A MOF-based material with enzyme-mimicking activity and its preparation method and application

Abstract

The present invention discloses a method for rapidly preparing an ultra-small cerium-based MOF nanozyme with enzyme-mimicking activity and its application, which belongs to the technical field of biomedical materials; the method comprises adding a metal salt, a bidentate ligand, and a regulator into water, performing a microwave reaction, and obtaining the product by centrifugation and washing after the reaction is completed; wherein the bidentate ligand is a dicarboxylic acid containing a benzene ring; the regulator is a mixture of a monodentate ligand and dimethylimidazole, and the monodentate ligand is a monocarboxylic acid. The MOF preparation method provided by the present invention is simple, low-cost, and environmentally friendly, avoiding the use of organic solvents that are costly and have great environmental hazards. The prepared ultra-small hierarchical porous cerium-based MOF material has a high specific surface area and a large number of defect sites, can effectively load cinnamic acid with the function of regulating PTEN expression, and can be used as a drug for the treatment of diabetic nephropathy; the ultra-small cerium-based metal-organic framework with enzyme-mimicking activity can delay glomerular sclerosis and achieve the treatment of diabetic nephropathy.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and in particular relates to a MOF-based material with simulated enzyme activity, a preparation method and an application thereof. Background Art

[0002] Diabetic nephropathy (DN) is the most common and severe renal microvascular complication of diabetes mellitus (DN) and the leading cause of end-stage renal disease (ESRD) worldwide. According to global epidemiological surveys, DN endangers the lives and health of 425 million adults worldwide, and the number of DN patients is expected to reach 629 million by 2045. Studies indicate that 30%-40% of DN patients will eventually progress to ESRD and require renal replacement therapy, which will have a significant negative impact on global public health and significantly increase the global healthcare burden. DN is known to manifest as a chronic loss of renal function caused by long-term poor glycemic control, characterized by pathological proteinuria and a progressive decrease in glomerular filtration rate (GFR). The progression of DN can further affect cardiovascular and cerebrovascular systems, significantly increasing the risk of related cardiovascular events.

[0003] The main pathogenesis of DN includes tubulointerstitial fibrosis (TIF) and epithelial-mesenchymal transition (EMT). EMT is a key transdifferentiation process in fibrosis, during which epithelial cells lose their original adhesion ability and instead express a variety of mesenchymal characteristics, including invasion and migration. Tubular epithelial cells undergo partial EMT dedifferentiation to secrete various cytokines and chemokines to promote fibroblast proliferation, while aggravating the inflammatory response, promoting renal fibrosis, and leading to renal failure. Glomerulosclerosis is an important feature of the renal pathological changes in DN. Various pathological injuries such as mesangial expansion, glomerular basement membrane thickening, and podocyte damage will eventually lead to glomerular sclerosis. Therefore, specifically improving glomerular sclerosis is a new treatment for diabetic nephropathy.

[0004] Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a tumor suppressor gene with biological functions primarily manifested in the regulation of cell growth, proliferation, differentiation, migration, and apoptosis. PTEN is a key inhibitor of the PI3K-AKT signaling pathway, which regulates EMT and exerts anti-fibrotic effects. It negatively regulates the PI3K-AKT signaling pathway, thereby stimulating podocyte autophagy and slowing the progression of diabetic nephropathy. However, there are currently few studies investigating drugs that inhibit glomerulosclerosis in diabetic nephropathy by regulating PTEN expression to exert renal protective effects, and the underlying therapeutic mechanisms are unclear.

