Defective metal organic framework antioxidant nano-enzyme as well as preparation method and application thereof

Abstract

The invention discloses a defective metal organic framework antioxidant nano-enzyme as well as a preparation method and application thereof, and belongs to the technical field of nano-biology. The preparation method comprises the following steps: dissolving a copper source, a zirconium source and an organic ligand containing pyrazole and carboxyl in an organic solvent, then adding a competitive ligand, uniformly mixing, and carrying out solvothermal reaction at 80-120 DEG C to prepare the defective metal organic framework; after a defective metal organic framework aqueous solution and a kidney targeting molecular solution are uniformly mixed, the defective metal organic framework anti-oxidation nano-enzyme is obtained through coordination and electrostatic adsorption effects. The organic ligand containing pyrazole and carboxyl, the competitive ligand containing carboxylic acid and the organic solvent are matched to adjust the particle size and the defect, the metal organic framework with the small particle size and the defect is prepared, the antioxidant activity is improved, kidney targeting molecules are loaded to obtain the defect metal organic framework antioxidant nano-enzyme, and the kidney targeting effect is achieved.

Description

Technical Field

[0001] The present invention relates to the field of nanobiotechnology, and more specifically to a defective metal organic framework antioxidant nanozyme and a preparation method and application thereof. Background Art

[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 kidney is the main factor inducing AKI. So far, there are no effective preventive and therapeutic drugs in clinical practice except for supportive treatments such as water supplementation, diuresis and dialysis.

[0003] With the continuous development and progress of nanotechnology, the emergence of antioxidant nanozymes has undoubtedly provided possibilities for the prevention and treatment of AKI. Nanozymes are a type of nanomaterials with enzyme-like catalytic activity, which can catalyze enzyme substrates, produce catalytic reactions similar to natural enzymes, and have characteristics such as enzymatic reaction kinetics. They belong to a new type of mimetic enzymes. Similar to natural enzymes, nanozymes can efficiently catalyze enzyme substrates under mild physiological conditions. They have the advantages of adjustable catalytic activity, high stability under harsh conditions, flexibility in composition and structural design, and excellent biocompatibility.

[0004] The nanozymes with antioxidant activity reported so far mainly include cerium dioxide, manganese oxide, Prussian blue, carbon nanoparticles and metal organic frameworks (MOFs). Among them, MOFs antioxidant nanozymes have become one of the best choices for the treatment of AKI due to their high specific surface area, high porosity, adjustable structure, easy functionalization and good biocompatibility. However, the application of MOFs in the treatment of AKI is severely limited by the factors such as the large size, limited antioxidant activity and no kidney targeting of the MOFs antioxidant nanozymes reported so far. Summary of the invention

[0005] In response to the above problems, the present invention provides a defective metal-organic framework antioxidant nanozyme and a preparation method and application thereof. The particle size and defects are adjusted by combining organic ligands containing pyrazole and carboxyl groups, competitive ligands containing carboxylic acids and organic solvents to prepare a metal-organic framework with small particle size and defects, thereby improving the antioxidant activity, and loading kidney-targeting molecules to obtain a defective metal-organic framework antioxidant nanozyme, thereby achieving a targeted effect on the kidney.

[0006] The first object of the present invention is to provide a method for preparing a defective metal organic framework antioxidant nanozyme, comprising the following steps:

[0007] A copper source, a zirconium source and an organic ligand containing pyrazole and carboxyl are dissolved in an organic solvent, and then a competitive ligand is added, mixed evenly, and then a solvent thermal reaction is carried out at 80°C to 120°C, wherein the organic ligand containing pyrazole and carboxyl regulates the speed and number of nucleation, the competitive ligand containing carboxylic acid regulates the crystal growth speed and crystallinity, and the organic solvent regulates the number of crystal nuclei, and the particle size and defects are adjusted by combining the organic ligand containing pyrazole and carboxyl, the competitive ligand containing carboxylic acid and the organic solvent to prepare a defective metal organic framework; the ratio of the organic ligand containing pyrazole and carboxyl to the competitive ligand containing carboxylic acid is 20mg to 64mg: 0μL to 120μL; the ratio of the organic ligand containing pyrazole and carboxyl to the organic solvent is 20mg to 64mg: 10mL to 30mL;

