Defective metal-organic framework antioxidant nanoszyme and preparation method and application thereof

Abstract

The application discloses a kind of defective metal organic framework antioxidant nanoenzymes and preparation method and application thereof, belong to nanobiotechnology field.The preparation method is that copper source, zirconium source and organic ligand containing pyrazole and carboxyl are dissolved in organic solvent, then competitive ligand is added, after being mixed uniformly, solvent thermal reaction is carried out at 80 DEG C~120 DEG C, and defective metal organic framework is prepared;Defective metal organic framework aqueous solution and kidney targeting molecule solution are mixed uniformly, and through coordination and electrostatic adsorption, defective metal organic framework antioxidant nanoenzyme is obtained.The application is prepared by adjusting particle size and defect by organic ligand containing pyrazole and carboxyl, competitive ligand containing carboxylic acid and organic solvent, and metal organic framework with small particle size and containing defect is prepared, antioxidant activity is improved, and kidney targeting molecule is loaded to obtain defective metal organic framework antioxidant nanoenzyme, to realize the targeting effect of kidney.

Description

Technical Field

[0001] This invention relates to the field of nanobiotechnology, and more specifically to a defective metal-organic framework antioxidant nanozyme, its preparation method, and its application. Background Technology

[0002] Acute kidney injury (AKI) is a common critical illness in clinical practice, with high morbidity and mortality. Studies have found that abnormal reactive oxygen species (ROS) in the kidneys are a major contributing factor to AKI. To date, apart from supportive treatments such as rehydration, diuresis, and dialysis, there are no effective preventative or therapeutic drugs in clinical practice.

[0003] With the continuous development and advancement of nanotechnology, the emergence of antioxidant nanozymes undoubtedly offers possibilities for the prevention and treatment of AKI. Nanozymes are a class of enzyme-like catalytic nanomaterials capable of catalyzing enzyme substrates, producing catalytic reactions similar to those of natural enzymes, and exhibiting characteristics such as enzyme-catalyzed reaction kinetics. They belong to a novel class of enzyme mimics. Similar to natural enzymes, nanozymes can efficiently catalyze enzyme substrates under mild physiological conditions, possessing advantages such as tunable catalytic activity, high stability under harsh conditions, flexibility in composition and structural design, and excellent biocompatibility.

[0004] Currently reported nanozymes with antioxidant activity mainly include cerium dioxide, manganese oxide, Prussian blue, carbon nanoparticles, and metal-organic frameworks (MOFs). Among them, MOF-based antioxidant nanozymes have become one of the best choices for treating AKI due to their advantages such as high specific surface area, high porosity, tunable structure, easy functionalization, and good biocompatibility. However, the application of MOF-based antioxidant nanozymes in the treatment of AKI is severely limited by factors such as excessive size, limited antioxidant activity, and lack of renal targeting. Summary of the Invention

[0005] To address the above problems, this invention provides a defective metal-organic framework antioxidant nanozyme, its preparation method, and its application. By adjusting the particle size and defects through the combination of pyrazole and carboxyl-containing organic ligands, carboxylic acid-containing competitive ligands, and organic solvents, a metal-organic framework with small particle size and defects is prepared, which improves antioxidant activity. Furthermore, by loading kidney-targeting molecules, a defective metal-organic framework antioxidant nanozyme is obtained, achieving kidney-targeting action.

[0006] The first objective of this invention is to provide a method for preparing defective metal-organic framework antioxidant nanozymes, comprising the following steps:

[0007] A copper source, a zirconium source, and organic ligands containing pyrazole and carboxyl groups were dissolved in an organic solvent. Then, competing ligands were added, and the mixture was thoroughly mixed. A solvothermal reaction was then carried out at 80℃–120℃. The organic ligands containing pyrazole and carboxyl groups controlled the rate and number of nucleations, the competing ligands containing carboxylic acids controlled the crystal growth rate and crystallinity, and the organic solvent controlled the number of crystal nuclei. By adjusting the particle size and defects through the combination of the organic ligands containing pyrazole and carboxyl groups, the competing ligands containing carboxylic acids, and the organic solvent, defective metal-organic frameworks were prepared. The ratio of the organic ligands containing pyrazole and carboxyl groups to the competing ligands containing carboxylic acids was 20 mg–64 mg: 0 μL–120 μL; the ratio of the organic ligands containing pyrazole and carboxyl groups to the organic solvent was 20 mg–64 mg: 10 mL–30 mL.

[0008] For example, the ratio of organic ligands containing pyrazole and carboxyl groups to competing ligands containing carboxylic acid groups is 20 mg: 30 μL, 20 mg: 60 μL, 20 mg: 90 μL, 20 mg: 120 μL, 40 mg: 40 μL, 40 mg: 80 μL, 40 mg: 100 μL, 64 mg: 30 μL, 64 mg: 50 μL, 64 mg: 70 μL, 64 mg: 90 μL, 64 mg: 110 μL, etc.

[0009] The ratio of organic ligands containing pyrazole and carboxyl groups to organic solvents is 20mg:10mL, 20mg:20mL, 20mg:30mL, 30mg:10mL, 30mg:20mL, 40mg:10mL, 40mg:30mL, 50mg:10mL, 50mg:20mL, 50mg:30mL, etc.; however, it is not limited to the values ​​listed above, and other unlisted values ​​within the above range are also applicable.

