obviously, those skilled in the art can make various modifications and variations to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention also intends to include these modifications and variations.

Claims

1. A method for preparing a multifunctional nanozyme, characterized in that, Including the following steps: Step 01: Dissolve cerium acetate Ce(CH3COO)3·xH2O and sodium hydroxide solution NaOH in deionized water at a molar ratio of 2:1; Step 02: Add the mixture obtained in Step 01 to the hydrothermal reactor and hydrothermally react at 180°C for 24 hours; Step 03: The precipitated reaction product obtained in Step 02 is centrifuged and filtered, and then washed with deionized water; Step 04: The solid product washed in step 03 is dried in a vacuum drying oven at 60°C to obtain CeO2 nanoparticles. Step 05: Dissolve the CeO2 nanoparticles obtained in step 04 and polyvinylpyrrolidone (PVP) in methanol and mix at 25°C for 24 hours. Step 06: Centrifuge the mixture obtained in Step 05, and then mix it with zinc nitrate hexahydrate Zn(NO3)2·6H2O and 2-methylimidazolium 2-MIM in methanol solvent, and react at room temperature for 30 min. Step 07: After centrifugation and washing of the mixture obtained in step 06, CeO2@ZIF-8 composite material is obtained; Step 08: Dissolve CeO2@ZIF-8 and chloroauric acid HAuCl4 obtained in step 07 in methanol; Step 09: Add sodium borohydride aqueous solution dropwise to the mixed solution obtained in step 08, and stir the mixture at room temperature for 1 hour. Step 10: After centrifuging and washing the mixture obtained in step 09, the final product CeO2@ZIF-8@Au is obtained.

2. The method for preparing multifunctional nanozymes according to claim 1, characterized in that, In step 03, the centrifugal filtration conditions are 10,000 rpm / min for 10 minutes.

3. The method for preparing multifunctional nanozymes according to claim 1, characterized in that, In step 05, when CeO2 nanoparticles and polyvinylpyrrolidone (PVP) are mixed and dissolved, the mass ratio of CeO2 nanoparticles to PVP is (0.03-0.1):

1.

4. The method for preparing multifunctional nanozymes according to claim 1, characterized in that, In step 05, the molecular weight of polyvinylpyrrolidone (PVP) is 4000-10000 g / mol.

5. The method for preparing multifunctional nanozymes according to claim 1, characterized in that, In step 06, the molar ratio of zinc nitrate hexahydrate Zn(NO3)2·6H2O to 2-methylimidazole 2-MIM is 1:

1.

6. The method for preparing multifunctional nanozymes according to claim 1, characterized in that, In step 08, the concentration of chloroauric acid HAuCl4 is 0.05-0.20M.

7. The method for preparing multifunctional nanozymes according to claim 1, characterized in that, In step 09, the concentration of the sodium borohydride aqueous solution is 0.1M.

8. The application of a multifunctional nanozyme in the preparation of diabetes treatment drugs, characterized in that, Application of CeO2@ZIF-8@Au, prepared by the method of any one of claims 1 to 7, in the preparation of drugs for repairing bone defects in diabetic patients.

Images