Application of Fe3O4-coated ZIF-8 ultra-small nano enzyme in myocardial injury

Abstract

The invention discloses an application of a Fe3O4 (at) ZIF-8 nano-enzyme in preparation of a medicine for treating myocardial injury, the Fe3O4 (at) ZIF-8 nano-enzyme is of a core-shell structure which takes Fe3O4 as a nano-enzyme and is externally coated with ZIF-8, the overall average particle size of the nano-enzyme is (12-22) nm, it can be ensured that the ultra-small nano-enzyme has good dispersity and myocardial tissue accessibility, and the nano-enzyme can be used as a medicine for treating myocardial injury. And a ZIF-8 shell layer is utilized to stabilize the enzyme-like activity of Fe3O4 and regulate and control the microenvironment, so that additional oxidative damage caused by free iron is reduced, and the material as a main body plays a multi-dimensional mechanism of oxidation resistance, inflammation resistance and mitochondria protection. According to the invention, the Fe3O4 ZIF-8 nano-enzyme is applied to drug-induced myocardial injury for the first time, and it is found that the nano-enzyme shows a remarkable heart protection effect. The Fe3O4 ZIF-8 nano-enzyme can be prepared on a large scale, is good in biocompatibility, and has a wide application prospect in drug cardiotoxicity intervention and other ROS / inflammation related heart diseases.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of biological medicine, and particularly relates to an application of Fe3O4@ZIF-8 ultra-small nanoscale enzyme in myocardial injury. BACKGROUND

[0002] Drug-induced myocardial injury, also known as drug-induced cardiomyopathy, refers to myocardial damage caused by drug toxicity, which leads to myocardial hypertrophy, cardiac enlargement or abnormal cardiac function. Common drugs include antitumor drugs, antipsychotic drugs, cardiovascular drugs and antiparasitic drugs. As a widely used antitumor drug, the dose-related cardiotoxicity of doxorubicin has become a major factor limiting its clinical use. At present, there is a lack of effective and safe treatment for doxorubicin-induced myocardial injury, and the clinical treatment mainly relies on symptomatic supportive treatment, which cannot directly improve the pathological process.

[0003] The existing nanomaterial intervention strategies mainly include two categories: one is to use nanomaterials as drug carriers to encapsulate antioxidant or anti-inflammatory drugs, but there are problems such as unstable drug encapsulation efficiency, uncontrollable release and insufficient heart targeting; the other is a composite material based on metal organic framework or inorganic nanoscale enzyme, which improves heart function by removing free radicals through the composite material.

[0004] Patent CN121059822A discloses the application of nano-iron (PEI@Fe3O4) in vascular endothelial cells, which is a composite material formed by combining nanoscale polyethyleneimine and nanoscale iron oxide. It discloses the application of PEI@Fe3O4 in promoting the regeneration of vascular endothelial cells. However, the above-mentioned nano-iron does not involve the application of drug-induced myocardial injury; and like the existing nanoscale enzyme system, there are problems such as unstable particle size, potential toxicity caused by iron ion leakage and limited antioxidant effect in vivo. SUMMARY

[0005] In view of the above existing technical problems, the primary object of the present application is to provide the application of Fe3O4@ZIF-8 nanoscale enzyme in the preparation of drugs for treating and / or preventing myocardial injury.

[0006] The second object of the present application is to provide the application of Fe3O4@ZIF-8 nanoscale enzyme in the preparation of drugs for treating and / or preventing myocardial fibrosis.

[0007] The third object of the present application is to provide the application of Fe3O4@ZIF-8 nanoscale enzyme in the preparation of drugs for improving cardiac dysfunction.

[0008] In order to achieve the above-mentioned objects, the present application is realized by the following technical solutions:

[0009] The application claims the use of Fe3O4@ZIF-8 nanoscale enzyme in the preparation of a drug for treating and / or preventing myocardial injury, wherein the Fe3O4@ZIF-8 nanoscale enzyme is a core-shell structure with Fe3O4 as a nanocore and ZIF-8 as an outer coating; and the average particle size of the Fe3O4@ZIF-8 nanoscale enzyme is (12-22) nm.

[0010] The application provides a Fe3O4@ZIF-8 nanoscale enzyme with Fe3O4 as a nanocore and ZIF-8 as an outer coating, and an average particle size of (12-22) nm. The ultra-small nanoscale enzyme with a specific particle size has good dispersibility and easy access to myocardial tissue, and the ZIF-8 shell layer stabilizes the Fe3O4 enzyme-like activity, regulates the microenvironment, thereby reducing the additional oxidative damage caused by free iron, so that the material itself as the main body plays a multi-dimensional mechanism of antioxidant, anti-inflammatory and mitochondrial protection. The application first applies the Fe3O4@ZIF-8 nanoscale enzyme to drug-induced myocardial injury, and finds that the nanoscale enzyme exhibits significant cardiac protection. The Fe3O4 nanoscale enzyme prepared by the application has smaller particle size, is more stable, has good biocompatibility, and has excellent therapeutic effect on drug toxicity-induced myocardial injury.

[0011] Preferably, the average particle size of the Fe3O4@ZIF-8 nanoscale enzyme is (16-18) nm.

[0012] Preferably, the myocardial injury is drug-induced myocardial injury caused by oxidative stress and / or inflammation imbalance.

[0013] More preferably, the drug is at least one selected from adriamycin, cisplatin, fluorouracil, cyclophosphamide, daunorubicin, epirubicin, and pirarubicin.