[0010] After uniformly mixing the aqueous solution of the defective metal-organic framework and the solution of the kidney-targeting molecule, the antioxidant nanozyme of the defective metal-organic framework was obtained through coordination and electrostatic adsorption.

[0011] In a preferred embodiment of the present invention, the ratio of the organic ligand containing pyrazole and carboxyl groups to the organic solvent is 48 mg: 20 mL.

[0012] In a preferred embodiment of the present invention, the ratio of the organic ligand containing pyrazole and carboxyl groups to the competing ligand containing carboxylic acid is 48 mg: 30 μL.

[0013] In a preferred embodiment of the present invention, the mass ratio of the organic ligand containing pyrazole and carboxyl groups to the copper source is 1:2 to 6; and the mass ratio of the zirconium source to the copper source is 1:2 to 4.

[0014] For example, the mass ratio of organic ligands containing pyrazole and carboxyl groups to copper sources is 1:2, 1:3, 1:4, 1:5, 1:6, etc.

[0015] The mass ratio of zirconium source to copper source is 1:2, 1:3, 1:4, etc.; however, it is not limited to the listed values, and other unlisted values ​​within the above range also apply.

[0016] In a preferred embodiment of the present invention, the reaction time of the solvothermal reaction is 6h to 10h, for example, the reaction time of the solvothermal reaction is 6h, 7h, 8h, 9h, 10h, etc.; but it is not limited to the listed values, and other unlisted values ​​within the above range are also applicable.

[0017] In a preferred embodiment of the present invention, the mass ratio of the defective metal-organic framework to the kidney-targeting molecule is 10:0.1 to 0.5, for example, the mass ratio of the defective metal-organic framework to the kidney-targeting molecule is 10:0.1, 10:0.2, 10:0.3, 10:0.4, 10:0.5, etc.; but it is not limited to the listed values, and other unlisted values ​​within the above range are also applicable.

[0018] In a preferred embodiment of the present invention, the reaction temperature for coordination and electrostatic adsorption is 20°C to 40°C, and the reaction time is 8h to 28h.

[0019] For example, the reaction temperatures for coordination and electrostatic adsorption reactions are 20℃, 25℃, 30℃, 35℃, 40℃, etc., and the reaction times are 8h, 12h, 16h, 20h, 24h, 28h, etc.; however, they are not limited to the values ​​listed above, and other unlisted values ​​within the above range are also applicable.

[0020] In a preferred embodiment of the present invention, the copper source is copper nitrate or copper nitrate hydrate;

[0021] The zirconium source is zirconium oxychloride octahydrate or zirconium oxide;

[0022] Organic ligands containing pyrazole and carboxyl groups are 1H-pyrazole-4-carboxylic acid, 3-methylpyrazole-4-carboxylic acid, 3,5-dimethyl-4-pyrazole carboxylic acid, or 5-phenyl-1H-pyrazole-4-carboxylic acid;

[0023] The competing ligands are trifluoroacetic acid or acetic acid;

[0024] The kidney-targeting molecules are L-serine or hyaluronic acid.

[0025] A second objective of this invention is to provide a defective metal-organic framework antioxidant nanozyme prepared by the above-described preparation method.

[0026] A third objective of this invention is to provide the application of the aforementioned defective metal-organic framework antioxidant nanozymes in the preparation of drugs for treating ROS-mediated kidney diseases.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) This invention employs a solvothermal method to prepare defective metal-organic frameworks with small size and high defects by controlling the content of organic ligands containing pyrazole and carboxyl groups, competing ligands containing carboxylic acids, and the volume of the reaction solvent. Due to the reduced particle size and increased defects, these frameworks have a larger specific surface area and pore size, thereby exposing more catalytic active sites and improving their antioxidant performance. Furthermore, by modifying the surface of the defective metal-organic framework enzyme with the kidney-targeting molecule L-serine through post-modification, a defective metal-organic framework antioxidant nanozyme with kidney-targeting function is obtained, ultimately achieving effective treatment of ROS-mediated kidney diseases.

[0029] (2) The synthesis process of this invention is simple, highly operable, and suitable for high-efficiency and large-scale production.

[0030] (3) The NMOF-Ls prepared in this invention have a particle size of 20-100 nm, which is essential for their kidney targeting and accumulation.

[0031] (4) The final product NMOF-Ls obtained in this invention can achieve the therapeutic effect of cisplatin-induced AKI by actively targeting the kidney. Attached Figure Description

[0032] Figure 1 This is a transmission electron microscope image of the NMOF in Example 12.

[0033] Figure 2 This is a powder X-ray diffraction pattern of NMOF in Example 12.

[0034] Figure 3 The results show the specific surface area and porosity of the NMOF in Example 12.

[0035] Figure 4 The images show the UV-Vis absorption spectra of different materials in Example 12.

[0036] Figure 5 The image shows a transmission electron microscope image of NMOF-Ls from Example 12.

[0037] Figure 6 The image shows the X-ray photoelectron spectrum of NMOF-Ls in Example 12.

[0038] Figure 7 The results show the SOD and CAT activity test results of different materials in Example 12.

[0039] Figure 8 This is a biological transmission electron microscope image of NMOF-Ls taken up by cells in Example 12.