[0005] Current drug delivery platforms for treating diabetic nephropathy also suffer from a series of drawbacks that urgently need to be addressed: 1. Direct injection of drug delivery systems fails to effectively accumulate in the kidneys, and renal cell uptake of drug-loaded nanoparticles is low, making it difficult to achieve effective targeted treatment for diabetic nephropathy. 2. Existing drug delivery platforms are prone to accumulation in lysosomes, where various enzymes degrade the nanoparticles and their loaded drugs, thereby reducing the drug's therapeutic efficacy for diabetic nephropathy. Summary of the Invention

[0006] In view of this, the main purpose of the present invention is to provide a method for preparing a MOF-based material with enzyme-mimicking activity. The preparation method of the present invention is simple, low-cost, green and environmentally friendly, and avoids the use of high-cost and environmentally harmful organic solvents. The prepared MOF-based material with hierarchical porous structure and enzyme-mimicking activity can accurately and safely treat diabetic nephropathy mainly based on: (1) The MOF-based material has a high specific surface area and a large number of defect sites, and 4+ and Ce 3+ The results show that the MOF-based material exhibits excellent SOD activity under the synergistic effect of the reversible transformation between the two, which can clear the excess reactive oxygen species (ROS) in podocytes induced by high glucose levels, thereby protecting podocytes from oxidative stress damage. (2) The porous structure of the MOF-based material can serve as an efficient drug delivery platform, effectively loading cinnamic acid, which has the function of regulating PTEN expression, downregulating the expression of PTEN protein in diabetic mice and delaying glomerular sclerosis. (3) The MOF-based material was repackaged using erythrocyte membranes extracted from homologous erythrocytes, showing significant human renal cell targeting ability. In summary, the enzyme-mimicking MOF-based material can be used to target renal cells in a high glucose environment for drug delivery and cell protection, and is a nano-platform with potential clinical application prospects.

[0007] To achieve the above object, the technical solution of the present invention is as follows:

[0008] A method for preparing a MOF-based material with enzyme-mimicking activity, comprising adding a metal salt, a bidentate ligand, and a regulator to water, performing a microwave reaction, and obtaining the material by centrifugation and washing after the reaction is complete.

[0009] Wherein, the bidentate ligand is a dicarboxylic acid containing a benzene ring;

[0010] The regulator is a mixture of a monodentate ligand and dimethylimidazole, and the monodentate ligand is a monocarboxylic acid;

[0011] The metal salt is a cerium salt.

[0012] Furthermore, the cerium salt is CeCl3.

[0013] Furthermore, the bidentate ligand is at least one of 2-aminoterephthalic acid, biphenyldicarboxylic acid, and 4,4'-terphenyldicarboxylic acid.

[0014] Furthermore, the monodentate ligand is at least one of benzoic acid, formic acid, acetic acid, propionic acid, butyric acid, valeric acid, and hexanoic acid.

[0015] Furthermore, the usage of the metal salt, bidentate ligand, monodentate ligand, dimethylimidazole, and deionized water is 1 mmol: (1-6) mmol: (4-50) mmol: (1-5) g: (10-25) mL.

[0016] Furthermore, the microwave reaction time is 10-130 min and the temperature is 50-150°C.

[0017] A second object of the present invention is to provide a MOF-based material having enzyme-mimicking activity prepared by any of the above preparation methods.

[0018] The present invention also aims to provide the use of the above-mentioned MOF-based material with simulated enzyme activity as a drug delivery platform for the targeted treatment of diabetic nephropathy.

[0019] Furthermore, the simulated enzyme activity is a simulated superoxide dismutase activity, and the drug is a drug that regulates PTEN expression.

[0020] Furthermore, the dosage ratio of the MOF-based material having enzyme-mimicking activity to the drug for regulating PTEN is (50-300) mg: (5-25) mg.

[0021] Furthermore, the targeting effect comes from red blood cell membrane (RBCM) extracted from homologous red blood cells. When the red blood cell membrane is packaged with a MOF-based material having simulated enzyme activity, the usage ratio of the red blood cell membrane and the MOF-based material is (1-20) mg: (0.5-15) mg.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] (1) The present invention allows for the rapid preparation of enzyme-mimicking MOF-based materials with hierarchical porous structures in aqueous solvents through microwave heating and modulator induction. The preparation method is simple, low-cost, and environmentally friendly, avoiding the use of costly and environmentally hazardous organic solvents.