[0014] Most preferably, the drug is adriamycin. The Fe3O4@ZIF-8 nanoscale enzyme provided by the application exhibits significant cardiac protection in myocardial injury, especially adriamycin-induced myocardial injury, including improving cardiac volume reduction, reducing cardiac fibrosis, reducing myocardial injury marker levels, and restoring myocardial mitochondrial structural integrity under transmission electron microscopy, showing obvious advantages in stability and safety over existing materials. It can be seen that the Fe3O4@ZIF-8 nanoscale enzyme provided by the application has a broad application prospect in adriamycin myocardial toxicity intervention and other ROS / inflammation-related heart diseases.

[0015] Based on this, the drug-induced myocardial injury includes at least one of adriamycin-induced cardiotoxicity, cardiac atrophy, cardiac dysfunction, cardiac fibrosis, myocardial cell ferroptosis, myocardial cell mitochondrial dysfunction, and myocardial cell reactive oxygen species accumulation.

[0016] Specifically, the preparation of the Fe3O4@ZIF-8 nanoscale enzyme comprises the following steps: Fe3O4 nanocore, zinc salt, 2-methyl imidazole are added into methanol and mixed, and the mixture is uniformly mixed at 3-5 ℃ to obtain the Fe3O4@ZIF-8 nanoscale enzyme.

[0017] Preferably, the preparation of the Fe3O4 nanocore comprises the following steps: iron salt is added into a solvent and mixed, the temperature is raised to 55-65 ℃ under a nitrogen atmosphere, a base solution is added, and the temperature is raised to 75-85 ℃, and the Fe3O4 nanocore is obtained. Compared with the traditional solvothermal method, the temperature required for the reaction in the preparation of the Fe3O4 nanocore is significantly reduced, the energy consumption is reduced, the environmental risks such as solvent volatilization caused by high temperature are avoided, and the particle size is reduced while the crystal form is maintained.

[0018] Preferably, the molar ratio of the Fe3O4 nanocore to the zinc salt is (2-4):1. More preferably, the molar ratio of the Fe3O4 nanocore to the zinc salt is (2-3):1. Most preferably, the molar ratio of the Fe3O4 nanocore to the zinc salt is 2.2:1, which is conducive to the stable growth of the ZIF-8 shell.

[0019] Further, the Fe3O4 nanocore is a surfactant-modified Fe3O4 nanocore; and the preparation method of the surfactant-modified Fe3O4 nanocore comprises the following steps: Fe3O4 nanocore and surfactant are added into a solvent and mixed, and the surfactant-modified Fe3O4 nanocore is obtained. The surfactant can effectively improve the colloidal stability of Fe3O4 and provide a negatively charged surface ligand, which is conducive to the growth of the subsequent ZIF-8 shell.

[0020] Preferably, the molar ratio of the iron salt to the surfactant is 1:(1.5-2.5). More preferably, the molar ratio of the iron salt to the surfactant is 1:2.

[0021] Preferably, the surfactant is at least one selected from the group consisting of sodium citrate, oleic acid, oleylamine, polyethylene glycol, sodium dodecyl sulfate, and polyacrylic acid. More preferably, the surfactant is sodium citrate. The surfactant can effectively improve the colloidal stability of Fe3O4 and provide a negatively charged surface ligand, which is conducive to the growth of the subsequent ZIF-8 shell.

[0022] Preferably, the mass-volume ratio of the iron salt to the solvent is (1-3) g:(40-60) mL. More preferably, the mass-volume ratio of the iron salt to the solvent is 1.5 g:50 mL.

[0023] Specifically, the iron salt is composed of at least one of trivalent iron salt and divalent iron salt.

[0024] Preferably, the ferric salt is selected from at least one of ferric chloride, ferric sulfate, ferric nitrate, ferric oxalate, ferric citrate, ferric acetylacetone. More preferably, the ferric salt is ferric chloride. Preferably, the ferrous salt is selected from at least one of ammonium ferrous sulfate, ferrous sulfate, ferrous chloride. More preferably, the ferrous salt is ammonium ferrous sulfate. Compared with ferrous chloride, ammonium ferrous sulfate has higher oxidation stability and more controllable Fe 2+ The release function is conducive to the formation of uniform and ultra-small Fe3O4 nanoparticles.

[0025] Preferably, the molar ratio of the ferric salt and the ferrous salt is (1.5~2.5):1. More preferably, the molar ratio of the ferric salt and the ferrous salt is 2:1. The preferred ratio is conducive to the formation of a stable inverse spinel structure, and if the ratio deviates, impurities will be introduced, reducing the enzyme-like activity of the product. Excessive ferrous salt will cause imbalance in the nucleation and growth process, leading to uneven distribution of surface charges and enhanced magnetic interaction of the nanoparticles, thereby exacerbating nanoparticle agglomeration. It can be seen that the present application provides a Fe3O4@ZIF-8 nanoscale enzyme which can be prepared on a large scale and has good biocompatibility by controlling the preparation conditions.

[0026] Preferably, the concentration of the Fe3O4@ZIF-8 nanoscale enzyme is (25~200) µg / mL. When the concentration of the Fe3O4@ZIF-8 nanoscale enzyme is at the cell level, the solvent is PBS buffer; when the concentration of the Fe3O4@ZIF-8 nanoscale enzyme is at the animal level, the solvent is physiological saline. It is found that the Fe3O4@ZIF-8 nanoscale enzyme has little effect on cell viability, and as the concentration of Fe3O4@ZIF-8 increases, the cell viability decreases slightly, but still maintains a high cell viability at a concentration of 200 µg / mL. At the same time, the ability of Fe3O4@ZIF-8 to catalytically decompose H2O2 to produce oxygen increases with the increase of the concentration of Fe3O4@ZIF-8, indicating that the Fe3O4@ZIF-8 nanoscale enzyme has good catalytic ability.