[0040] Figure 9 Laser scanning confocal microscopy images showing the materials prepared for Comparative Example 1(a) and Example 12(b) achieving HK-2 cell targeting function.

[0041] Figure 10 The results show the characterization of the antioxidant properties of the different materials in Example 12.

[0042] Figure 11 The results show the creatinine and urea nitrogen content of AKI mice after treatment with different materials in Example 12. Detailed Implementation

[0043] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0044] A method for preparing a defective metal-organic framework antioxidant nanozyme, characterized by comprising the following steps:

[0045] Defective metal-organic frameworks were prepared by dissolving copper source, zirconium source, and organic ligands containing pyrazole and carboxyl groups in an organic solvent, then adding competing ligands, mixing thoroughly, and carrying out a solvothermal reaction at 80℃~120℃. The ratio of organic ligands containing pyrazole and carboxyl groups to competing ligands containing carboxyl groups was 20~64mg:0μL~120μL; the ratio of organic ligands containing pyrazole and carboxyl groups to organic solvent was 20mg~64mg:10mL~20mL.

[0046] After uniformly mixing the aqueous solution of the defective metal-organic framework and the aqueous solution of the kidney-targeting molecule, an antioxidant nanozyme with kidney-targeting function was obtained through coordination and electrostatic adsorption.

[0047] At the initial stage of the reaction, a suitable increase in the amount of organic ligands significantly increases the rate and number of NMOF nuclei, which plays a crucial role in reducing the NMOF particle size. Furthermore, during the reaction, competing ligands regulate the crystal growth rate and crystallinity of NMOFs. If the amount of competing ligands is appropriately reduced, the crystal growth rate of NMOFs accelerates, making it easier to form short-range disordered structures, thereby increasing the number of defects in the NMOFs.

[0048] Within a certain range, a decrease in the volume of organic solvent will increase the concentration of MOF precursors, which will form a large number of crystal nuclei in the short time at the beginning of the reaction, and then grow into NMOFs with smaller particle size.

[0049] During the preparation process, the mass ratio of the defective metal-organic framework to the kidney-targeting molecule was 10:0.1–0.5. The coordination and electrostatic adsorption reactions were carried out at temperatures ranging from 20°C to 40°C for 8–28 hours.

[0050] It is understandable that during the preparation process, the mass ratio of the defective metal-organic framework and the kidney-targeting molecule, as well as the reaction parameters, need to be carried out according to the above conditions. If the above parameter range is exceeded, the amount of kidney-targeting molecule is insufficient or the reaction time is too short, which will result in a lower content of it on the NMOF surface, thereby affecting its targeting function on HK-2 cells.

[0051] In a preferred embodiment of the present invention, the copper source is copper nitrate or copper nitrate hydrate, and the zirconium source is zirconium oxychloride octahydrate or zirconium oxide.

[0052] In a preferred embodiment of the present invention, the organic ligand containing pyrazole and carboxyl groups is 1H-pyrazole-4-carboxylic acid (H2PyC), 3-methylpyrazole-4-carboxylic acid, 3,5-dimethyl-4-pyrazole carboxylic acid, or 5-phenyl-1H-pyrazole-4-carboxylic acid;

[0053] The competing ligands are trifluoroacetic acid (CF3COOH) or acetic acid;

[0054] The kidney-targeting molecules are L-serine or hyaluronic acid.

[0055] This invention first prepares a defective metal-organic framework, denoted as NMOF. NMOF is an amorphous structure with a size of approximately 20-100 nm, exhibiting increased specific surface area and pore size. The presence of defective structures in NMOF, along with the increased specific surface area and pore size, leads to an increase in catalytic active sites, ultimately enhancing its antioxidant performance. Then, based on NMOF, it is compounded with kidney-targeting molecules. The resulting defective metal-organic framework antioxidant nanozyme can achieve kidney-targeting function through targeting kidney injury markers, effectively improving therapeutic efficacy. This defective metal-organic framework antioxidant nanozyme can be used to prepare drugs for treating ROS-mediated kidney diseases, such as acute kidney injury, chronic kidney injury, and renal fibrosis.

[0056] Example 1

[0057] Step 1: Dissolve 124 mg of Cu(NO3)2·3H2O, 42.5 mg of ZrOCl2·8H2O, 32.5 mg of H2PyC, and 120 μL of CF3COOH in 10 mL of DMF using ultrasonication. Then transfer the solution to a 50 mL hydrothermal reactor and react at 100 °C for 8 h. After naturally cooling to room temperature, centrifuge at 12000 rpm for 5 min and wash three times with DMF. Disperse the resulting 50 mg of NMOF in 5 mL of deionized water for later use.

[0058] Step 2: Take 1 mL of the above-obtained NMOF aqueous solution and mix it with 1 mL of L-serine aqueous solution with a concentration of 0.5 mg / mL. Stir and react at 25°C for 12 h. Then centrifuge at 14000 rpm for 20 min and wash with deionized water 3 times. Disperse the obtained product NMOF-Ls in 2 mL of deionized water for later use.

[0059] Example 2

[0060] Step 1: Dissolve 124 mg of Cu(NO3)2·3H2O, 42.5 mg of ZrOCl2·8H2O, 32.5 mg of H2PyC, and 120 μL of CF3COOH in 15 mL of DMF using ultrasonication. Then transfer the solution to a 50 mL hydrothermal reactor and react at 100 °C for 8 h. After naturally cooling to room temperature, centrifuge at 12000 rpm for 5 min and wash three times with DMF. Disperse the resulting 50 mg of NMOF in 5 mL of deionized water for later use.