[0008] For example, the ratio of the organic ligand containing pyrazole and carboxyl groups to the competitive ligand containing carboxylic acid 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 the organic ligand containing pyrazole and carboxyl groups to the organic solvent is 20 mg:10 mL, 20 mg:20 mL, 20 mg:30 mL, 30 mg:10 mL, 30 mg:20 mL, 40 mg:10 mL, 40 mg:30 mL, 50 mg:10 mL, 50 mg:20 mL, 50 mg:30 mL, etc.; but is not limited to the listed values, and other values ​​not listed within the above numerical range are also applicable.

[0010] After the defective metal organic framework aqueous solution and the kidney-targeting molecule solution are evenly mixed, the defective metal organic framework antioxidant nanozyme is 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 competitive 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 to the copper source is 1:2-6; the mass ratio of the zirconium source to the copper source is 1:2-4.

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

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

[0016] In a preferred embodiment of the present invention, the reaction time of the solvothermal reaction is 6 h to 10 h. For example, the reaction time of the solvothermal reaction is 6 h, 7 h, 8 h, 9 h, 10 h, etc.; but it is not limited to the listed values, and other unlisted values ​​within the above numerical 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 10:0.1-0.5. 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 numerical range are also applicable.

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

[0019] For example, the reaction temperature of the coordination and electrostatic adsorption reactions is 20°C, 25°C, 30°C, 35°C, 40°C, etc., and the reaction time is 8h, 12h, 16h, 20h, 24h, 28h, etc.; but it is not limited to the listed values, and other unlisted values ​​within the above numerical 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] The organic ligand containing pyrazole and carboxyl groups is 1H-pyrazole-4-carboxylic acid, 3-methylpyrazole-4-carboxylic acid, 3,5-dimethyl-4-pyrazolecarboxylic acid or 5-phenyl-1H-pyrazole-4-carboxylic acid;

[0023] The competing ligand was trifluoroacetic acid or acetic acid;

[0024] The kidney-targeting molecule is L-serine or hyaluronic acid.

[0025] The second object of the present invention is to provide a defective metal organic framework antioxidant nanozyme prepared by the above preparation method.

[0026] The third object of the present invention is to provide the use of the above-mentioned defective metal organic framework antioxidant nanozyme 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) The present invention adopts a solvothermal method to prepare a defective metal organic framework with small size and high defects by regulating the content of organic ligands containing pyrazole and carboxyl groups, competitive ligands containing carboxylic acids, and the volume of the reaction solvent. The defective metal organic framework has a larger specific surface area and pore size due to the reduced particle size and increased defects, thereby exposing more catalytic active sites and improving its antioxidant performance. In addition, the kidney-targeting molecule L-serine is modified on the surface of the defective metal organic framework enzyme by a post-modification method to obtain a defective metal organic framework antioxidant nanozyme with kidney-targeting function, ultimately achieving effective treatment of ROS-mediated kidney diseases.

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

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

[0031] (4) The final product NMOF-Ls obtained in the present invention can achieve the therapeutic effect of AKI caused by cisplatin by actively targeting the kidneys. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

[0035] Figure 4 : These are the UV-visible absorption spectra of different materials in Example 12.

[0036] Figure 5 This is a transmission electron microscope image of NMOF-Ls in Example 12.

[0037] Figure 6 This is the X-ray photoelectron spectrum of NMOF-Ls in Example 12.

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

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

[0040] Fig. 9 Laser scanning confocal microscopy images of the materials prepared in Comparative Example 1(a) and Example 12(b) achieving targeting function on HK-2 cells.

[0041] Fig.10 These are the characterization results of the antioxidant properties of different materials in Example 12.