[0024] (2) Cerium is a star element in the rare earth field. The ultra-small cerium-based MOF nanozyme prepared rapidly by microwave has Ce 3+ and Ce 4+The reversible conversion characteristics between the two, as well as the high specific surface area and a large number of defect sites, give the cerium-based MOF nanozyme excellent SOD activity. This mechanism reduces the ROS level of podocytes induced by high sugar, protects mitochondrial damage, and restores mitochondrial energy metabolism.

[0025] (3) MOF-based materials with hierarchical porous structures can be used as a high-drug loading platform for cinnamic acid, and have excellent sustained-release effects in actual treatment. The loaded cinnamic acid has the function of regulating PTEN expression, and with the help of the MOF drug loading platform, it can act as a protective layer to inhibit drug degradation caused by lysosomes, thereby significantly improving the therapeutic effect of diabetic nephropathy.

[0026] (4) Using homologous red blood cell membranes to encapsulate drug-loaded ultrasmall cerium-based MOF nanozymes, they were specifically targeted to the glomeruli and podocytes of the kidney. After targeting, the nanoplatform sustained-released cinnamic acid, significantly downregulated PTEN expression, and delayed glomerular sclerosis. This design provides a safe and effective strategy for the treatment of diabetic nephropathy. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a scanning electron microscope image of HP-Ce-UiO-66 prepared in the present invention.

[0028] Figure 2 3 is a nitrogen adsorption-desorption curve of HP-Ce-UiO-66 prepared in the present invention; wherein A is the adsorption isotherm of the sample, and B is the pore size distribution diagram of the BJH model.

[0029] Figure 3 It is a performance diagram of the SOD activity test of HP-Ce-UiO-66 and Ce-UiO-66 prepared by the present invention.

[0030] Figure 4 This is an X-ray energy spectrum analysis diagram of Cinn@HP-Ce-UiO-66@RBCM prepared by the present invention.

[0031] Figure 5 shows changes in body weight, blood glucose, and kidney weight / body weight in DN mice after intraperitoneal injection of Cinn@HP-Ce-UiO-66@RBCM and Cinn prepared according to the present invention. (A) Weekly changes in body weight. (B) Weekly changes in fasting blood glucose. (C) Results of an oral glucose tolerance test. (D) Changes in kidney weight / body weight. **p<0.01 compared to the control group; #p<0.05 compared to the DN+Cinn group.

[0032] Figure 6Figure 2. Changes in renal function and renal pathology in DN mice following intraperitoneal administration of Cinn and Cinn@HP-Ce-UiO-66@RBCM. (A) Changes in blood urea nitrogen, serum creatinine, and urine albumin / creatinine ratio. **p<0.01 compared to the control group; ##p<0.01 compared to the DN+Cinn group. (B) HE staining. (C) PAS staining. (D) Masson staining.

[0033] Figure 7 After DN mice were intraperitoneally injected with Cinn@HP-Ce-UiO-66@RBCM and Cinn prepared in the present invention, the expression of PTEN was verified by Western blotting. DETAILED DESCRIPTION

[0034] The following description sets forth numerous specific details to facilitate a thorough understanding of the present invention. However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art may make similar modifications without departing from the scope of the present invention. Therefore, the present invention is not limited to the specific implementations disclosed below.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in this specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0036] Example 1 Preparation of Cinn Drug-Loaded MOF

[0037] A method for preparing a Cinn drug-loaded MOF comprises the following steps:

[0038] 1. Preparation of HP-Ce-UiO-66: 1 mmol CeCl3, 1 mmol biphenyldicarboxylic acid, 10 mmol butyric acid, and 2.5 g dimethylimidazole were added to 12 mL deionized water. The mixture was microwaved at 100°C for 100 min. The product was centrifuged, washed, and dried to obtain HP-Ce-UiO-66. Figure 1 The scanning electron microscope image of the prepared HP-Ce-UIO-66 shows that the prepared HP-Ce-UIO-66 has a polyhedral shape as a whole and a very rough surface. This rough surface is mainly because butyric acid and dimethylimidazole were introduced as regulators during the preparation process. During the nucleation and growth of HP-Ce-UiO-66, these regulators compete with the ligands and coordinate with the metal center, thereby interfering with the crystallization and growth of HP-Ce-UiO-66, forming HP-Ce-UiO-66 rich in defects and multi-level channels, and showing a rough surface structure. Further nitrogen adsorption and desorption experiments ( Figure 2) showed that the specific surface area of ​​the prepared HP-Ce-UiO-66 was 249.25 m 2 / g. From Figure 2 A and Figure 2 As can be seen from Figure B, HP-Ce-UiO-66 is rich in micropores and mesoporous structures, which is beneficial for drug loading and delivery. Figure 3 It can be seen that compared with Ce-UiO-66 prepared without adding a regulator in Comparative Example 1, the ultra-small cerium-based MOF material HP-Ce-UiO-66 prepared by dual regulators exhibits excellent scavenging performance for superoxide anions (O2•-), which can greatly reduce the oxidative stress of the kidneys.

[0039] Preparation of Cinn@ HP-Ce-UiO-66: Disperse 95 mg of dried HP-Ce-UiO-66 and 5 mg of cinnamic acid in 10 mL of ethanol and stir at room temperature until the solvent evaporates to obtain Cinn@ HP-Ce-UiO-66.

[0040] 3. Preparation of Cinn@HP-Ce-UIO-66@RBCM: The mixed solution of erythrocyte membrane and Cinn@HP-Ce-UiO-66 was co-incubated at 4°C for 4 hours. Then, the mixed solution was sonicated using an ultrasonic probe in an ice bath for 5 seconds on and 5 seconds off, for a total of 4 minutes at 100W. Finally, the obtained Cinn@HP-Ce-UiO-66@RBCM was centrifuged at 4°C (8000r / min, 10min) and washed three times. Figure 4 X-ray energy spectrum analysis shows that the surface of the sample has a high content of P element and trace S element, which mainly comes from RBCM. This verifies that RBCM successfully encapsulates Cinn@HP-Ce-UiO-66, improving the biocompatibility of the overall material while also helping the drug to target the kidneys.

[0041] Comparative Example 1 Preparation of Cinn Drug-Loaded MOF

[0042] A method for preparing a Cinn drug-loaded MOF comprises the following steps:

[0043] 1. Preparation of Ce-UiO-66: 1 mmol CeCl3 and 2.5 g dimethylimidazole were added to 12 mL deionized water. The mixture was microwaved at 100°C for 100 min. The product was centrifuged, washed, and dried to obtain Ce-UiO-66.

[0044] 2. Preparation of Cinn@Ce-UiO-66: Same as Example 1.

[0045] 3. Preparation of Cinn@Ce-UiO-66@RBCM: Same as Example 1.

[0046] Example 2 In vivo experiment in DN mice

[0047] 1. Experimental grouping, modeling, and drug administration

[0048] 1.1 Experimental Grouping and Modeling

[0049] A streptozotocin-induced DN mouse model was established for in vivo experimental verification. A control group, a DN+Cinn group (intraperitoneal injection of Cinn), and a Cinn@HP-Ce-UiO-66@RBCM group (intraperitoneal injection of Cinn@HP-Ce-UiO-66@RBCM) were set up.

[0050] The specific procedure was to acclimate 18 healthy male mice to the DN+Cinn group for one week and then randomly divide them into three groups: control, DN+Cinn, and DN+Cinn@HP-Ce-UiO-66@RBCM (n=6). After a 12-hour fast, mice in the DN+Cinn and DN+Cinn@HP-Ce-UiO-66@RBCM groups were intraperitoneally injected with 80 mg / kg of 1% streptozotocin (STZ, Sigma, USA) to induce DN mouse models. Mice in the control group received an equal volume of 0.1 mmol / L sodium citrate buffer intraperitoneally for five consecutive days. Tail vein blood was collected 72 hours after injection. A positive urine protein test indicated successful DN mouse model establishment when the blood glucose level was >16.7 mmol / L.