[0061] Step 2: Take 1 mL of the above-obtained NMOF aqueous solution and mix it with 1 mL of L-serine aqueous solution with a concentration of 0.5 mg / mL. Stir and react at 25°C for 12 h. Then centrifuge at 14000 rpm for 20 min and wash with deionized water 3 times. Disperse the obtained product NMOF-Ls in 2 mL of deionized water for later use.

[0062] Example 3

[0063] Step 1: Dissolve 124 mg of Cu(NO3)2·3H2O, 42.5 mg of ZrOCl2·8H2O, 32.5 mg of H2PyC, and 120 μL of CF3COOH in 20 mL of DMF using ultrasonication. Then transfer the solution to a 50 mL hydrothermal reactor and react at 100 °C for 8 h. After naturally cooling to room temperature, centrifuge at 12000 rpm for 5 min and wash three times with DMF. Disperse the resulting 50 mg of NMOF in 5 mL of deionized water for later use.

[0064] Step 2: Take 1 mL of the above-obtained NMOF aqueous solution and mix it with 1 mL of L-serine aqueous solution with a concentration of 0.5 mg / mL. Stir and react at 25°C for 12 h. Then centrifuge at 14000 rpm for 20 min and wash with deionized water 3 times. Disperse the obtained product NMOF-Ls in 2 mL of deionized water for later use.

[0065] Example 4

[0066] Step 1: Dissolve 124 mg of Cu(NO3)2·3H2O, 42.5 mg of ZrOCl2·8H2O, 32.5 mg of H2PyC, and 120 μL of CF3COOH in 25 mL of DMF using ultrasonication. Then transfer the solution to a 50 mL hydrothermal reactor and react at 100 °C for 8 h. After naturally cooling to room temperature, centrifuge at 12000 rpm for 5 min and wash three times with DMF. Disperse the resulting 50 mg of NMOF in 5 mL of deionized water for later use.

[0067] Step 2: Take 1 mL of the above-obtained NMOF aqueous solution and mix it with 1 mL of L-serine aqueous solution with a concentration of 0.5 mg / mL. Stir and react at 25°C for 12 h. Then centrifuge at 14000 rpm for 20 min and wash with deionized water 3 times. Disperse the obtained product NMOF-Ls in 2 mL of deionized water for later use.

[0068] Example 5

[0069] Step 1: Dissolve 124 mg of Cu(NO3)2·3H2O, 42.5 mg of ZrOCl2·8H2O, 32.5 mg of H2PyC, and 120 μL of CF3COOH in 30 mL of DMF using ultrasonication. Then transfer the solution to a 50 mL hydrothermal reactor and react at 100 °C for 8 h. After naturally cooling to room temperature, centrifuge at 12000 rpm for 5 min and wash three times with DMF. Disperse the resulting 50 mg of NMOF in 5 mL of deionized water for later use.

[0070] Step 2: Take 1 mL of the above-obtained NMOF aqueous solution and mix it with 1 mL of L-serine aqueous solution with a concentration of 0.5 mg / mL. Stir and react at 25°C for 12 h. Then centrifuge at 14000 rpm for 20 min and wash with deionized water 3 times. Disperse the obtained product NMOF-Ls in 2 mL of deionized water for later use.

[0071] Example 6

[0072] Step 1: Dissolve 124 mg of Cu(NO3)2·3H2O, 42.5 mg of ZrOCl2·8H2O, 32.5 mg of H2PyC, and 0 μL of CF3COOH in 20 mL of DMF using ultrasonication. Then transfer the solution to a 50 mL hydrothermal reactor and react at 100 °C for 8 h. After naturally cooling to room temperature, centrifuge at 12000 rpm for 5 min and wash three times with DMF. Disperse the resulting 50 mg of NMOF in 5 mL of deionized water for later use.

[0073] Step 2: Take 1 mL of the above-obtained NMOF aqueous solution and mix it with 1 mL of L-serine aqueous solution with a concentration of 0.5 mg / mL. Stir and react at 25°C for 12 h. Then centrifuge at 14000 rpm for 20 min and wash with deionized water 3 times. Disperse the obtained product NMOF-Ls in 2 mL of deionized water for later use.

[0074] Example 7

[0075] Step 1: Dissolve 124 mg of Cu(NO3)2·3H2O, 42.5 mg of ZrOCl2·8H2O, 32.5 mg of H2PyC, and 30 μL of CF3COOH in 20 mL of DMF using ultrasonication. Then transfer the solution to a 50 mL hydrothermal reactor and react at 100 °C for 8 h. After naturally cooling to room temperature, centrifuge at 12000 rpm for 5 min and wash three times with DMF. Disperse the resulting 50 mg of NMOF in 5 mL of deionized water for later use.

[0076] Step 2: Take 1 mL of the above-obtained NMOF aqueous solution and mix it with 1 mL of L-serine aqueous solution with a concentration of 0.5 mg / mL. Stir and react at 25°C for 12 h. Then centrifuge at 14000 rpm for 20 min and wash with deionized water 3 times. Disperse the obtained product NMOF-Ls in 2 mL of deionized water for later use.