[0042] Fig.11 These are the results of creatinine and urea nitrogen levels in AKI mice after they were treated with different materials in Example 12. DETAILED DESCRIPTION

[0043] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0044] A method for preparing a defective metal organic framework antioxidant nanozyme, characterized in that it comprises the following steps:

[0045] The copper source, the zirconium source and the organic ligand containing pyrazole and carboxyl are dissolved in an organic solvent, and then the competitive ligand is added, mixed evenly, and then a solvent thermal reaction is carried out at 80°C to 120°C to prepare a defective metal organic framework; the ratio of the organic ligand containing pyrazole and carboxyl to the competitive ligand containing carboxyl is 20 to 64 mg: 0 μL to 120 μL; the ratio of the organic ligand containing pyrazole and carboxyl to the organic solvent is 20 mg to 64 mg: 10 mL to 20 mL;

[0046] After the defective metal organic framework aqueous solution and the kidney targeting molecule aqueous solution are evenly mixed, the defective metal organic framework antioxidant nanozyme with kidney targeting function is obtained through coordination and electrostatic adsorption.

[0047] At the beginning of the reaction, the amount of organic ligands increases appropriately, which greatly increases the speed and number of NMOF nucleation, which plays a key role in reducing the particle size of NMOF. In addition, during the reaction, the competing ligands regulate the crystal growth rate and crystallinity of NMOF. If the amount of competing ligands decreases appropriately, the nucleus growth rate of NMOF will accelerate, making it easier to form a short-range disordered structure, which in turn increases the defects of NMOF.

[0048] Within a certain range, the reduction in the volume of the organic solvent will increase the concentration of the MOFs precursor, which will form a large number of crystal nuclei in a short period of 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 is 10:0.1-0.5. The reaction temperature of the coordination and electrostatic adsorption reaction is 20°C-40°C, and the reaction time is 8h-28h.

[0050] It is understandable that during the preparation process, the mass ratio of the defective metal organic framework and the kidney targeting molecule and the reaction parameters need to be carried out according to the above conditions. If the above parameter range is exceeded, the amount of the kidney targeting molecule is insufficient or the reaction time is too short, which will lead to a small content of the kidney targeting molecule on the NMOF surface, thereby affecting its targeting function to 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-pyrazolecarboxylic acid or 5-phenyl-1H-pyrazole-4-carboxylic acid;

[0053] The competing ligand was trifluoroacetic acid (CF3COOH) or acetic acid;

[0054] The kidney-targeting molecule is L-serine or hyaluronic acid.

[0055] The present invention first prepares a defective metal organic framework, denoted as NMOF, which is an amorphous structure with a size of about 20-100nm, and its specific surface area and pore size are increased. The presence of defective structures in NMOF and the increase in specific surface area and pore size increase the number of active sites for its catalysis, and ultimately effectively improve its antioxidant properties. Then, based on NMOF, NMOF is compounded with kidney-targeting molecules, and the obtained defective metal organic framework antioxidant nanozyme can achieve kidney-targeting function through targeting of kidney injury markers, effectively improving its therapeutic effect. Defective metal organic framework antioxidant nanozymes 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 by ultrasonic. Then transfer them to a 50 mL hydrothermal reactor, react at 100 °C for 8 h, cool naturally to room temperature, centrifuge at 12000 rpm for 5 min, wash three times with DMF, and then disperse the resulting 50 mg of NMOF in 5 mL of deionized water for later use.

[0058] Step 2: Take 1 mL of the aqueous solution of the NMOF obtained above and mix it with 1 mL of an aqueous solution of L-serine with a concentration of 0.5 mg / mL, stir and react at 25°C for 12 hours, then centrifuge at 14000 rpm for 20 minutes, and wash it with deionized water three times before dispersing the resulting 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 by ultrasonic. Then transfer them to a 50 mL hydrothermal reactor, react at 100 °C for 8 h, cool naturally to room temperature, centrifuge at 12000 rpm for 5 min, wash three times with DMF, and then disperse the resulting 50 mg of NMOF in 5 mL of deionized water for later use.

