Improved ruthenium-manganese nanosheets, and methods of making and using the same

Abstract

The application provides an improved ruthenium-manganese nanosheet, and the periphery of the ruthenium-manganese nanosheet is coated with phospholipid. The application also provides a preparation method of the improved ruthenium-manganese nanosheet, wherein solid powders of triruthenium dodecacarbonyl, dimanganese decacarbonyl and salicylic acid are mixed, dissolved in oleylamine, heated in a protective atmosphere to obtain a ruthenium-manganese nanosheet product with an initial snowflake-like morphology, and after cooling to room temperature, the product is washed with a cyclohexane and anhydrous ethanol mixture, and centrifuged to collect the product; the obtained product is dispersed in an organic solvent together with phospholipid, then the organic solvent is removed, ultrapure water is added, ultrasonic oscillation is performed, isosmotic liquid is used for washing, and centrifugation is performed to obtain the phospholipid-coated ruthenium-manganese nanosheet. The application also provides application of the improved ruthenium-manganese nanosheet in preparation of a drug for treating myocardial ischemia-reperfusion injury. The ruthenium-manganese nanosheet provided by the application can realize catalytic elimination of ROS function, inhibit release of inflammatory factors and invasion of neutrophils, so as to achieve the purpose of treating myocardial ischemia-reperfusion injury.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to an improved method for preparing ruthenium manganese nanosheets and their applications. Background Technology

[0002] Cardiovascular disease remains a leading cause of death worldwide, accounting for 31.5% of all deaths annually, with approximately 7.4 million deaths related to myocardial infarction. Stenosis or occlusion of the coronary arteries restricts blood flow to the heart, causing myocardial damage and acute myocardial infarction. Currently, timely cardiac reperfusion is considered the primary means of saving lives in acute myocardial infarction. Clinically, medications or revascularization are commonly used to restore coronary blood flow to ischemic areas of the myocardium. However, ischemic myocardium, upon receiving reperfusion, suffers ischemia-reperfusion injury, thus limiting the effectiveness of reperfusion therapy and even increasing the risk of heart failure. Unfortunately, there is still no effective treatment for myocardial ischemia-reperfusion injury.

[0003] Myocardial ischemia-reperfusion injury is a complex process mediated by multiple factors and mechanisms; oxidative stress and inflammatory response are important pathological mechanisms triggering myocardial ischemia-reperfusion injury. During cardiac reperfusion, the mitochondrial electron transport chain generates a large number of reactive oxygen species (ROS), leading to mitochondrial damage. Subsequently, cytochrome C is released from the mitochondria, activating caspase and further inducing cardiomyocyte apoptosis. Under the influence of ROS oxidative damage, damaged cardiomyocytes can release a large number of inflammatory factors, thereby exacerbating the inflammatory response and promoting the recruitment of neutrophils to the heart. Neutrophils invading myocardial tissue are activated under the stimulation of inflammatory factors, and then further aggravate myocardial damage by releasing proteases, producing ROS, and secreting inflammatory factors. However, the short half-life, poor stability, low bioavailability, and adverse clinical reactions of small molecule drugs in the treatment of myocardial ischemia-reperfusion injury severely limit the efficacy and application of these drugs. For example, while cyclosporine A can protect mitochondria by inhibiting the opening of the mitochondrial permeability transition pore and preventing calcium ion influx, phase III clinical trials have shown that cyclosporine A does not alleviate myocardial ischemia-reperfusion injury. Metoprolol can provide cardiac protection by reducing neutrophil-mediated microvascular embolism, but unfortunately, phase III clinical trials also failed to show that metoprolol effectively reduces myocardial ischemia-reperfusion injury. Therefore, there is an urgent need to find effective methods and drugs for treating myocardial ischemia-reperfusion injury.