[0027] More preferably, the concentration of the Fe3O4@ZIF-8 nanoscale enzyme is (25~200) µg / mL. Further preferably, the concentration of the Fe3O4@ZIF-8 nanoscale enzyme is (90~150) µg / mL. It is found that the inhibition rate of the Fe3O4@ZIF-8 nanoscale enzyme to superoxide anion can reach 50%~75% within the concentration range of (100~150) µg / mL.

[0028] The present application also provides the use of the Fe3O4@ZIF-8 nanoscale enzyme in the preparation of a drug for treating and / or preventing myocardial fibrosis.

[0029] The present application also provides the use of the Fe3O4@ZIF-8 nanoscale enzyme in the preparation of a drug for improving cardiac dysfunction.

[0030] Compared with the prior art, the application has the following beneficial effects: The application provides application of Fe3O4@ZIF-8 nanozyme in preparation of a drug for treating and / or preventing myocardial injury, the Fe3O4@ZIF-8 nanozyme has good dispersity and myocardial tissue easy access, and utilizes the ZIF-8 shell layer to stabilize the peroxidase / superoxide dismutase-like activity of Fe3O4, regulates the microenvironment, thereby reducing the additional oxidative damage caused by free iron, so that the material itself as the main body plays a multi-dimensional mechanism of antioxidant, anti-inflammatory and mitochondrial protection. The Fe3O4@ZIF-8 nanozyme is applied to drug-induced myocardial injury for the first time, and it is found that the nanozyme exhibits significant cardiac protection. The Fe3O4@ZIF-8 nanozyme provided by the application can realize stable peroxidase / superoxide dismutase-like activity, and the antioxidant and anti-inflammatory capacity is also significantly enhanced. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 The figure is a result graph of characterization and activity test of Fe3O4@ZIF-8 prepared in Example 1. (A) Transmission electron microscope (TEM) images show the morphology of Fe3O4 nanoparticles (NPs) and Fe3O4@ZIF-8; (B) X-ray diffraction (XRD) patterns compare the crystal structures of Fe3O4 and Fe3O4@ZIF-8; (C) CCK-8 experiments show the influence of different concentrations of Fe3O4@ZIF-8 on cell viability; (D) Inhibition rate of different concentrations of Fe3O4@ZIF-8 on superoxide anion (·O2 - ); (E) H2O2 bubble experiment shows the ability of different concentrations of Fe3O4@ZIF-8 to catalyze the decomposition of H2O2 to produce oxygen.

[0032] Figure 2 The figure is a result graph of influence of Fe3O4@ZIF-8 prepared in Example 1 on DOX-induced cardiac histopathological changes. (A) Appearance of the heart of mice in different treatment groups; (B) Histological sections (H&E staining) of the heart of mice in each group; (C) Histological sections (Masson staining) of the heart of mice in each group; (D) Histological sections (transmission electron microscope images) of the heart of mice in each group.

[0033] Figure 3The effect of Fe3O4@ZIF-8 prepared in Example 1 on DOX-induced cardiotoxicity is shown in the figure. (A) Echocardiography showing left ventricular function in mice in different treatment groups. (A) Quantitative analysis of left ventricular end-diastolic diameter (LVIDd) and left ventricular end-systolic diameter (LVIDs); (B) Quantitative analysis of left ventricular ejection fraction (LVEF); (C) Quantitative analysis of left ventricular fractional shortening (LVFS); (D) Quantitative analysis of left ventricular end-diastolic diameter (LVIDd); (E) Quantitative analysis of left ventricular end-systolic diameter (LVIDs); (F) Quantitative analysis of left ventricular posterior wall diastolic thickness (LVPWd); (G) Quantitative analysis of left ventricular posterior wall systolic thickness (LVPWs); (H) Quantitative analysis of left ventricular anterior wall diastolic thickness (LVAWd); (I) Quantitative analysis of left ventricular anterior wall systolic thickness (LVAWs); (J) Quantitative analysis of heart weight / body weight ratio (HW / BW); (K) Quantitative analysis of serum creatine kinase isoenzyme (CK-MB); (L) Quantitative analysis of serum lactate dehydrogenase (LDH) activity; (M) Quantitative analysis of serum cardiac troponin I (cTnI) levels. * p <0.05,** p <0.01, *** p <0.001, **** p <0.0001; # p <0.05, ## p <0.01, ### p <0.001, #### p <0.0001; ns (not significant) indicates that the difference between the two groups of data is not statistically significant. p >0.05). Detailed Implementation

[0034] The present invention will be further described below with reference to the specification and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0035] Example 1 This invention provides a method for preparing Fe3O4@ZIF-8 nanozymes, specifically including the following steps: Preparation of S1 and Fe3O4 nanonuclei Take 1.15 g of iron (III) chloride hexahydrate (FeCl3·6H2O) and 0.93 g of ferrous ammonium sulfate hexahydrate (Fe(NH4)2(SO4)2·6H2O), dissolve in 50 mL of double distilled water, and mix uniformly under magnetic stirring. Nitrogen gas is introduced into the system for 5 min to remove dissolved oxygen in the solution. Then the reaction system is heated to 60 ℃, and 22 mL of deoxygenated 1M NaOH solution is quickly added, and the reaction is maintained for 15 min. Continue to heat to 80 ℃ for 30 min to promote the formation and growth of Fe3O4 crystal nuclei. After the reaction is completed, the precipitate is collected by centrifugation at 12000 g for 2 h, and washed twice with double distilled water. The obtained solid is freeze-dried to obtain Fe3O4 nanonuclei.

[0036] S2, sodium citrate modification of Fe3O4 surface Take 1 g of Fe3O4 nanonuclei obtained in step (1) and disperse in an appropriate amount of double distilled water, add 3.8 g of sodium citrate dihydrate in 200 mL of double distilled water, and react under mechanical stirring for 5 h. After the reaction is completed, the solid is separated by centrifugation at 12000 g for 2 h, washed twice with double distilled water, and freeze-dried to obtain sodium citrate-modified Fe3O4 nanonuclei.