[0051] 1.2 Administration

[0052] Mice in the DN+Cinn group received Cinn (5 mg / kg / day) via intraperitoneal injection. Mice in the DN+Cinn@HP-Ce-UiO-66@RBCM group received Cinn@HP-Ce-UIO-66@RBCM (100 mg / kg / day) via intraperitoneal injection. Mice in the control group received normal saline (5 mg / kg) via intraperitoneal injection. Dosing frequency was once daily for 6 weeks.

[0053] 2. Data and specimen collection:

[0054] Mice were weighed weekly using an electronic scale, and fasting blood glucose (FBG) levels were assessed weekly for 6 weeks, with corresponding experimental data recorded. After the intervention, all mice underwent an oral glucose tolerance test (OGTT). Urine was collected from the mice using metabolic cages for 24 hours the day before the end of the experiment. After urine collection, eye blood samples were collected under sodium pentobarbital anesthesia for subsequent renal function testing. Finally, kidneys were collected under anesthesia and weighed using an electronic scale. Relative kidney weight (kidney weight (g) / body weight (g), KW / BW) was calculated and recorded. Left kidney tissue samples were fixed and embedded for subsequent renal histopathological examination, while right kidney tissue was directly frozen and stored at -80°C.

[0055] 3. Mouse body weight, FBG and OGTT detection

[0056] The weight of mice was measured using an electronic scale every week, and the fasting blood glucose of mice was evaluated every week. The mice were fasted for 6 hours before the test, the end of the tail of the mouse was disinfected, 2 mm of the tail tip was cut off, and blood was collected to measure the fasting blood glucose of the mice using a blood glucose meter (Roche, Germany). After 6 weeks of treatment under different dosing conditions, all mice were subjected to OGTT testing. The mice were fasted for 6 hours before the OGTT test. First, the tail vein blood was collected to evaluate the fasting blood glucose level of the mice. After the fasting blood glucose test, each mouse was given 20% glucose solution (1 g / kg) by gavage according to its body weight, and then the blood glucose level of the mouse was measured and recorded at different times such as 30 minutes, 60 minutes, 90 minutes and 120 minutes using a blood glucose meter. According to the weight measurement results of the mice ( Figure 5 (A) Compared with the control group, the DN+Cinn mice significantly lost weight after Cinn treatment (p<0.01). Compared with the DN+Cinn group, the DN+Cinn@HP-Ce-UiO-66@RBCM group significantly recovered their weight after Cinn@HP-Ce-UIO-66@RBCM treatment (p<0.05). The FBG test results of the mice showed that ( Figure 5 In Figure B), the fasting blood glucose of DN+Cinn mice was significantly higher than that of the control group (p<0.01), and the FBG level increased from less than 10mmol / L to nearly 25mmol / L. After treatment with Cinn@HP-Ce-UiO-66@RBCM, the fasting blood glucose of mice in the DN+Cinn@HP-Ce-UiO-66@RBCM group was significantly lower than that of mice in the DN+Cinn group, and the hyperglycemia of mice in the DN+Cinn@HP-Ce-UiO-66@RBCM group was significantly improved (p<0.05). OGTT testing can evaluate the body's pancreatic islet cell function and its ability to regulate blood glucose. The results are shown in the figure below. Figure 5 As shown in Figure C, blood glucose levels in DN+Cinn mice were significantly higher than those in the control group at various time points, including fasting, 30 minutes, 60 minutes, 90 minutes, and 120 minutes (p<0.01). Compared with mice treated with Cinn, blood glucose levels in the DN+Cinn@HP-Ce-UiO-66@RBCM-treated mice were significantly improved (p<0.05). These results demonstrate that, compared with Cinn, Cinn@HP-Ce-UiO-66@RBCM significantly improves blood glucose levels in DN mice and maintains blood glucose homeostasis.