[0004] The pathogenesis of myocardial ischemia-reperfusion injury revealed above shows that the large amount of ROS generated during reperfusion can cause damage to mitochondria and myocardium, triggering an inflammatory cascade and promoting neutrophil invasion into the reperfused myocardium, further aggravating myocardial damage. Therefore, eliminating ROS generated during cardiac reperfusion can reduce oxidative stress in cardiomyocytes, inhibit downstream inflammatory cascades and neutrophil infiltration, providing a potential approach for treating myocardial ischemia-reperfusion injury. Ruthenium and manganese have good biocompatibility and wide applications in biomedical imaging. Furthermore, because ruthenium and manganese have multiple valence states, their valence state changes can mimic the function of biological enzymes, achieving catalytic decomposition and elimination of ROS. Moreover, the introduction of manganese can regulate the valence state of ruthenium, and the pinning effect of ruthenium sites can maintain the long-term stability of the catalytic reaction, ensuring high catalytic activity under universal pH conditions. This can solve the problem of catalytic reactions under different pH conditions, such as in biological fluid environments and lysosomes. Furthermore, the topological configuration of nanomaterials can improve their catalytic performance. In heterogeneous catalysis, snowflake-shaped nanomaterials have a large contact surface area with the substrate, which can enhance catalytic efficiency. Therefore, by adjusting the topological structure of nanomedicines to improve their catalytic performance, the principle of topological catalysis can be used to guide the treatment of myocardial ischemia-reperfusion injury.

[0005] Currently, the preparation of ruthenium and manganese nanomaterials uses a high-temperature oil-phase method, which results in the nanomaterials themselves being highly hydrophobic, making them difficult to disperse in aqueous solutions and thus unsuitable for in vivo applications. Phospholipids are amphoteric molecules with good biocompatibility, consisting of a hydrophilic end containing a nitrogen or phosphorus group and a hydrophobic tail composed of a fatty acid chain. By coating ruthenium and manganese nanomaterials with phospholipids, the hydrophilic end of the phospholipid faces outward, contacting the aqueous solution, while the hydrophobic tail faces inward, contacting the ruthenium and manganese nanomaterials. This improves the hydrophilicity of the ruthenium and manganese nanomaterials, enabling them to disperse well in aqueous solutions. This provides assistance for the clinical translation of ruthenium and manganese nanomaterials for the treatment of myocardial ischemia-reperfusion injury. Summary of the Invention

[0006] To address the aforementioned technical problems in the prior art, this invention provides an improved ruthenium manganese nanosheet (RuMnBNSP), its preparation method, and its uses. This improved ruthenium manganese nanosheet, its preparation method, and its uses aim to solve the technical problem of poor therapeutic effects of existing drugs on myocardial ischemia-reperfusion injury.

[0007] The present invention provides an improved ruthenium manganese nanosheet, wherein the outer periphery of the ruthenium manganese nanosheet is coated with phospholipids.

[0008] This invention also provides a method for preparing the above-mentioned improved ruthenium manganese nanosheets, comprising the following steps:

[0009] S1) A mixture of dodecacarbonyltriruthenium, decacarbonyldimanganese, and salicylic acid solid powders was dissolved in oleylamine and heated under a protective atmosphere to obtain ruthenium-manganese nanosheets with an initial snowflake-like morphology. After cooling to room temperature, the nanosheets were reacted with cyclohexane and anhydrous ethanol.

[0010] Wash the mixture and collect the product by centrifugation;

[0011] S2) Disperse the product obtained in step 1) with phospholipids in an organic solvent, then remove the organic solvent and add ultrapure water.

[0012] The product was subjected to ultrasonic oscillation, washed with isotonic liquid, and centrifuged to collect the final product, yielding phospholipid-coated ruthenium manganese nanomaterials.

[0013] Furthermore, in S1), the mass ratio of dodecyltriruthenium carbonyl, decacarbonyldimanganese, and salicylic acid is 8:(1-3):10. The purity of the oleylamine is 70%-90%; and the amount of oleylamine added maintains the salicylic acid concentration at 1 mg / mL. -1 ~4mg / mL -1 ;

[0014] The protective atmosphere is provided by argon or nitrogen. The volume ratio of the cyclohexane and anhydrous ethanol mixture is 1:(1-3). The washing is performed 2-4 times. The centrifugation collection requires a centrifugation speed of 8000g-12000g, a time of 5min-15min, and a temperature of 24℃-26℃.