[0037] S3, in-situ growth of ZIF-8 shell Take 0.5 g of the above sodium citrate-modified Fe3O4 nanomaterials, and add a mixed methanol solution (total volume 24 mL) of 0.2975 g of zinc nitrate hexahydrate (Zn(NO3)2·6H2O) and 0.3285 g of 2-methylimidazole. Continue to mechanically mix at 4 ℃ for 8 h, and control the low temperature in-situ growth to promote uniform nucleation of ZIF-8 and form a complete core-shell structure on the surface of Fe3O4. After the reaction is completed, the product is collected by centrifugation at 12000 g for 2 h, and washed twice with ethanol to remove unreacted zinc nitrate hexahydrate and 2-methylimidazole.

[0038] The final product is freeze-dried to obtain a black powder, which is Fe3O4@ZIF-8 core-shell nanoscale enzyme.

[0039] Comparative Example 1 Fe3O4 nanoscale enzyme is prepared by step S1 in Example 1, and is denoted as Fe3O4 NPs.

[0040] Test Example 1 The Fe3O4@ZIF-8 nanoscale enzyme prepared in Example 1 is characterized by transmission electron microscopy (TEM) for the morphology of Fe3O4 nanoparticles (NPs) and Fe3O4@ZIF-8, and X-ray diffraction (XRD) pattern for comparison of the crystal structure of Fe3O4 and Fe3O4@ZIF-8.

[0041] Transmission electron microscopy images as shown in FIG. 1 Figure 1 As shown in FIG. 1 (A), it is observed that Fe3O4@ZIF-8 shows a more complex structure, indicating the successful coating of ZIF-8. X-ray diffraction patterns as shown in FIG. 1 Figure 1 (B), it is observed that Fe3O4@ZIF-8 adds characteristic peaks of ZIF-8 (marked with green arrows) on the basis of characteristic peaks of Fe3O4, confirming the successful coating of ZIF-8. At the same time, it can be seen that the Fe3O4@ZIF-8 prepared by the present application has an average particle size of 17.7 nm (measured using ImageJ), a core part of about 10 nm or less, uniform color, good dispersibility, and is suitable for biomedical use in vivo and in vitro.

[0042] Test Example 2 The activity of Fe3O4@ZIF-8 nanoszyme prepared in Example 1 was tested.

[0043] I. Experimental materials Before testing, different concentrations of Fe3O4@ZIF-8 were prepared, specifically: an appropriate amount of Fe3O4@ZIF-8 powder was weighed, and after adding 10 mL of PBS, it was ultrasonically dispersed. Fe3O4@ZIF-8 nanoszyme suspensions with concentrations of 3.125 µg / mL, 6.25 µg / mL, 12.5 µg / mL, 25 µg / mL, 50 µg / mL, 75 µg / mL, 100 µg / mL, 150 µg / mL, and 200 µg / mL were prepared.

[0044] II. Experimental methods The test method includes CCK-8 experiment, superoxide anion (·O2 - ) inhibition rate test, and bubble experiment.

[0045] 1. CCK-8 Assay. The CCK-8 assay evaluated the effect of different concentrations of Fe3O4@ZIF-8 on cell viability. The specific procedure for the CCK-8 assay was as follows: H9C2 cells were seeded at a density of 8,000 cells per well in 96-well plates and treated with different concentrations (3.125 µg / mL, 6.25 µg / mL, 12.5 µg / mL, 25 µg / mL, 50 µg / mL, 75 µg / mL, 100 µg / mL, 150 µg / mL, and 200 µg / mL) of Fe3O4@ZIF-8. Cell viability was then assessed using the Cell Counting Kit-8 (CCK-8). After treatment, 100 μL of CCK-8 solution (C0039, Beyotime) was added to each well, and the cells were incubated in a cell culture incubator for 2 h. Absorbance was measured at 450 nm using a Varioskan LUX multi-scan spectrometer (Thermo Fisher Scientific) and calculated as a percentage of the control group to indicate cell viability.

[0046] 2. For superoxide anions (·O2) - The inhibition rate of Fe3O4@ZIF-8 at different concentrations was tested. The inhibition rate of superoxide anion was measured using the Solarbio BC5165-100T superoxide dismutase (SOD) activity assay kit (WST-1 method). The SOD-like activity of Fe3O4@ZIF-8 at different concentrations (12.5 µg / mL, 25 µg / mL, 50 µg / mL, 100 µg / mL, 150 µg / mL) was evaluated, and the inhibition rate was calculated.

[0047] 3. Bubble Experiment. The H2O2 bubble experiment was used to evaluate the catalytic performance of Fe3O4@ZIF-8 at different concentrations. The specific procedure was as follows: 100 μL of Fe3O4@ZIF-8 ultrasonically dispersed suspensions at different concentrations (12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL) were mixed with 100 μL of 30% H2O2 solution and incubated at room temperature for 6 hours. The bubble generation was then observed.

[0048] III. Experimental Results like Figure 1 As shown in (C), the results of the CCK-8 experiment showed that cell viability decreased slightly with increasing Fe3O4@ZIF-8 concentration, but remained high at a concentration of 200 µg / mL.

[0049] like Figure 1As shown in FIG. 8 (D), the results showed that the inhibitory effect of Fe3O4@ZIF-8 on superoxide anion gradually increased with the increase of the concentration of Fe3O4@ZIF-8. When the concentration was 100 µg / mL and 150 µg / mL, the inhibitory effect of Fe3O4@ZIF-8 on superoxide anion was more significant, which was 50% and 75% respectively. It can be seen that in the concentration range of (100~150) µg / mL, Fe3O4@ZIF-8 had good inhibitory effect on superoxide anion.