[0061] Step 2: Take 1 mL of the aqueous solution of the NMOF obtained above and mix it with 1 mL of an aqueous solution of L-serine with a concentration of 0.5 mg / mL, stir and react at 25°C for 12 hours, then centrifuge at 14000 rpm for 20 minutes, and wash it with deionized water three times before dispersing the resulting 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 by ultrasonic. Then transfer them to a 50 mL hydrothermal reactor, react at 100 °C for 8 h, cool naturally to room temperature, centrifuge at 12000 rpm for 5 min, wash three times with DMF, and then disperse the resulting 50 mg of NMOF in 5 mL of deionized water for later use.

[0064] Step 2: Take 1 mL of the aqueous solution of the NMOF obtained above and mix it with 1 mL of an aqueous solution of L-serine with a concentration of 0.5 mg / mL, stir and react at 25°C for 12 hours, then centrifuge at 14000 rpm for 20 minutes, and wash it with deionized water three times before dispersing the resulting 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 by ultrasonic. Then transfer them to a 50 mL hydrothermal reactor, react at 100 °C for 8 h, cool naturally to room temperature, centrifuge at 12000 rpm for 5 min, wash three times with DMF, and then disperse the resulting 50 mg of NMOF in 5 mL of deionized water for later use.

[0067] Step 2: Take 1 mL of the aqueous solution of the NMOF obtained above and mix it with 1 mL of an aqueous solution of L-serine with a concentration of 0.5 mg / mL, stir and react at 25°C for 12 hours, then centrifuge at 14000 rpm for 20 minutes, and wash it with deionized water three times before dispersing the resulting 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 by ultrasonic. Then transfer them to a 50 mL hydrothermal reactor, react at 100 °C for 8 h, cool naturally to room temperature, centrifuge at 12000 rpm for 5 min, wash three times with DMF, and then disperse the resulting 50 mg of NMOF in 5 mL of deionized water for later use.

[0070] Step 2: Take 1 mL of the aqueous solution of the NMOF obtained above and mix it with 1 mL of an aqueous solution of L-serine with a concentration of 0.5 mg / mL, stir and react at 25°C for 12 hours, then centrifuge at 14000 rpm for 20 minutes, and wash it with deionized water three times before dispersing the resulting 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 by ultrasonic. Then transfer them to a 50 mL hydrothermal reactor, react at 100 °C for 8 h, cool naturally to room temperature, centrifuge at 12000 rpm for 5 min, wash three times with DMF, and then disperse the resulting 50 mg of NMOF in 5 mL of deionized water for later use.

[0073] Step 2: Take 1 mL of the aqueous solution of the NMOF obtained above and mix it with 1 mL of an aqueous solution of L-serine with a concentration of 0.5 mg / mL, stir and react at 25°C for 12 hours, then centrifuge at 14000 rpm for 20 minutes, and wash it with deionized water three times before dispersing the resulting 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 by ultrasonic. Then transfer them to a 50 mL hydrothermal reactor, react at 100 °C for 8 h, cool naturally to room temperature, centrifuge at 12000 rpm for 5 min, wash three times with DMF, and then disperse the resulting 50 mg of NMOF in 5 mL of deionized water for later use.

[0076] Step 2: Take 1 mL of the aqueous solution of the NMOF obtained above and mix it with 1 mL of an aqueous solution of L-serine with a concentration of 0.5 mg / mL, stir and react at 25°C for 12 hours, then centrifuge at 14000 rpm for 20 minutes, and wash it with deionized water three times before dispersing the resulting 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 by ultrasonic. Then transfer them to a 50 mL hydrothermal reactor, react at 100 °C for 8 h, cool naturally to room temperature, centrifuge at 12000 rpm for 5 min, wash three times with DMF, and then disperse the resulting 50 mg of NMOF in 5 mL of deionized water for later use.