[0077] Example 8

[0078] Step 1: Dissolve 124 mg of Cu(NO3)2·3H2O, 42.5 mg of ZrOCl2·8H2O, 32.5 mg of H2PyC, and 60 μL of CF3COOH in 20 mL of DMF using ultrasonication. Then transfer the solution to a 50 mL hydrothermal reactor and react at 100 °C for 8 h. After naturally cooling to room temperature, centrifuge at 12000 rpm for 5 min and wash three times with DMF. Disperse the resulting 50 mg of NMOF in 5 mL of deionized water for later use.

[0079] Step 2: Take 1 mL of the above-obtained NMOF aqueous solution and mix it with 1 mL of L-serine aqueous solution with a concentration of 0.5 mg / mL. Stir and react at 25°C for 12 h. Then centrifuge at 14000 rpm for 20 min and wash with deionized water 3 times. Disperse the obtained product NMOF-Ls in 2 mL of deionized water for later use.

[0080] Example 9

[0081] Step 1: Dissolve 124 mg of Cu(NO3)2·3H2O, 42.5 mg of ZrOCl2·8H2O, 32.5 mg of H2PyC, and 90 μL of CF3COOH in 20 mL of DMF using ultrasonication. Then transfer the solution to a 50 mL hydrothermal reactor and react at 100 °C for 8 h. After naturally cooling to room temperature, centrifuge at 12000 rpm for 5 min and wash three times with DMF. Disperse the resulting 50 mg of NMOF in 5 mL of deionized water for later use.

[0082] Step 2: Take 1 mL of the above-obtained NMOF aqueous solution and mix it with 1 mL of L-serine aqueous solution with a concentration of 0.5 mg / mL. Stir and react at 25°C for 12 h. Then centrifuge at 14000 rpm for 20 min and wash with deionized water 3 times. Disperse the obtained product NMOF-Ls in 2 mL of deionized water for later use.

[0083] Example 10

[0084] Step 1: Dissolve 124 mg of Cu(NO3)2·3H2O, 42.5 mg of ZrOCl2·8H2O, 20 mg of H2PyC, and 30 μL of CF3COOH in 20 mL of DMF using ultrasonication. Then transfer the solution to a 50 mL hydrothermal reactor and react at 100 °C for 8 h. After naturally cooling to room temperature, centrifuge at 12000 rpm for 5 min and wash three times with DMF. Disperse the resulting 50 mg of NMOF in 5 mL of deionized water for later use.

[0085] Step 2: Take 1 mL of the above-obtained NMOF aqueous solution and mix it with 1 mL of L-serine aqueous solution with a concentration of 0.5 mg / mL. Stir and react at 25°C for 12 h. Then centrifuge at 14000 rpm for 20 min and wash with deionized water 3 times. Disperse the obtained product NMOF-Ls in 2 mL of deionized water for later use.

[0086] Example 11

[0087] Step 1: Dissolve 124 mg of Cu(NO3)2·3H2O, 42.5 mg of ZrOCl2·8H2O, 32 mg of H2PyC, and 30 μL of CF3COOH in 20 mL of DMF using ultrasonication. Then transfer the solution to a 50 mL hydrothermal reactor and react at 100 °C for 8 h. After naturally cooling to room temperature, centrifuge at 12000 rpm for 5 min and wash three times with DMF. Disperse the resulting 50 mg of NMOF in 5 mL of deionized water for later use.

[0088] Step 2: Take 1 mL of the above-obtained NMOF aqueous solution and mix it with 1 mL of L-serine (Ls) aqueous solution with a concentration of 0.5 mg / mL. Stir and react at 25 °C for 12 h. Then centrifuge at 14000 rpm for 20 min and wash with deionized water 3 times. Disperse the obtained product NMOF-Ls in 2 mL of deionized water for later use.

[0089] Example 12

[0090] Step 1: Dissolve 124 mg of Cu(NO3)2·3H2O, 42.5 mg of ZrOCl2·8H2O, 48 mg of H2PyC, and 30 μL of CF3COOH in 20 mL of DMF using ultrasonication. Then transfer the solution to a 50 mL hydrothermal reactor and react at 100 °C for 8 h. After naturally cooling to room temperature, centrifuge at 12000 rpm for 5 min and wash three times with DMF. Disperse the resulting 50 mg of NMOF in 5 mL of deionized water for later use.

[0091] Step 2: Take 1 mL of the above-obtained NMOF aqueous solution and mix it with 1 mL of L-serine aqueous solution with a concentration of 0.5 mg / mL. Stir and react at 25°C for 12 h. Then centrifuge at 14000 rpm for 20 min and wash with deionized water 3 times. Disperse the obtained product NMOF-Ls in 2 mL of deionized water for later use.

[0092] Example 13

[0093] Step 1: Dissolve 124 mg of Cu(NO3)2·3H2O, 42.5 mg of ZrOCl2·8H2O, 64 mg of H2PyC, and 30 μL of CF3COOH in 20 mL of DMF using ultrasonication. Then transfer the solution to a 50 mL hydrothermal reactor and react at 100 °C for 8 h. After naturally cooling to room temperature, centrifuge at 12000 rpm for 5 min and wash three times with DMF. Disperse the resulting 50 mg of NMOF in 5 mL of deionized water for later use.