[0050] As shown in FIG. 8 (D), the results showed that the inhibitory effect of Fe3O4@ZIF-8 on superoxide anion gradually increased with the increase of the concentration of Fe3O4@ZIF-8. When the concentration was 100 µg / mL and 150 µg / mL, the inhibitory effect of Fe3O4@ZIF-8 on superoxide anion was more significant, which was 50% and 75% respectively. It can be seen that in the concentration range of (100~150) µg / mL, Fe3O4@ZIF-8 had good inhibitory effect on superoxide anion. Figure 1 As shown in FIG. 8 (E), the H2O2 bubble experiment showed the ability of different concentrations of Fe3O4@ZIF-8 to catalyze the decomposition of H2O2 to produce oxygen. It was found that the amount of bubbles produced increased significantly with the increase of the concentration of Fe3O4@ZIF-8, indicating that Fe3O4@ZIF-8 had good catalytic performance.

[0051] Test Example 3 The influence of Fe3O4@ZIF-8 nanoszyme prepared in Test Example 1 and Fe3O4 NPs prepared in Comparative Example 1 on DOX (doxorubicin) induced cardiac histopathology changes.

[0052] I. Experimental materials and grouping Twenty-five C57BL / 6J mice (purchased from Guangdong Medical Laboratory Animal Center) were randomly divided into the following 5 groups, 5 mice in each group: 1. Saline group: that is, the control group, physiological saline was injected into the tail vein of the mice on the 0 / 7 / 14 / 21 days (the drug volume was calculated as 10 μL / g according to the body weight); 2. DOX group: model group, physiological saline was injected into the tail vein of the mice on the 0 / 7 / 14 / 21 days (the drug volume was calculated as 10 μL / g according to the body weight); 3. Fe3O4@ZIF-8 group: Fe3O4@ZIF-8 nanoszyme treatment group, 1.25 mg / kg (according to the body weight) of 0.5 mg / mL Fe3O4@ZIF-8 nanoszyme solution was injected into the tail vein of the mice on the 0 / 7 / 14 / 21 days, and the drug volume was calculated as 10 μL / g according to the body weight.

[0053] 4. DXZ (dexrazoxane, purchased from Shanghai Taotuo Biotechnology Co., Ltd.) group: DXZ treatment group, 5 mg / kg (according to the body weight) of 0.5 mg / mL DXZ solution was injected into the abdominal cavity of the mice on the 0 / 7 / 14 / 21 days, and the drug volume was calculated as 10 μL / g according to the body weight.

[0054] 5. Fe3O4 NPs group: Fe3O4 nanoscale enzyme treatment group, on the 0 / 7 / 14 / 21 day, 1.25 mg / kg (dose by weight) of 0.5 mg / mL Fe3O4 nanoscale enzyme was injected into the tail vein of mice, and the volume of the drug was 10 μL / g according to the body weight.

[0055] The experiment started after the first administration and lasted for 28 days.

[0056] II. Experimental methods After the start of the experiment, the following experiments were performed.

[0057] 1. Establishing a DOX-induced myocardial injury mouse model A DOX-induced myocardial injury model was established using a multiple low-dose cumulative method: DOX (doxorubicin) was dissolved in physiological saline with a concentration of 0.5 mg / mL, and on the 1st / 8th / 15th / 22nd day (i.e. 24 h after each treatment), the mice were injected intraperitoneally (dose by weight, 5 mg / kg), and the cumulative dose reached 20 mg / kg.

[0058] The mice in the Saline group were injected intraperitoneally with the same volume of physiological saline at the same time points.

[0059] On the 7th day after the last DOX injection, the mice were qualitatively evaluated for the model, and compared with normal mice in the Saline group, those that met the following characteristics were considered to have successfully established a DOX-induced myocardial injury model: Changes in cardiac function: left ventricular ejection fraction (LVEF) and shortening fraction (LVFS) were significantly decreased; left ventricular end-diastolic diameter (LVIDd) and / or left ventricular end-systolic diameter (LVIDs) were abnormally changed.

[0060] Elevated serum myocardial injury markers: CK-MB, LDH, c-TnI, etc. were significantly elevated.

[0061] Cardiopulmonary pathology: H&E staining showed disordered myocardial fiber arrangement, vacuolar degeneration of cells; Masson staining showed increased collagen deposition; accompanied by mitochondrial ultrastructure damage.

[0062] 2. Detection method (1) Take the mouse heart: anesthetize the mouse by intraperitoneal injection of 50 mg / kg 3% sodium pentobarbital, cut the mouse chest with surgical scissors and expose the heart, insert a syringe into the mouse heart apex and push (5-10) mL of 0.1% potassium chloride solution to stop the heart in diastole and flush the residual blood in the heart, use surgical forceps and surgical scissors to cut off the aorta and other connecting tissues, remove the heart, then use ophthalmic forceps to squeeze the heart in pre-cooled physiological saline, completely remove the blood stains and dry the surface with filter paper, and observe the appearance of the mouse heart in different treatment groups.

[0063] (2) Quantitative analysis of left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS): According to the measured LVIDd and LVIDs, the left ventricular ejection fraction was calculated according to the formula LVEF = (1 - (LVIDs 3 / LVIDd 3 )) x 100%. According to the measured LVIDd and LVIDs, the left ventricular fractional shortening was calculated according to the formula LVFS = ((LVIDd - LVIDs) / LVIDd) x 100%.

[0064] (3) Measurement of left ventricular end-diastolic diameter (LVIDd) and end-systolic diameter (LVIDs): After anesthesia, the mouse was fixed, and the high-frequency echocardiogram was used to take the left ventricular long-axis section, record the M-mode ultrasound image, and measure the left ventricular end-diastolic diameter and end-systolic diameter.