[0057] 4. Renal function test

[0058] The mice were fasted but not watered and placed in metabolic cages for normal feeding. Urine of the mice in the cages was collected after 24 hours. After anesthesia with 2% sodium pentobarbital, blood was collected from the mouse eyeballs and allowed to stand at room temperature for 2 hours to obtain serum. Serum and urine were centrifuged at 3000 r / min for 10 minutes. The levels of blood creatinine (CREA), blood urea nitrogen (BUN) and urine albumin creatinine ratio (UACR) were detected using a blood creatinine detection kit (Shanghai ELISA Biotechnology Co., Ltd.), a urea nitrogen detection kit (Shanghai ELISA Biotechnology Co., Ltd.) and a urine albumin creatinine ratio detection kit (BioVision, USA). The KW / BW results of mice are shown in the figure. Figure 5 Figure D shows that compared with the control group, DN+Cinn mice treated with Cinn showed a significant increase in the KW / BW ratio (p<0.01), suggesting the presence of renal dysfunction. The KW / BW ratio of DN mice was significantly improved after treatment with Cinn@HP-Ce-UiO-66@RBCM (p<0.05). Blood creatinine, blood urea nitrogen and urine albumin-creatinine ratio are key markers for evaluating renal function and can intuitively reflect the damage to renal function. Compared with the control group mice, the blood creatinine, blood urea nitrogen and urine albumin-creatinine ratio of DN+Cinn mice were significantly increased (p<0.01). After treatment with Cinn@HP-Ce-UiO-66@RBCM, the blood creatinine, blood urea nitrogen and urine albumin-creatinine ratio of Cinn@HP-Ce-UiO-66@RBCM mice were significantly reduced (p<0.01, Figure 6 The above results indicate that compared with Cinn, Cinn@HP-Ce-UiO-66@RBCM can improve renal function damage and reduce the excretion of proteinuria in DN mice.

[0059] 5. Preparation of pathological tissue specimens and pathological staining

[0060] 5.1 Preparation of Kidney Pathological Sections

[0061] Kidney tissue samples were fixed in 4% paraformaldehyde at room temperature for 24 hours; the fixed pathological tissue was slowly rinsed with running water for 5 minutes; the tissue was dehydrated by soaking in 70% ethanol, 80% ethanol, 95% ethanol, 95% ethanol, anhydrous ethanol, and anhydrous ethanol for 20 minutes respectively; anhydrous ethanol and xylene were mixed in equal volumes, and the dehydrated kidney pathological tissue was placed in it and soaked for 30 minutes, and then soaked again in xylene solution for 30 minutes each time; finally, the transparent kidney tissue was immersed in paraffin liquid for 2 hours for embedding; after the paraffin solidified, it was continuously sectioned with a microtome, and the tissue block was cut into pathological tissue sections with a thickness of 4μm. The sections were placed in a 42℃ water bath for rinsing, and the slides were quickly picked up to adhere to the slides, and the slides were placed in a 58℃ oven for 60 minutes.

[0062] 5.2 HE staining

[0063] The tissue sections were sequentially immersed in the following solutions for dewaxing: xylene × 10 min, xylene × 10 min, anhydrous ethanol × 1 min, anhydrous ethanol × 1 min, 95% ethanol × 1 min, 95% ethanol × 1 min, 80% ethanol × 1 min, 75% ethanol × 1 min, and rinsed with running water for 1-2 min; after staining with hematoxylin staining solution for 8 min, the excess staining solution was rinsed with running water; differentiated with differentiation solution for 10 s, rinsed with running water to return to blue; stained with eosin staining solution for 4 min, the excess washing solution was rinsed with running water; the tissue sections were sequentially immersed in different concentrations of ethanol solutions for gradient dehydration: 75% ethanol × 3 min, 80% ethanol × 5 min, 95% ethanol × 10 min, anhydrous ethanol × 10 min, anhydrous ethanol × 10 min; xylene × 10 min, transparent twice, remove the excess xylene around the sections, air dry, and seal with neutral gum. According to the results of kidney pathology examination, HE staining ( Figure 6 In Figure B), the glomerular and tubular structures of the control mice were normal, while the renal damage in the Cinn-treated DN+Cinn mice was primarily manifested by glomerular structural disorder, inflammatory cell infiltration, and tubular dilation. Treatment with Cinn@HP-Ce-UiO-66@RBCM significantly ameliorated renal damage in the DN+Cinn@HP-Ce-UiO-66@RBCM mice.