[0094] Step 2: Take 1 mL of the above-obtained NMOF aqueous solution and mix it with 1 mL of L-serine aqueous solution with a concentration of 0.5 mg / mL. Stir and react at 25°C for 12 h. Then centrifuge at 14000 rpm for 20 min and wash with deionized water 3 times. Disperse the obtained product NMOF-Ls in 2 mL of deionized water for later use.

[0095] Example 14

[0096] Step 1: Dissolve 64 mg of Cu(NO3)2·3H2O, 10.7 mg of ZrOCl2·8H2O, 32 mg of 3-methylpyrazole-4-carboxylic acid, and 30 μL of glacial acetic acid in 20 mL of DMF by sonication. Then transfer the solution to a 50 mL hydrothermal reactor and react at 80 °C for 10 h. After cooling naturally to room temperature, centrifuge at 12000 rpm for 5 min and wash three times with DMF. Disperse the resulting 50 mg of NMOF in 5 mL of deionized water for later use.

[0097] Step 2: Take 1 mL of the above-obtained NMOF aqueous solution and mix it with 1 mL of hyaluronic acid aqueous solution with a concentration of 1.5 mg / mL. Stir and react at 20°C for 28 h. Then centrifuge at 14000 rpm for 20 min and wash with deionized water 3 times. Disperse the obtained product NMOF-Ls in 2 mL of deionized water for later use.

[0098] Example 15

[0099] Step 1: Dissolve 128 mg of Cu(NO3)2·3H2O, 64 mg of ZrOCl2·8H2O, 32 mg of 3,5-dimethyl-4-pyrazolecarboxylic acid, and 30 μL of CF3COOH in 20 mL of DMF by sonication. Then transfer the solution to a 50 mL hydrothermal reactor and react at 120 °C for 6 h. After naturally cooling to room temperature, centrifuge at 12000 rpm for 5 min and wash three times with DMF. Disperse the resulting 50 mg of NMOF in 5 mL of deionized water for later use.

[0100] Step 2: Take 1 mL of the above-obtained NMOF aqueous solution and mix it with 1 mL of hyaluronic acid aqueous solution with a concentration of 2.5 mg / mL. Stir and react at 40°C for 8 h. Then centrifuge at 14000 rpm for 20 min and wash with deionized water 3 times. Disperse the obtained product NMOF-Ls in 2 mL of deionized water for later use.

[0101] Example 16

[0102] Step 1: Dissolve 124 mg of Cu(NO3)2, 42.5 mg of zirconium oxide, 48 mg of 5-phenyl-1H-pyrazole-4-carboxylic acid, and 30 μL of CF3COOH in 20 mL of DMF by sonication. Then transfer the solution to a 50 mL hydrothermal reactor and react at 100 °C for 6 h. After cooling naturally to room temperature, centrifuge at 12000 rpm for 5 min and wash three times with DMF. Disperse the resulting 50 mg of NMOF in 5 mL of deionized water for later use.

[0103] Step 2: Take 1 mL of the above-obtained NMOF aqueous solution and mix it with 1 mL of L-serine aqueous solution with a concentration of 0.5 mg / mL. Stir and react at 30°C for 20 h. Then centrifuge at 14000 rpm for 20 min and wash with deionized water 3 times. Disperse the obtained product NMOF-Ls in 2 mL of deionized water for later use.

[0104] Comparative Example 1

[0105] 124 mg of Cu(NO3)2·3H2O, 42.5 mg of ZrOCl2·8H2O, 32.5 mg of 1H-pyrazole-4-carboxylic acid (H2PyC), and 120 μL of trifluoroacetic acid (CF3COOH) were ultrasonically dissolved in 20 mL of DMF. The solution was then transferred to a 50 mL polytetrafluoroethylene autoclave and reacted at 100 °C for 8 h. After naturally cooling to room temperature, the solution was centrifuged and washed with DMF and ethanol to obtain defect-free MOF.

[0106] The morphology of the NMOF prepared in Example 12 was characterized by transmission electron microscopy, and the results are as follows: Figure 1 As shown, NMOF consists of irregular particles with a particle size of approximately 50 nm.

[0107] Example 12: The crystal structure of the prepared NMOF was characterized by powder X-ray diffraction, and the results are as follows: Figure 2 As shown, the X-ray diffraction peaks of NMOF are not sharp and have bulging peaks, indicating that the prepared NMOF has an amorphous structure.

[0108] Example 12: The specific surface area and pore size of the prepared NMOF were characterized by a specific surface area and porosity analyzer, and the results are as follows: Figure 3 As shown, compared with MOF, NMOF has increased specific surface area and pore size.

[0109] Example 12: The optical properties of the prepared NMOF-Ls were characterized by UV-Vis absorption spectroscopy, and the results are as follows: Figure 4 As shown, the absorption peak of NMOF is not obvious after modification with L-serine, but it has a significant absorption peak after modification with L-serine. This is because the modification with L-serine changes the chemical environment of NMOF, which in turn changes its optical properties. This provides a reliable basis for the successful preparation of NMOF-Ls.

[0110] The morphology of the NMOF-Ls prepared in this embodiment was characterized by transmission electron microscopy, and the results are as follows: Figure 5 As shown, NMOF-Ls are also irregular particles with a particle size of approximately 50 nm.