[0065] (4) Quantitative analysis of creatine kinase isoenzyme (CK-MB) in serum: The mouse was taken from the orbit, and the serum was separated by centrifugation; the creatine kinase isoenzyme detection kit was used to determine the content of CK-MB in serum according to the instruction manual.

[0066] (5) Quantitative analysis of lactate dehydrogenase (LDH) activity in serum: The separated serum was used to determine the LDH activity using the lactate dehydrogenase activity detection kit according to the kit instruction manual.

[0067] (6) Quantitative analysis of serum cardiac troponin I (cTnI) level: The serum sample was taken, and the cardiac troponin I detection kit (such as ELISA method) was used to determine the serum cTnI level according to the kit instruction manual.

[0068] (7) H&E staining: After baking, the paraffin-embedded tissue section (thickness generally (3-5) μm) was placed in xylene for deparaffinization, and then gradually hydrated in gradient ethanol (100%, 95%, 80%, 70%) to distilled water. The section was placed in hematoxylin staining solution for nuclear staining, so that the cell nucleus was blue-violet, and after sufficient water washing, it was differentiated with hydrochloric acid alcohol to remove background staining, and then returned to blue with tap water to enhance the contrast of the nucleus. Then the section was transferred into eosin staining solution for cytoplasm and extracellular matrix staining, which showed pink to red. After staining, the section was dehydrated with gradient ethanol, transparent treated with xylene, and finally sealed with neutral resin to obtain a section that can be used for histological morphological observation.

[0069] (8) Masson staining: This can show the distribution of collagen fibers and reflect the degree of cardiac fibrosis. First, paraffin-embedded tissue sections are baked, then dewaxed with xylene and hydrated with a gradient of ethanol to distilled water. The sections are first treated with modified Bouin solution to enhance tissue structure and staining stability, followed by hematoxylin staining to reveal cell nuclei. After rinsing with running water, the sections are placed in Ponceau S staining solution to stain cytoplasm, muscle fibers, and other structures, and then differentiated with phosphomolybdic acid to selectively remove non-collagenous components of the dye. The sections are then transferred to aniline blue staining solution to specifically stain collagen fibers, making them blue. After staining, the sections are treated with glacial acetic acid to stabilize the staining effect, followed by gradient ethanol dehydration, xylene clearing, and mounting, thereby achieving a clear display of collagen fiber deposition and the degree of fibrosis in the tissue.

[0070] (9) Histological sections of the heart of each group of mice were observed using a transmission electron microscope, mainly to observe the morphology, size and arrangement of mitochondria.

[0071] III. Experimental Results like Figure 2 As shown in (A) and (B), the hearts of mice in the control group (Saline) are bright red and of normal size, indicating healthy hearts. The hearts of mice in the DOX-treated group are darker and smaller, possibly due to DOX-induced myocardial damage and cardiac atrophy. The hearts of mice in the Fe3O4@ZIF-8, Fe3O4NPs, and DXZ-treated groups showed improved color and texture, more closely resembling the control group, indicating that these three treatments can improve DOX-induced cardiac damage. The heart appearance of the Fe3O4@ZIF-8 group is closest to the control group, showing a stronger cardioprotective effect.

[0072] like Figure 2 As shown in (C), Masson staining results indicate that collagen fibers are evenly distributed in the heart tissue of the control group, with no obvious signs of fibrosis. The DOX group may show more blue collagen fibers in its heart tissue, indicating increased cardiac fibrosis. The Fe3O4@ZIF-8 group, Fe3O4NPs group, and DXZ treatment group show reduced collagen fiber distribution and decreased fibrosis. The Fe3O4@ZIF-8 group exhibits the lowest degree of fibrosis, demonstrating a stronger anti-fibrotic effect.

[0073] like Figure 2As shown in FIG. 13D, the transmission electron microscope can be used to observe the ultrastructure changes of the myocardial cells. The myocardial cell structure of the control group is complete, and the organelles such as mitochondria and myofibrils are clearly visible. The myocardial cells of the DOX group have ultrastructure damage such as mitochondrial swelling and myofibril rupture. The ultrastructure of the myocardial cells of the Fe3O4@ZIF-8 group, the Fe3O4 NPs group and the DXZ treatment group is improved, and the structures of the mitochondria and the myofibrils are more complete. The ultrastructure change of the Fe3O4@ZIF-8 group is the smallest, indicating that the Fe3O4@ZIF-8 has a stronger effect on protecting the myocardial cells.

[0074] Test Example 4 Effect of the Fe3O4@ZIF-8 prepared in Test Example 1 on DOX-induced cardiotoxicity.

[0075] The experimental grouping and the modeling method are the same as those in Test Example 3.

[0076] The experimental method is as follows: (1) Quantitative analysis of left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS): the same as Test Example 3.

[0077] (2) Measurement of left ventricular internal diameter at end diastole (LVIDd) and left ventricular internal diameter at end systole (LVIDs): the same as Test Example 3.

[0078] (3) Measurement of left ventricular posterior wall and anterior wall thickness: after the mice are anesthetized and fixed, the high-frequency echocardiogram is used to obtain the left ventricular long-axis section, and the sampling line is vertically placed at the papillary muscle level under the M-mode ultrasound; left ventricular posterior wall diastolic thickness (LVPWd): the vertical distance from the endocardium to the epicardium of the posterior wall at the end diastole (the top of the R wave of the electrocardiogram) is measured; left ventricular posterior wall systolic thickness (LVPWs): the vertical distance from the endocardium to the epicardium of the posterior wall at the end systole (the end of the T wave of the electrocardiogram) is measured; left ventricular anterior wall diastolic thickness (LVAWd): the vertical distance from the endocardium to the epicardium of the anterior wall (the side away from the probe) at the end diastole (the top of the R wave) is measured; left ventricular anterior wall systolic thickness (LVAWs): the vertical distance from the endocardium to the epicardium of the anterior wall at the end systole (the end of the T wave) is measured.