[0015] Further, the phospholipid in S2) is any one or a combination of two or more of dipalmitoylphosphatidylcholine, distearylphosphatidylcholine, lecithin, distearylphosphatidylethanolamine-methoxy polyethylene glycol, distearylphosphatidylethanolamine-amino polyethylene glycol, and distearylphosphatidylethanolamine-carboxylated polyethylene glycol, wherein the polyethylene glycol has a molecular weight of 1000 Da to 5000 Da, and the organic solvent is chloroform or dichloromethane, and the added organic solvent liquid volume is... The mass percentage concentration ratio of ruthenium manganese nanosheets to phospholipids in an organic solvent is 1:(2-4). The ultrasonic oscillation is continuous ultrasonic oscillation with a power of 60W-180W and a time of 10min-20min. The isotonic liquid is physiological saline or PBS solution. The washing is performed 2-4 times. The centrifugation collection requires a centrifugation speed of 8000g-12000g, a time of 5min-15min, and a temperature of 24℃-26℃.

[0016] This invention also provides the application of the above-mentioned improved ruthenium manganese nanosheets in the preparation of medicaments for treating myocardial ischemia-reperfusion injury.

[0017] This invention also provides the application of the above-described improved ruthenium manganese nanosheets in the preparation of topological catalysts.

[0018] This invention provides a method for preparing snowflake-shaped RuMn BNSP agents, elucidating that these nanosheet agents possess topological catalytic functions, can eliminate excess ROS generated during myocardial ischemia-reperfusion, and can inhibit the release of inflammatory factors and neutrophil invasion in the cardiac reperfusion zone. This invention details and demonstrates the biological mechanism of snowflake-shaped RuMn BNSP agents in treating myocardial ischemia-reperfusion injury, providing a novel nanosheet agent for the effective treatment of myocardial ischemia-reperfusion injury.

[0019] Compared with existing technologies, the technical effects of this invention are positive and significant. This invention provides a method for preparing a RuMn BNSP agent with a snowflake-like morphology. Due to the introduction of manganese, the valence state of ruthenium can be controlled, maintaining the pinning effect of ruthenium sites. This allows it to catalyze the elimination of ROS under different pH conditions, such as in biological fluid environments and lysosomes. Furthermore, its snowflake-like topological morphology provides a larger contact reaction area with the substrate ROS. By controlling the topological morphology to improve catalytic reaction efficiency, and through efficient ROS elimination, the release of inflammatory factors and neutrophil invasion are inhibited, thereby achieving the treatment of myocardial ischemia-reperfusion injury. Attached Figure Description

[0020] Figure 1 The images shown are transmission electron microscopy (a) and energy-dispersive X-ray spectral elemental mapping (b) of the RuMn BNSP agent with a snowflake-like morphology obtained in Example 1 of this invention.

[0021] Figure 2 The RuMn BNSP reagent with snowflake-like morphology in Example 2 of this invention can achieve the catalytic decomposition of H2O2.

[0022] Figure 3 This invention demonstrates the scavenging effect of the snowflake-shaped RuMn BNSP agent on hydroxyl radicals (·OH) in Example 3 of this invention.

[0023] Figure 4 In Example 4 of this invention, the snowflake-shaped RuMn BNSP agent is used to target superoxide anion radicals (O2·). - The removal effect.

[0024] Figure 5 This invention relates to Example 5, which describes the effect of different concentrations of RuMn BNSP agents with snowflake-like morphology on the viability of normal cardiac fibroblasts.

[0025] Figure 6In Example 6 of this invention, when cardiac fibroblasts are under oxidative stress, the snowflake-shaped RuMn BNSP agent can improve the cell viability of cardiac fibroblasts. Figure 6 a) and can eliminate ROS in cardiac fibroblasts. Figure 6 b and 6c).