[0079] Step 2: Take 1 mL of the aqueous solution of the NMOF obtained above and mix it with 1 mL of an aqueous solution of L-serine with a concentration of 0.5 mg / mL, stir and react at 25°C for 12 hours, then centrifuge at 14000 rpm for 20 minutes, and wash it with deionized water three times before dispersing the resulting 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 by ultrasonic. Then transfer them to a 50 mL hydrothermal reactor, react at 100 °C for 8 h, cool naturally to room temperature, centrifuge at 12000 rpm for 5 min, wash three times with DMF, and then disperse the resulting 50 mg of NMOF in 5 mL of deionized water for later use.

[0082] Step 2: Take 1 mL of the aqueous solution of the NMOF obtained above and mix it with 1 mL of an aqueous solution of L-serine with a concentration of 0.5 mg / mL, stir and react at 25°C for 12 hours, then centrifuge at 14000 rpm for 20 minutes, and wash it with deionized water three times before dispersing the resulting 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 by ultrasonic. Then transfer them to a 50 mL hydrothermal reactor, react at 100 °C for 8 h, cool naturally to room temperature, centrifuge at 12000 rpm for 5 min, wash three times with DMF, and then disperse the resulting 50 mg of NMOF in 5 mL of deionized water for later use.

[0085] Step 2: Take 1 mL of the aqueous solution of the NMOF obtained above and mix it with 1 mL of an aqueous solution of L-serine with a concentration of 0.5 mg / mL, stir and react at 25°C for 12 hours, then centrifuge at 14000 rpm for 20 minutes, and wash it with deionized water three times before dispersing the resulting product NMOF-Ls in 2 mL of deionized water for later use.

[0086] Embodiment 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 by ultrasonic. Then transfer them to a 50 mL hydrothermal reactor, react at 100 °C for 8 h, cool naturally to room temperature, centrifuge at 12000 rpm for 5 min, wash three times with DMF, and then disperse the resulting 50 mg of NMOF in 5 mL of deionized water for later use.

[0088] Step 2: Take 1 mL of the aqueous solution of the NMOF obtained above and mix it with 1 mL of an aqueous solution of L-serine (Ls) with a concentration of 0.5 mg / mL, stir the reaction at 25°C for 12 hours, then centrifuge at 14000 rpm for 20 minutes, and wash it with deionized water three times before dispersing the resulting 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 by ultrasonic. Then transfer them to a 50 mL hydrothermal reactor, react at 100 °C for 8 h, cool naturally to room temperature, centrifuge at 12000 rpm for 5 min, wash three times with DMF, and then disperse the resulting 50 mg of NMOF in 5 mL of deionized water for later use.

[0091] Step 2: Take 1 mL of the aqueous solution of the NMOF obtained above and mix it with 1 mL of an aqueous solution of L-serine with a concentration of 0.5 mg / mL, stir and react at 25°C for 12 hours, then centrifuge at 14000 rpm for 20 minutes, and wash it with deionized water three times before dispersing the resulting product NMOF-Ls in 2 mL of deionized water for later use.

[0092] Embodiment 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 by ultrasonic. Then transfer them to a 50 mL hydrothermal reactor, react at 100 °C for 8 h, cool naturally to room temperature, centrifuge at 12000 rpm for 5 min, wash three times with DMF, and then disperse the resulting 50 mg of NMOF in 5 mL of deionized water for later use.

[0094] Step 2: Take 1 mL of the aqueous solution of the NMOF obtained above and mix it with 1 mL of an aqueous solution of L-serine with a concentration of 0.5 mg / mL, stir and react at 25°C for 12 hours, then centrifuge at 14000 rpm for 20 minutes, and wash it with deionized water three times before dispersing the resulting product NMOF-Ls in 2 mL of deionized water for later use.

[0095] Embodiment 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 ultrasonication, then transfer them to a 50 mL hydrothermal reactor, react at 80°C for 10 h, cool naturally to room temperature, centrifuge at 12000 rpm for 5 min, wash three times with DMF, and then disperse the resulting 50 mg of NMOF in 5 mL of deionized water for later use.