[0064] 5.3 PAS staining

[0065] The tissue sections were sequentially immersed in the following solutions for dewaxing: xylene × 10 min, xylene × 10 min, anhydrous ethanol × 1 min, anhydrous ethanol × 1 min, 95% ethanol × 1 min, 95% ethanol × 1 min, 80% ethanol × 1 min, 75% ethanol × 1 min; oxidized with periodic acid solution for 20 min, rinsed with running water and air-dried; stained with Schiff stain in a dark room at room temperature for 1 h, rinsed with running water to remove the staining solution; stained with hematoxylin for 3-5 min, then rinsed with running water to remove excess staining solution; differentiated with differentiation solution for 3 s, rinsed with running water to reverse blue; tissue sections were sequentially immersed in ethanol solutions of different concentrations for gradient dehydration: 75% ethanol × 3 min, 80% ethanol × 5 min, 95% ethanol × 10 min, anhydrous ethanol × 10 min, anhydrous ethanol × 10 min; xylene × 10 min for clearing twice, then excess xylene around the sections was removed, air-dried, and sealed with neutral gum. PAS staining ( Figure 6 Figure (C) shows characteristic pathological changes in the kidneys of DN+Cinn mice, including thickening of the glomerular basement membrane and proliferation of the glomerular mesangial matrix. These pathological changes were significantly reduced in the DN+Cinn@HP-Ce-UiO-66@RBCM group compared to the DN+Cinn group.

[0066] 5.4 Masson staining

[0067] Part of the kidney tissue was fixed in Bouin's solution, rinsed with running water overnight, dehydrated and embedded as usual, and the sections were dewaxed to water; stained with Weigert's iron hematoxylin (Weigert's iron hematoxylin A and B solutions mixed in equal proportions) for 5-10 minutes, rinsed with running water; differentiated with differentiation solution for 1 minute, rinsed with running water; stained with Ponceau acid fuchsin solution for 5-10 minutes, rinsed with running water; treated with 1% phosphomolybdic acid solution for 5 minutes, without rinsing with running water, and directly counterstained with aniline blue staining solution for 5 minutes. Treated with 1% glacial acetic acid for 1 minute, and treated in the following solutions: dehydrated twice with 95% ethanol for 5 minutes, dehydrated twice with 100% ethanol for 5 minutes, and transparentized twice with xylene for 5 minutes, and sealed with neutral gum. Masson staining results ( Figure 6 Figure (D) shows increased glomerular collagen deposition and fibrosis in the DN+Cinn group compared to the control group. However, these findings were significantly improved in the DN+Cinn@HP-Ce-UiO-66@RBCM group, indicating that Cinn@HP-Ce-UiO-66@RBCM treatment significantly alleviated glomerulosclerosis in DN mice. Renal function tests and pathological staining analysis demonstrated that Cinn@HP-Ce-UiO-66@RBCM improved renal function and reduced renal tissue damage in DN mice.

[0068] 6. Western blot detection

[0069] 6.1. Kidney Tissue Protein Extraction

[0070] Take a small piece of kidney tissue, add 200 μL of lysis buffer prepared by 1 ml RIPA lysis buffer, 10 μL protease inhibitor, 10 μL phosphatase inhibitor and grinding beads, grind thoroughly at low temperature, homogenize with a homogenizer for 1 minute, and place on ice for lysis for 30 minutes; take the homogenate and centrifuge at 12000 rpm for 10 minutes at 4°C, take part of the protein supernatant for subsequent protein concentration determination, and package the remaining supernatant, denature at 95°C for 10 minutes, and store in a -20°C refrigerator.