[0026] Figure 7 In Example 7 of this invention, when cardiac fibroblasts are under oxidative stress, the snowflake-shaped RuMn BNSP agent can reduce the inflammatory response of cardiac fibroblasts and inhibit the release of inflammatory factors, including IL-6. Figure 7 a) CCL2 ( Figure 7 b) and CXCL2 Figure 7 c).

[0027] Figure 8 In Example 8 of this invention, the snowflake-shaped RuMn BNSP agent can improve cardiac function in mice with a myocardial ischemia-reperfusion model.

[0028] Figure 9 In Example 9 of this invention, the snowflake-shaped RuMn BNSP agent can reduce the infarct area in a mouse model of myocardial ischemia-reperfusion.

[0029] Figure 10 In Example 10 of this invention, during myocardial ischemia-reperfusion, the snowflake-shaped RuMn BNSP agent can inhibit IL-6 (…). Figure 10 a) CCL2 ( Figure 10 b) and CXCL2 Figure 10 c) Release of inflammatory factors, eliminating ROS in the myocardial reperfusion zone. Figure 10 d), and inhibiting neutrophil invasion of myocardial tissue ( Figure 10 e).

[0030] Figure 11 In Example 11 of this invention, the biosafety of the RuMn BNSP agent with snowflake-like morphology was evaluated in live animals. Detailed Implementation

[0031] The following detailed description, in conjunction with the accompanying drawings, will help to understand the present invention, but does not limit the scope of the invention.

[0032] Example 1

[0033] Weigh 8 mg of dodecacarbonyltriruthenium, 1.5 mg of decacarbonyldimanganese, and 10 mg of salicylic acid, and dissolve them in 5 mL of oleylamine. Then, heat the above liquid to 230 °C under an argon atmosphere and react for 3 h. After the reaction is complete, cool the liquid to room temperature, wash it three times with a mixture of cyclohexane and ethanol (volume ratio 1:2), centrifuge at 10000 g for 10 min, and collect the product.

[0034] Next, 1 mg of the above product and 2 mg of distearate phosphatidylethanolamine-methoxy polyethylene glycol (polyethylene glycol molecular weight 2000 Da) were dispersed in 1 mL of chloroform. At this point, the mass percentage concentration ratio of ruthenium manganese nanosheets to phospholipids in chloroform was 1:2, thus modifying the surface of the ruthenium manganese nanosheets. Subsequently, the chloroform was removed by rotary evaporator under vacuum. After the chloroform was removed, ultrapure water was added, and the mixture was ultrasonically vibrated at 120 W for 15 min until the solid product was uniformly dispersed in the liquid. Finally, the mixture was centrifuged at 10000 g for 10 min, and washed three times with PBS to obtain the improved ruthenium manganese nanosheets of this invention.

[0035] Transmission electron microscopy revealed that the nanosheet drug prepared in this invention has a snowflake-like layered structure, specifically as follows: Figure 1 The nanosheet drug prepared according to the present invention exhibits a snowflake-like layered structure as observed by transmission electron microscopy. Figure 1 a) The elemental mapping diagram of the energy-dispersive X-ray spectroscopy shows that the snowflake-shaped RuMn BNSP reagent prepared in this invention contains ruthenium and manganese. Figure 1 b).

[0036] Example 2

[0037] Before evaluating the snowflake-like morphology of the RuMn BNSP reagent (the improved ruthenium manganese nanosheets of Example 1) to catalytically decompose H2O2, dissolved oxygen in the ultrapure water needs to be removed. This is done by repeating vacuuming and nitrogen bubbling five times each, for a total of 30 minutes. Then, 100 μg mL -1 Snowflake-shaped RuMn BNSP reagent and 5 mM H2O2 were mixed in degassed ultrapure water. Oxygen generation in the degassed ultrapure water was continuously monitored using a dissolved oxygen analyzer. A control group was set up containing 5 mM H2O2 (without the snowflake-shaped RuMn BNSP reagent). Details are as follows... Figure 2 As described above, the snowflake-shaped RuMnBNSP agent can catalyze the decomposition of H2O2 to generate O2, causing the O2 content in the solution to increase with the extension of reaction time.