[0079] (4) Quantitative analysis of mouse body weight (BW) and heart weight (HW): the mouse is directly weighed on the electronic balance in a conscious state. After the mouse is sacrificed, the heart is quickly dissected and separated, washed with physiological saline to remove blood stains, the surface water is absorbed with filter paper, and the electronic balance is used for weighing. The measured heart weight is compared with the mouse body weight to calculate the ratio.

[0080] (5) Quantitative analysis of cardiac injury markers (CK-MB, LDH, cTnI): Same as test case 3.

[0081] The experimental results are as follows: like Figure 3 As shown in Figure A, echocardiographic results showed that, compared with the DOX treatment group, the left ventricular function of mice in the Fe3O4@ZIF-8 group, Fe3O4NPs group, and DXZ treatment group was improved, as evidenced by a decrease in the left ventricular end-diastolic diameter (LVIDd) and end-systolic diameter (LVIDs), indicating that cardiac systolic and diastolic functions were restored to some extent, with the improvement being more significant in the Fe3O4@ZIF-8 group.

[0082] like Figure 3 As shown in Figure B, the quantitative analysis of left ventricular ejection fraction (LVEF) showed that DOX treatment significantly reduced cardiac pumping function in mice, while Fe3O4@ZIF-8, Fe3O4NPs, and DXZ treatments all improved LVEF levels to varying degrees, indicating that cardiac pumping function was improved, with the Fe3O4@ZIF-8 group showing the most significant improvement.

[0083] like Figure 3 As shown in Figure C, the quantitative analysis results of left ventricular shortening fraction (LVFS) showed that Fe3O4@ZIF-8, Fe3O4NPs and DXZ treatments could all reverse the DOX-induced decrease in LVFS to some extent, further confirming the improvement in cardiac systolic function. Among them, the Fe3O4@ZIF-8 group showed the most significant improvement.

[0084] like Figure 3 As shown in Figure D, the quantitative analysis results of left ventricular end-diastolic diameter (LVIDd) showed that Fe3O4@ZIF-8, Fe3O4NPs and DXZ treatments could alleviate DOX-induced left ventricular dilation to some extent. Although the Fe3O4NPs group showed a certain advantage in terms of numerical values, the difference between it and the Fe3O4@ZIF-8 group did not reach the level of statistical significance.

[0085] like Figure 3 As shown in E, quantitative analysis of left ventricular end-systolic diameters (LVIDs) revealed that Fe3O4@ZIF-8, Fe3O4NPs, and DXZ treatments could all alleviate DOX-induced left ventricular dilation to some extent, with the Fe3O4@ZIF-8 group showing the most significant improvement.

[0086] like Figure 3As shown in FIG. F, the results of the analysis of left ventricular posterior wall diastolic thickness (LVPWd) showed that DOX treatment caused the ventricular wall to thin, while Fe3O4@ZIF-8, Fe3O4NPs and DXZ treatment all improved this change to varying degrees, and the Fe3O4@ZIF-8 group showed a more obvious improvement trend.

[0087] As shown in FIG. G, the results of the quantitative analysis of left ventricular posterior wall systolic thickness (LVPWs) showed that Fe3O4@ZIF-8, Fe3O4NPs and DXZ all reversed the DOX-induced weakening of ventricular wall systolic function to a certain extent. Although the Fe3O4NPs group showed a certain advantage in numerical value, the difference between it and the Fe3O4@ZIF-8 group did not reach a statistically significant level. Figure 3 As shown in FIG. H, the results of the quantitative analysis of left ventricular anterior wall diastolic thickness (LVAWd) showed that DOX treatment significantly reduced the anterior wall thickness, while Fe3O4@ZIF-8, Fe3O4NPs and DXZ treatment all had a certain improvement effect on this index, and the Fe3O4@ZIF-8 group improved more obviously.

[0088] Figure 3 As shown in FIG. I, the results of the analysis of left ventricular anterior wall systolic thickness (LVAWs) showed that Fe3O4@ZIF-8, Fe3O4NPs and DXZ treatment all reversed the DOX-induced decline in anterior wall systolic function to varying degrees, and the Fe3O4@ZIF-8 group showed a more significant improvement effect.

[0089] As shown in FIG. J, the results of the analysis of heart weight / body weight ratio (HW / BW) showed that DOX treatment significantly reduced HW / BW, while Fe3O4@ZIF-8, Fe3O4NPs and DXZ treatment all alleviated this change to a certain extent, and the DXZ group was similar in numerical value to the Fe3O4@ZIF-8 group, the difference did not reach a statistically significant level, suggesting that the protective effect of Fe3O4@ZIF-8 on heart weight / body weight ratio was similar to that of the positive drug DXZ. Figure 3 As shown in FIG. K, the serum CK-MB level was significantly increased in the DOX treatment group, while Fe3O4@ZIF-8, Fe3O4NPs and DXZ treatment all reduced the CK-MB level to varying degrees, and the Fe3O4@ZIF-8 and Fe3O4NPs groups both significantly reduced the CK-MB level, and the Fe3O4NPs group showed a certain advantage in numerical value, but the difference between it and the Fe3O4@ZIF-8 group did not reach a statistically significant level.

[0090] Figure 3 As shown in FIG. L, the results of the analysis of serum AST level showed that DOX treatment significantly increased the serum AST level, while Fe3O4@ZIF-8, Fe3O4NPs and DXZ treatment all reduced the AST level to varying degrees, and the Fe3O4@ZIF-8 and Fe3O4NPs groups both significantly reduced the AST level, and the Fe3O4NPs group showed a certain advantage in numerical value, but the difference between it and the Fe3O4@ZIF-8 group did not reach a statistically significant level.