[0071] 6.2. Protein Concentration Determination (BCA Method)

[0072] Thoroughly mix BCA reagent A:B at a ratio of 50:1 to prepare BCA working solution. Add 20 mg of protein standard (BSA) to 800 μL of protein standard solution to prepare BSA standard. Dilute 20 μL of BSA standard to a concentration of 0.5 mg / mL. Add 0, 2, 4, 8, 12, 16, and 20 μL of diluted protein standard and 5 μL of the sample to be tested to a 96-well plate. Add 200 μL of BCA working solution to each well. Incubate at 37°C in the dark for 30 minutes. Measure absorbance at 562 nm with a microplate reader. Draw a standard curve and calculate the protein concentration of the sample to be tested.

[0073] 6.3 Electrophoresis, transfer and antibody incubation

[0074] Prepare a separating gel of appropriate concentration according to the kit instructions and protein molecular weight. Pour the separating gel into the glass plate and press the gel. Let it stand at room temperature until the separating gel solidifies. Then, pour the stacking gel and quickly insert a comb. Let it stand at room temperature until the stacking gel solidifies, then remove the comb. Load 25µg of protein sample onto the polyacrylamide gel and add a pre-stained protein marker. Stop electrophoresis when the bromophenol blue band reaches the bottom of the gel plate. Immediately transfer the gel to a PVDF membrane. Block the PVDF membrane in 5% skim milk for 1 hour at room temperature. Add antibodies to PTEN and GAPDH and incubate overnight at 4°C. Wash the membrane three times with TBST (10 minutes each). Apply an HRP-conjugated secondary antibody to the membrane and incubate it at room temperature for 1 hour. Wash again with TBST (10 minutes each). In a dark room, the A and B reagents in the ECL kit were mixed in equal volumes to prepare a working solution, which was evenly dropped onto the membrane. The strips were automatically exposed using Amersham Imager 600, and the grayscale values ​​of the strips were analyzed. The results showed that Figure 7 A and Figure 7As shown in Figure B, compared with the control group, the brightness of the PTEN band in the experimental group treated with Cinn@HP-Ce-UiO-66@RBCM was significantly enhanced, indicating that PTEN played an important role in the protective effect of Cinn@HP-Ce-UiO-66@RBCM in alleviating DN.

[0075] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0076] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A method for preparing a MOF-based material with simulated enzyme activity, characterized in that: The method comprises adding a metal salt, a bidentate ligand and a regulator into water, performing microwave reaction, and after the reaction is completed, centrifuging and washing to obtain the product; Wherein, the bidentate ligand is biphenyldicarboxylic acid; The regulator is a mixture of a monodentate ligand and dimethylimidazole, and the monodentate ligand is butyric acid; The metal salt is a cerium salt; The metal salt, bidentate ligand, monodentate ligand, dimethylimidazole, and deionized water are used in amounts of 1 mmol: (1-6) mmol: (4-50) mmol: (1-5) g: (10-25) mL; The microwave reaction time is 10-130 min, and the temperature is 50-150°C.

2. The preparation method according to claim 1, characterized in that The cerium salt is CeCl3.

3. A MOF-based material having enzyme-mimicking activity prepared by the preparation method according to any one of claims 1-2.

4. Use of the MOF-based material with enzyme-mimicking activity according to claim 3 as a drug delivery platform in the preparation of drugs for the targeted treatment of diabetic nephropathy.

5. The use according to claim 4, characterized in that The simulated enzyme activity is superoxide dismutase activity, the drug is a drug for regulating PTEN expression, and the targeting effect is derived from the erythrocyte membrane extracted from homologous erythrocytes.

6. The use according to claim 5, characterized in that The dosage ratio of the MOF-based material having simulated enzyme activity to the drug for regulating PTEN expression is (50-300) mg: (5-25) mg.

7. The use according to claim 5, characterized in that When the red blood cell membrane is used to package the MOF-based material having simulated enzyme activity, the usage ratio of the red blood cell membrane to the MOF-based material is (1-20) mg: (0.5-15) mg.

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