[0038] Example 3

[0039] The reaction system for evaluating the snowflake-like morphology of RuMn BNSP reagent (the improved ruthenium manganese nanosheets of Example 1) catalyzing the decomposition of hydroxyl radicals (·OH) contained 200 μM FeSO4, 400 μM H2O2, 24.5 μM crystal violet, and different concentrations of snowflake-like morphology RuMn BNSP reagent (0, 5, 10, 20, 40, and 80 μg mL). -1 FeSO4 reacts with H2O2 via a Fenton reaction to produce ·OH; crystal violet can be oxidized and degraded by ·OH, resulting in a decrease in absorbance at 583 nm; therefore, crystal violet can reflect the content of ·OH. The above reaction system needs to be carried out in a dark environment. After reacting for 30 minutes, the centrifuge speed is set to 20000g, and centrifuged for 5 minutes. The supernatant is collected, and its absorption spectrum is detected and recorded using a UV-Vis spectrometer. (Details follow...) Figure 3 As the concentration of RuMn BNSP reagent with snowflake-like morphology increases, more ·OH can be removed, resulting in reduced crystal violet degradation and an increase in the absorbance of the solution at 583 nm.

[0040] Example 4

[0041] The activity of snowflake-shaped RuMn BNSP reagent (modified ruthenium manganese nanosheets from Example 1) against superoxide anion radicals (O2·) was detected using a superoxide dismutase (SOD) activity assay kit produced by Shanghai Beyotime Biotechnology Co., Ltd. - The scavenging ability of SOD was assessed. First, 151 μL of SOD detection buffer, 8 μL of WST-8 chromogenic solution, and 1 μL of enzyme solution were mixed to obtain the WST-8 / enzyme working solution. Then, 20 μL of RuMn BNSP reagent with different concentrations (0, 5, 10, 20, 40, and 80 μg / mL) was taken. -1 Mix 160 μL of WST-8 / enzyme working solution with 20 μL of reaction starter solution and incubate at 37°C in the dark for 30 min. Set up a corresponding control group according to the kit instructions. Finally, measure the absorbance of the solution using a microplate reader. (Details follow...) Figure 4 The snowflake-shaped RuMn BNSP agent is effective against O2· - The removal of O2 is concentration-dependent, meaning that as the concentration of the snowflake-shaped RuMn BNSP reagent of the present invention increases, the O2· - The clearance rate also increased.

[0042] Example 5

[0043] Cardiac fibroblasts were donated by the Translational Medicine Research Center of Tongji University Affiliated Oriental Hospital. They were cultured in DMEM medium containing 10% fetal bovine serum, 1% streptomycin, and 1% penicillin. The cell culture incubator temperature was set at 37°C, and 5% CO2 gas was introduced. The cytotoxicity of the snowflake-shaped RuMnBNSP agent (the modified ruthenium manganese nanosheets of Example 1) to the cells was evaluated using a CCK-8 assay. First, cardiac fibroblasts were cultured at 1.5 × 10⁻⁶ cells / year. 4 Cells were seeded at a density of 10 cells / well into 96-well flat-bottomed culture plates and cultured in a cell incubator for 24 h. Then, the culture medium was aspirated, and RuMn BNSP reagent containing different concentrations of snowflake-like morphology (0, 5, 10, 20, 40, and 60 μg / mL) was added again. -1 Fresh culture medium was transferred to a cell culture incubator and cultured for 24 hours. Then, the culture medium was aspirated, and the cells were washed three times with pre-warmed PBS. Cell culture medium containing CCK-8 was added, and the 96-well plate was transferred to a cell culture incubator for 1 hour of incubation. Finally, the absorbance of the 96-well plate was recorded using a microplate reader. (Details follow...) Figure 5 The concentration of RuMn BNSP agent with snowflake-like morphology, co-incubated with cardiac fibroblasts, was as high as 60 μg / mL. -1 However, the cell viability of cardiac fibroblasts remained above 95%, indicating that the snowflake-shaped RuMn BNSP agent did not produce significant toxicity to cardiac fibroblasts, confirming its good biocompatibility with cardiac fibroblasts.