[0091] As shown in FIG. M, the results of the analysis of serum ALT level showed that DOX treatment significantly increased the serum ALT level, while Fe3O4@ZIF-8, Fe3O4NPs and DXZ treatment all reduced the ALT level to varying degrees, and the Fe3O4@ZIF-8 and Fe3O4NPs groups both significantly reduced the ALT level, and the Fe3O4NPs group showed a certain advantage in numerical value, but the difference between it and the Fe3O4@ZIF-8 group did not reach a statistically significant level. Figure 3 As shown in FIG. N, the results of the analysis of serum BUN level showed that DOX treatment significantly increased the serum BUN level, while Fe3O4@ZIF-8, Fe3O4NPs and DXZ treatment all reduced the BUN level to varying degrees, and the Fe3O4@ZIF-8 and Fe3O4NPs groups both significantly reduced the BUN level, and the Fe3O4NPs group showed a certain advantage in numerical value, but the difference between it and the Fe3O4@ZIF-8 group did not reach a statistically significant level.

[0092] As shown in FIG. O, the results of the analysis of serum Cr level showed that DOX treatment significantly increased the serum Cr level, while Fe3O4@ZIF-8, Fe3O4NPs and DXZ treatment all reduced the Cr level to varying degrees, and the Fe3O4@ZIF-8 and Fe3O4NPs groups both significantly reduced the Cr level, and the Fe3O4NPs group showed a certain advantage in numerical value, but the difference between it and the Fe3O4@ZIF-8 group did not reach a statistically significant level.​​Figure 3 As shown in Fig. 5B, serum LDH levels were significantly increased in the DOX-treated group, while Fe3O4@ZIF-8, Fe3O4NPs and DXZ treatment could effectively reduce the levels of LDH, among which the Fe3O4@ZIF-8 group showed the most significant improvement effect.

[0093] As shown in Fig. 5B, serum LDH levels were significantly increased in the DOX-treated group, while Fe3O4@ZIF-8, Fe3O4NPs and DXZ treatment could effectively reduce the levels of LDH, among which the Fe3O4@ZIF-8 group showed the most significant improvement effect. ​ As shown in Fig. 5B, serum LDH levels were significantly increased in the DOX-treated group, while Fe3O4@ZIF-8, Fe3O4NPs and DXZ treatment could effectively reduce the levels of LDH, among which the Fe3O4@ZIF-8 group showed the most significant improvement effect.

[0094] In summary, Fe3O4@ZIF-8 nanoszyme can achieve stable peroxidase / superoxide dismutase-like activity, thereby significantly enhancing its antioxidant and anti-inflammatory capacity, and thus has good therapeutic effect on diseases with oxidative stress and / or inflammation imbalance, and has broad application prospects in drug, especially doxorubicin cardiotoxicity intervention and other ROS / inflammation-related heart diseases.

[0095] The foregoing examples are illustrative only and are not intended to limit the scope of the methods described herein. The appended claims are intended to claim as broad a range as is allowed under the rules. The examples presented herein are presented by way of illustration only and are not intended to limit the scope of the application. Thus, applicants intend that the appended claims are not limited to the choice of examples presented herein. Some of the ranges in the claims are intended to cover sub-ranges within the range. The ranges in the claims are intended to cover all possible sub-ranges. The application is intended to cover all possible combinations of the elements in the claims.

Claims

1. The application of Fe3O4@ZIF-8 nanozyme in the preparation of drugs for treating and / or preventing myocardial injury, characterized in that, The Fe3O4@ZIF-8 nanozyme has a core-shell structure with Fe3O4 as the nanocore and ZIF-8 as the outer coating; the average particle size of the Fe3O4@ZIF-8 nanozyme is (12~22) nm.

2. The application according to claim 1, characterized in that, The myocardial injury is drug-induced myocardial injury caused by an imbalance of oxidative stress and / or inflammation.

3. The application according to claim 2, characterized in that, The drug is selected from at least one of doxorubicin, cisplatin, fluorouracil, cyclophosphamide, daunorubicin, epirubicin, and pirarubicin.

4. The application according to claim 2, characterized in that, The drug-induced myocardial injury includes at least one of the following: drug-induced cardiotoxicity, cardiac atrophy, cardiac dysfunction, cardiac fibrosis, cardiomyocyte ferroptosis, cardiomyocyte mitochondrial dysfunction, and accumulation of reactive oxygen species in cardiomyocytes.

5. The application according to any one of claims 1 to 4, characterized in that, The preparation of the Fe3O4@ZIF-8 nanozyme includes the following steps: Fe3O4 nanonucleus, zinc salt, and 2-methylimidazole are added to methanol and mixed, and then mixed at 3~5℃ to obtain Fe3O4@ZIF-8 nanozyme.

6. The application according to claim 5, characterized in that, The preparation of the Fe3O4 nanonucleus includes the following steps: adding iron salt to a solvent and mixing, heating to 55~65℃ under a nitrogen atmosphere, adding an alkaline solution, and heating to 75~85℃ to obtain the nanonucleus.

7. The application according to claim 5, characterized in that, The molar ratio of the Fe3O4 nanonucleus to the zinc salt is (2~4):

1.

8. The application according to claim 1, characterized in that, The concentration of the Fe3O4@ZIF-8 nanozyme is (25~200) µg / mL.

9. Application of Fe3O4@ZIF-8 nanozyme in the preparation of drugs for the treatment and / or prevention of myocardial fibrosis.

10. Application of Fe3O4@ZIF-8 nanozyme in the preparation of drugs to improve cardiac dysfunction.

Images