[0111] Example 12: The valence state of Cu in NMOF-Ls was characterized by X-ray photoelectron spectroscopy, and the results are as follows: Figure 6 As shown, NMOF contains Cu 2+ There is also Cu + In the NMOF prepared by this invention, copper ions are more likely to contact and react with the catalytic substrate. 2+ Cu + ROS can be converted into oxygen and water through redox reactions, ultimately achieving antioxidant properties.

[0112] Table 1 Results of varying NMOF particle size with different DMF addition levels

[0113] DMF (mL) Particle size (nm) Example 1 10 508 Example 2 15 320 Example 3 20 316 Example 4 25 372 Example 5 30 426

[0114] The particle sizes of the NMOFs prepared in Examples 1 to 5 of this invention, after varying the amount of DMF added, are shown in Table 1. As can be seen from Table 1, with increasing DMF content, the particle size of the prepared NMOFs exhibits a trend of first decreasing and then increasing. When the amount of DMF is 20 mL, the particle size of the prepared NMOFs is the smallest, at 316 nm. This is because, within a certain range, reducing the volume of DMF increases the concentration of the MOF precursor, thereby forming a large number of crystal nuclei in the initial short period of the reaction, thus reducing the particle size.

[0115] Table 2 Results of NMOF particle size variation with varying amounts of competing ligands

[0116] <![CDATA[C2HF3O2(μL)]]> Particle size (nm) Example 6 0 126 Example 7 30 101 Example 8 60 201 Example 9 90 405

[0117] The particle sizes of the NMOFs prepared in Examples 6 to 9 of this invention, after varying the amount of C2HF3O2 added, are shown in Table 2. Table 2 shows that as the amount of C2HF3O2 increases, the particle size of the prepared NMOFs first decreases and then increases. When the amount of C2HF3O2 is 30 μL, the particle size of the prepared NMOF is the smallest, at 101 nm. This indicates that within a certain range, an increase in competing ligands slows down the growth of crystal nuclei during the "Wald ripening" stage, resulting in a product with higher crystallinity and larger particle size.

[0118] Table 3. Results of NMOF particle size variation with different organic ligand addition levels.

[0119] <![CDATA[H2PyC(mg)]]> Particle size (nm) Example 10 20 103 Example 11 32 99 Example 12 48 85 Example 13 64 92

[0120] The particle sizes of the NMOFs prepared in Examples 10-13 of this invention, after varying the amount of H2PyC added, are shown in Table 2. Table 2 shows that as the amount of H2PyC increases, the particle size of the prepared NMOFs first decreases and then increases. When the amount of H2PyC is 48 mg, the particle size of the prepared NMOF is the smallest, at 85 nm. This indicates that within a certain range, the amount of organic ligand added will significantly increase the rate and number of NMOF nucleation, thereby reducing the NMOF particle size. It should be noted that the particle size of the NMOF prepared in Example 12, measured using a particle size analyzer, is larger than the particle size measured using TEM. This difference is usually due to the hydration layer of the particles in the solution. Specifically, dynamic light scattering (DLS) is used to measure the hydrated particle size. This method considers the movement of particles in the solution, including the hydration layer on the particle surface; therefore, the measured hydrated particle size is usually larger than the size of the dry sample directly observed by TEM.

[0121] The performance of the NMOF prepared in Example 12 is characterized below.

[0122] Example 12: The CAT and SOD antioxidant properties of NMOF-Ls were determined by dissolved oxygen meter and SOD kit, respectively. The results are as follows: Figure 7 As shown, the SOD and CAT performance of defective NMOFs were effectively improved, indicating that reducing the size and increasing defects will increase the specific surface area and pore size of NMOFs, thereby exposing more active sites and ultimately improving their catalytic performance.

[0123] Example 12: Cellular uptake of NMOF-Ls was observed using a biological transmission electron microscope. The results are as follows: Figure 8 As shown, NMOF-Ls are located in the cytoplasm of HK-2 cells, indicating that the final prepared NMOF-Ls can be effectively taken up by HK-2 cells, which lays the foundation for its intracellular ROS clearance.

[0124] Example 12: The cell targeting ability of NMOF-Ls was observed using a laser scanning confocal microscope. The results are as follows: Figure 9 As shown. To facilitate observation of the cell entry of different materials, rhodamine B, which has fluorescent properties, was loaded into the prepared materials. Furthermore, the Kim-1 antibody used in the experiment could effectively bind to Kim-1 overexpressed in cisplatin-induced HK-2 cells. When NMOF was not modified with Ls, the addition of the Kim-1 inhibitor had a negligible effect on the targeting of NMOF. However, when NMOF was modified with Ls, the group without the Kim-1 inhibitor showed superior fluorescence compared to the group with the inhibitor. These results indicate that the modification of NMOF with Ls makes it easier for it to bind to and enter the cells with Kim-1 overexpressed on the surface of HK-2 cells, thus achieving targeting of HK-2 cells.

[0125] Example 12: The intracellular ROS scavenging performance of NMOF-Ls was observed using laser scanning confocal microscopy. The results are as follows: Figure 10 As shown, cisplatin induces ROS production in cells, resulting in a strong green fluorescence signal from the ROS probe DCFH-DA. It was observed that all prepared materials exhibited a certain degree of ROS scavenging ability within cells. NMOF showed a particularly significant ROS scavenging ability compared to MOF, due to its smaller size and superior SOD-CAT activity, making it more easily taken up by HK-2 cells and effectively clearing ROS. NMOF-Ls demonstrated superior ROS scavenging performance compared to NMOF, because Ls increased the targeting and uptake of NMOF on HK-2 cells, ultimately achieving a superior ROS scavenging effect.