[0044] Example 6

[0045] To investigate the oxidative stress resistance and cardiac fibroblast protection effects of snowflake-shaped RuMn BNSP agents (the improved ruthenium manganese nanosheets of Example 1), cardiac fibroblasts were subjected to 6 × 10⁻⁶ ppm of urea nitrogen. 4 Seeds were generated at a density of 1.5 × 10⁶ cells / well in 24-well flat-bottomed culture plates, and cultured at a density of 1.5 × 10⁶ cells / well. 4 Cells were seeded at a density of 10 cells / well into 96-well flat-bottomed culture plates and cultured in a cell incubator for 24 h. Then, the culture medium was aspirated, and RuMn BNSP reagent containing different concentrations of snowflake-like morphology (0, 2.5, 5, and 10 μg / mL) was added again. -1 Fresh culture medium was transferred to a cell culture incubator and cultured for 30 min. Then, 800 μM H2O2 was added, and the cells were cultured for another 24 h to induce oxidative stress. Afterward, the culture medium was aspirated, and the cells were washed three times with pre-warmed PBS. Cell culture medium containing CCK-8 was added to 96-well plates and incubated for 1 h. The absorbance of the 96-well plates was recorded using a microplate reader. (Details are as follows...) Figure 6 As described in section a, with the increase of the concentration of RuMn BNSP agent with snowflake-like morphology, the cell viability of cardiac fibroblasts also increased, indicating that the RuMn BNSP agent with snowflake-like morphology can protect cardiac fibroblasts under oxidative stress and improve their cell viability. For cardiac fibroblasts in 24-well plates, the cells were washed three times with preheated HBSS. Then, HBSS containing the ROS fluorescent probe DCFH-DA (20 μM) and cardiac fibroblasts were co-incubated in a cell culture incubator for 30 min. After incubation, the cells were washed three times with preheated HBSS, and then photographed and recorded under an inverted fluorescence microscope; details are as follows. Figure 6 As described in b and 6c, the ROS fluorescence intensity decreased with the increase of the concentration of RuMn BNSP agent with snowflake-like morphology, indicating that RuMn BNSP agent with snowflake-like morphology can eliminate excess ROS in cells through topological catalysis, thereby achieving the purpose of protecting cardiac fibroblasts.

[0046] Example 7

[0047] To investigate the effect of snowflake-shaped RuMn BNSP agent (the improved ruthenium manganese nanosheets of Example 1) on inhibiting the release of inflammatory factors from cardiac fibroblasts, cardiac fibroblasts were first subjected to treatment at 1.5 × 10⁻⁶ ppm. 5 Cells were seeded at a density of 10 cells / well into 12-well flat-bottomed culture plates and cultured in a cell culture incubator for 24 h. Then, the culture medium was aspirated and replaced with RuMn BNSP reagent containing different concentrations of snowflake-like morphology (0, 2.5, 5, and 10 μg / mL). -1 Fresh culture medium was incubated in an incubator for 30 minutes, followed by the addition of 800 μM H2O2. The cells were then cultured in a cell culture incubator to induce oxidative stress in cardiac fibroblasts. After 12 hours of culture, the cardiac fibroblasts were centrifuged at 500 g for 5 minutes, and the supernatant was collected. Finally, inflammatory factors such as IL-6, CCL2, and CXCL2 were detected using an ELISA kit from Hangzhou Lianke Biotechnology Co., Ltd. (Details follow...) Figure 7 As the concentration of RuMn BNSP agent with snowflake-like morphology in the culture medium increases, the secreted inflammatory factor IL-6 (…) Figure 7 a) CCL2 ( Figure 7 b) and CXCL2 Figure 7 c) It shows a clear downward trend.