[0097] Step 2: Take 1 mL of the aqueous solution of the NMOF obtained above and mix it with 1 mL of an aqueous solution of hyaluronic acid with a concentration of 1.5 mg / mL, stir and react at 20°C for 28 hours, then centrifuge at 14000 rpm for 20 minutes, wash it with deionized water three times, and then disperse the resulting product NMOF-Ls in 2 mL of deionized water for use.

[0098] Embodiment 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 ultrasonic. Then transfer them to a 50 mL hydrothermal reactor, react at 120 °C for 6 h, cool naturally to room temperature, centrifuge at 12000 rpm for 5 min, wash three times with DMF, and then disperse the resulting 50 mg of NMOF in 5 mL of deionized water for later use.

[0100] Step 2: Take 1 mL of the aqueous solution of the NMOF obtained above and mix it with 1 mL of an aqueous solution of hyaluronic acid with a concentration of 2.5 mg / mL, stir and react at 40°C for 8 hours, then centrifuge at 14000 rpm for 20 minutes, wash it with deionized water three times, and then disperse the resulting product NMOF-Ls in 2 mL of deionized water for 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 ultrasonication, and then transfer them to a 50 mL hydrothermal reactor, react at 100 °C for 6 h, cool naturally to room temperature, centrifuge at 12000 rpm for 5 min, wash 3 times with DMF, and then disperse the resulting 50 mg of NMOF in 5 mL of deionized water for use.

[0103] Step 2: Take 1 mL of the aqueous solution of the NMOF obtained above and mix it with 1 mL of an aqueous solution of L-serine with a concentration of 0.5 mg / mL, stir and react at 30°C for 20 hours, then centrifuge at 14000 rpm for 20 minutes, and wash it with deionized water three times before dispersing the resulting 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 and then transferred to a 50 mL polytetrafluoroethylene autoclave, reacted at 100 °C for 8 h, naturally cooled to room temperature, centrifuged, and washed with DMF and ethanol to obtain a defect-free MOF.

[0106] Example 12 The morphology of the prepared NMOF was characterized by transmission electron microscopy. Figure 1 As shown, NMOF is irregular particles with a particle size of about 50 nm.

[0107] Example 12 The crystal structure of the prepared NMOF was characterized by powder X-ray diffractometer. Figure 2 As shown, the X-ray diffraction peak of NMOF is not sharp, and there are peaks with bulge morphology, which indicates 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 pore size analyzer. The results are as follows: Figure 3 As shown in Figure 2, the specific surface area and pore size of NMOF are increased compared with MOF.

[0109] Example 12 The optical properties of NMOF-Ls prepared were characterized by UV-visible absorption spectroscopy. Figure 4 As shown, it can be seen that after NMOF is modified with L-serine, its absorption peak is not obvious, but after modified with L-serine, it has an obvious absorption peak. This is because the modification of L-serine causes the chemical environment of NMOF to change, which in turn causes the optical properties to change. This provides a reliable basis for the successful preparation of NMOF-Ls.

[0110] The morphology of NMOF-Ls prepared in this example was characterized by transmission electron microscopy. Figure 5 As shown, NMOF-Ls are also irregular particles with a particle size of about 50 nm.

[0111] Example 12 The valence state of Cu element in the prepared NMOF-Ls was characterized by X-ray photoelectron spectroscopy. Figure 6 As shown, NMOF contains both Cu 2+ , there is also Cu + The copper ions in the NMOF prepared by the present invention 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 Particle size results of NMOF with different DMF addition amounts

[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] After changing the amount of DMF added in Examples 1 to 5 of the present invention, the particle size of the prepared NMOF is shown in Table 1. It can be seen from Table 1 that with the increase in the amount of DMF used, the particle size of the prepared NMOF shows a trend of first decreasing and then increasing. When the amount of DMF is 20 mL, the particle size of the prepared NMOF is the smallest, which is 316 nm. This is because within a certain range, the reduction in the volume of DMF increases the concentration of the MOFs precursor, thereby forming a large number of crystal nuclei in a short time at the beginning of the reaction and reducing the particle size.