[0126] Example 12: The therapeutic effect of NMOF-Ls in mice was characterized by measuring creatinine and blood urea nitrogen in mice. The results are as follows: Figure 11 As shown, mice treated with NMOF-Ls exhibited creatinine and blood urea nitrogen levels closest to normal, thus demonstrating the most outstanding performance in treating acute kidney injury (AKI). This is because, after intravenous injection via the tail vein, NMOF-Ls target the overexpression of Kim-1 on the surface of HK-2 cells, preferentially accumulating and entering the cells in the kidney region. Simultaneously, due to its excellent ROS scavenging properties, it efficiently eliminates ROS in HK-2 cells, ultimately restoring kidney function to normal.

[0127] In summary, the antioxidant properties of the defective NMOF constructed based on defect engineering were effectively enhanced. Post-modification with L-serine also enabled it to target the kidneys. Ultimately, the prepared NMOF-Ls achieved effective treatment of AKI in a mouse model through kidney targeting and antioxidant properties.

[0128] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention. Clearly, those skilled in the art can make various alterations and modifications to the invention without departing from its spirit and scope. Thus, if these modifications and modifications of the invention fall within the scope of the claims and their equivalents, the invention is also intended to include these modifications and modifications.

Claims

1. The application of a defective metal-organic framework antioxidant nanozyme in the preparation of drugs for treating ROS-mediated kidney diseases, characterized in that, Defective metal-organic framework antioxidant nanozymes were prepared according to the following steps: A copper source, a zirconium source, and the pyrazole- and carboxyl-containing organic ligand 1H-pyrazole-4-carboxylic acid were dissolved in the organic solvent N,N-dimethylformamide. Then, the competing ligand CF3COOH was added, and after thorough mixing, a solvothermal reaction was carried out at 80℃~120℃. During the reaction, 1H-pyrazole-4-carboxylic acid controlled the rate and number of nuclei, CF3COOH controlled the crystal growth rate and crystallinity, and N,N-dimethylformamide controlled the number of crystal nuclei. The particle size and defects were adjusted by the combination of 1H-pyrazole-4-carboxylic acid, CF3COOH, and N,N-dimethylformamide to prepare a defective metal-organic framework. The ratio of 1H-pyrazole-4-carboxylic acid to CF3COOH was 20mg~64mg:30μL; the ratio of 1H-pyrazole-4-carboxylic acid to N,N-dimethylformamide was 20mg~64mg:20mL. After uniformly mixing the aqueous solution of the defective metal-organic framework and the solution of the kidney-targeting molecule, the antioxidant nanozyme of the defective metal-organic framework was obtained through coordination and electrostatic adsorption. The ROS-mediated kidney disease is cisplatin-induced acute kidney injury. The kidney-targeting molecule is L-serine; Defective metal-organic framework antioxidant nanozymes achieve targeted targeting of HK-2 cells and eliminate ROS in HK-2 cells.

2. The application of the defective metal-organic framework antioxidant nanozyme according to claim 1 in the preparation of drugs for treating ROS-mediated kidney diseases, characterized in that, The ratio of 1H-pyrazole-4-carboxylic acid to N,N-dimethylformamide was 48 mg: 20 mL.

3. The application of the defective metal-organic framework antioxidant nanozyme according to claim 1 in the preparation of drugs for treating ROS-mediated kidney diseases, characterized in that, The ratio of 1H-pyrazole-4-carboxylic acid to CF3COOH was 48 mg: 30 μL.

4. The application of the defective metal-organic framework antioxidant nanozyme according to claim 1 in the preparation of drugs for treating ROS-mediated kidney diseases, characterized in that, The mass ratio of 1H-pyrazole-4-carboxylic acid to copper source is 1:2~6; the mass ratio of zirconium source to copper source is 1:2~4.

5. The application of the defective metal-organic framework antioxidant nanozyme according to claim 1 in the preparation of drugs for treating ROS-mediated kidney diseases, characterized in that, The reaction time for the solvothermal reaction is 6 to 10 hours.

6. The application of the defective metal-organic framework antioxidant nanozyme according to claim 1 in the preparation of drugs for treating ROS-mediated kidney diseases, characterized in that, The mass ratio of the defective metal-organic framework to the kidney-targeting molecule is 10:0.1~0.

5.

7. The application of the defective metal-organic framework antioxidant nanozyme according to claim 1 in the preparation of drugs for treating ROS-mediated kidney diseases, characterized in that, The reaction temperature for coordination and electrostatic adsorption is 20℃~40℃, and the reaction time is 8h~28h.

8. The application of the defective metal-organic framework antioxidant nanozyme according to claim 1 in the preparation of drugs for treating ROS-mediated kidney diseases, characterized in that, The copper source is copper nitrate or copper nitrate hydrate; The zirconium source is zirconium oxychloride octahydrate or zirconium oxide; The organic ligand containing pyrazole and carboxyl groups is 1H-pyrazole-4-carboxylic acid; The competing ligand is trifluoroacetic acid; The kidney-targeting molecule is L-serine.

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