[0115] Table 2 Particle size results of NMOF with changing the amount of competitive ligand added

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

[0117] After changing the amount of C2HF3O2 added in Examples 6 to 9 of the present invention, the particle size of the prepared NMOF is shown in Table 2. It can be seen from Table 2 that with the increase in the amount of C2HF3O2, the particle size of the prepared NMOF shows a trend of first decreasing and then increasing. When the amount of C2HF3O2 is 30 μL, the particle size of the prepared NMOF is the smallest, which is 101 nm. This shows that within a certain range, the increase in the number of competing ligands will cause the crystal nucleus to grow slower in the "Wald ripening" stage, and the final product will have a higher degree of crystallinity and a larger particle size.

[0118] Table 3 Particle size results of NMOF with changing amount of organic ligand added

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

[0120] After changing the amount of H2PyC added in Examples 10 to 13 of the present invention, the particle size of the prepared NMOF is shown in Table 2. It can be seen from Table 2 that with the increase in the amount of H2PyC, the particle size of the prepared NMOF shows a trend of first decreasing and then increasing. When the amount of H2PyC is 48 mg, the particle size of the prepared NMOF is the smallest, which is 85 nm. This shows that within a certain range, the amount of organic ligand added will greatly increase the speed and number of NMOF nucleation, thereby reducing the particle size of NMOF. It should be noted that the particle size of the NMOF prepared in Example 12 tested by a particle size analyzer is larger than the particle size tested by TEM, because the difference is usually due to the hydration layer of the particles in the solution. Specifically, dynamic light scattering technology is used to measure the hydrated particle size. This method takes into account the movement of particles in the solution, including the hydration layer on the surface of the particles, so 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 as follows.

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

[0123] Example 12 The cellular uptake of NMOF-Ls was observed by biological transmission electron microscopy. 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 removal of intracellular ROS.

[0124] Example 12 The cell targeting of NMOF-Ls was observed by laser scanning confocal microscopy. Fig. 9 As shown. In order to facilitate the observation of different materials entering cells, rhodamine B with fluorescent properties was loaded into the prepared material. In addition, the Kim-1 antibody used in the experiment can effectively bind to Kim-1 overexpressed in HK-2 cells induced by cisplatin. When NMOF is not modified with Ls, the effect of whether the Kim-1 inhibitor is added on the targeting of NMOF is negligible. After NMOF is modified with Ls, the group without the addition of Kim-1 inhibitor has better fluorescence than the group with the addition of Ls. The above results show that the modification of NMOF by Ls can make it easier to bind to Kim-1 overexpressed on the surface of HK-2 and enter the cell, thereby achieving targeting of HK-2 cells.

[0125] Example 12 The intracellular ROS scavenging performance of NMOF-Ls was observed by laser scanning confocal microscopy. Fig.10 As shown. Cisplatin can induce cells to produce ROS, causing the ROS probe DCFH-DA to show a strong green fluorescence signal. It can be observed that the prepared materials have a certain ability to scavenge ROS in cells. Compared with MOF, NMOF has a particularly significant ROS scavenging ability. This is because NMOF has a smaller size and better SOD-CAT activity, which is easier to be taken up by HK-2 cells and effectively remove ROS. NMOF-Ls shows better ROS scavenging performance than NMOF. This is because Ls increases the targeting and uptake of NMOF to HK-2 cells, ultimately achieving a better ROS scavenging effect.

[0126] Example 12 Preparation of NMOF-Ls The therapeutic effect at the mouse level was characterized by measuring the creatinine and urea nitrogen of the mice. The results are as follows Fig.11 As shown in the figure, the creatinine and urea nitrogen levels of mice treated with NMOF-Ls are closest to normal values, so it performs best in treating AKI. This is because after NMOF-Ls is injected through the tail vein, it can achieve a targeted effect on Kim-1 overexpressed on the surface of HK-2 cells through Ls, and preferentially accumulate in the kidney and enter the cells. At the same time, due to its excellent ROS scavenging performance, it can efficiently eliminate ROS in HK-2 cells, and ultimately restore kidney function to normal.

[0127] In summary, the antioxidant properties of the defective NMOFs constructed by our defect engineering have been effectively improved. The post-modification of L-serine also enables it to achieve kidney targeting function. The prepared NMOF-Ls ultimately achieved effective treatment of the mouse AKI model through kidney targeting and antioxidant properties.

[0128] Although preferred embodiments of the present invention have been described, additional changes and modifications may be made to these embodiments by those skilled in the art once the basic inventive concepts are known. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention. Obviously, those skilled in the art may make various changes and modifications 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 is also intended to include these modifications and variations.

Claims

1. A method for preparing a defective metal organic framework antioxidant nanozyme, characterized in that: The following steps are involved: A copper source, a zirconium source and an organic ligand containing pyrazole and carboxyl are dissolved in an organic solvent, and then a competitive ligand is added, mixed evenly, and then a solvent thermal reaction is carried out at 80°C to 120°C. During the reaction, the organic ligand containing pyrazole and carboxyl regulates the speed and number of nucleation, the competitive ligand containing carboxylic acid regulates the crystal growth speed and crystallinity, and the organic solvent regulates the number of crystal nuclei. The particle size and defects are adjusted by combining the organic ligand containing pyrazole and carboxyl, the competitive ligand containing carboxylic acid and the organic solvent to prepare a defective metal organic framework; the ratio of the organic ligand containing pyrazole and carboxyl to the competitive ligand containing carboxylic acid is 20mg to 64mg: 0μL to 120μL; the ratio of the organic ligand containing pyrazole and carboxyl to the organic solvent is 20mg to 64mg: 10mL to 30mL; After the defective metal organic framework aqueous solution and the kidney-targeting molecule solution are evenly mixed, the defective metal organic framework antioxidant nanozyme is obtained through coordination and electrostatic adsorption.

2. The method for preparing a defective metal organic framework antioxidant nanozyme according to claim 1, characterized in that: The ratio of the organic ligand containing pyrazole and carboxyl groups to the organic solvent is 48 mg:20 mL.

3. The method for preparing a defective metal organic framework antioxidant nanozyme according to claim 1, characterized in that: The ratio of the organic ligand containing pyrazole and carboxyl groups to the competing ligand containing carboxylic acid was 48 mg:30 μL.

4. The method for preparing a defective metal organic framework antioxidant nanozyme according to claim 1, characterized in that: The mass ratio of the organic ligand containing pyrazole and carboxyl to the copper source is 1:2-6; the mass ratio of the zirconium source to the copper source is 1:2-4.

5. The method for preparing a defective metal organic framework antioxidant nanozyme according to claim 1, characterized in that: The reaction time of the solvent thermal reaction is 6h to 10h.

6. The method for preparing a defective metal organic framework antioxidant nanozyme according to claim 1, characterized in that: The mass ratio of defective metal organic framework to kidney targeting molecule is 10:0.1~0.

5.

7. The method for preparing a defective metal organic framework antioxidant nanozyme according to claim 1, characterized in that: The reaction temperature of the coordination and electrostatic adsorption reaction is 20°C to 40°C, and the reaction time is 8h to 28h.

8. The method for preparing a defective metal organic framework antioxidant nanozyme according to claim 1, 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, 3-methylpyrazole-4-carboxylic acid, 3,5-dimethyl-4-pyrazolecarboxylic acid or 5-phenyl-1H-pyrazole-4-carboxylic acid; The competing ligand was trifluoroacetic acid or acetic acid; The kidney-targeting molecule is L-serine or hyaluronic acid.

9. A defective metal organic framework antioxidant nanozyme prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the defective metal organic framework antioxidant nanozyme according to claim 9 in the preparation of a drug for treating ROS-mediated kidney diseases.

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