[0048] Example 8

[0049] Healthy male wild-type C57 BL / 6J mice aged 8 to 10 weeks were anesthetized with isoflurane gas. Oxygen was continuously supplied to the mice via endotracheal intubation and a small animal ventilator. The body temperature of the mice was maintained at 35-36°C by controlling the temperature of the operating table. The chest was then prepared, and a small incision was made on the left side of the chest using microsurgical scissors to expose the heart. The left anterior descending coronary artery was ligated with 8-0 silk suture to induce myocardial ischemia for 45 minutes. The ligation was then released, restoring myocardial blood supply, and the chest incision was sutured. Finally, 100 μL of 2.5 μg g of oxygen was injected into the mice via the tail vein. -1 The RuMn BNSP agent exhibited a snowflake-like morphology (the improved ruthenium manganese nanosheets of Example 1). The sham-operated mice underwent the same procedure as above, except that the left anterior descending coronary artery was not ligated. After 48 hours, echocardiography was used to assess cardiac function in the mice; specifically as follows... Figure 8 Compared with the sham-operated group, the left ventricular anterior wall motion was significantly weakened in the myocardial ischemia-reperfusion group, indicating impaired cardiac systolic function. In the myocardial ischemia-reperfusion group, after treatment with snowflake-shaped RuMn BNSP via tail vein injection, small animal echocardiography showed significant improvement in left ventricular anterior wall motion, indicating that the snowflake-shaped RuMn BNSP can effectively improve cardiac function and reduce myocardial ischemia-reperfusion injury in the myocardial ischemia-reperfusion group.

[0050] Example 9

[0051] Forty-eight hours after reperfusion, the mice were anesthetized with isoflurane gas. The thoracic cavity was then opened, following the procedure in Example 8. The left anterior descending coronary artery was ligated again with 8-0 silk suture to block blood flow; the ligation site was the same as in Example 8. Next, 100 μL of 3% Evans blue solution was injected into the mice via the tail vein. The mouse heart was then quickly cut off and frozen in dry ice until completely frozen. Subsequently, the heart was sliced ​​into five 1 mm thick slices perpendicular to the long axis of the left ventricle. Finally, the heart slices were immersed in a 1% triphenyltetrazolium chloride (TTC) solution at 37°C. After 30 minutes, photographs were taken for recording the results. Figure 9 In the mouse model of myocardial ischemia-reperfusion, compared with the group injected with snowflake-shaped RuMn BNSP (the improved ruthenium manganese nanosheets of Example 1), the mice injected with saline had a larger myocardial infarction area, indicating that the snowflake-shaped RuMn BNSP can effectively reduce myocardial ischemia-reperfusion injury.

[0052] Example 10

[0053] A mouse model of myocardial ischemia-reperfusion injury was constructed using the same method as in Example 8. Forty-eight hours after reperfusion, mouse blood was collected in centrifuge tubes and allowed to agglutinate naturally at room temperature. After 30 minutes, the centrifuge was set to 1000g and centrifuged for 10 minutes, and serum was collected. Then, the inflammatory factors IL-6, CCL2, and CXCL2 were detected using an ELISA kit from Hangzhou Lianke Biotechnology Co., Ltd.; specifically as follows... Figure 10 As described in a, 10b, and 10c, compared to the saline treatment group, the snowflake-shaped RuMnBNSP agent treatment group (the modified ruthenium manganese nanosheets of Example 1) had lower IL-6 levels ( Figure 10 a) CCL2 ( Figure 10 b) and CXCL2 Figure 10 c) The significantly reduced relative expression level indicates that the nanosheet drug can inhibit the secretion of inflammatory factors during myocardial ischemia-reperfusion injury. Furthermore, mice were euthanized, their hearts were harvested, and frozen sections of myocardial tissue were prepared. These sections were then stained with the ROS fluorescent probe dihydroethidium (DHE), mounted, and photographed under a fluorescence microscope for recording. Specifically... Figure 10 As described in section d, myocardial sections in the saline group showed significant DHE fluorescence signals, indicating the generation of a large amount of ROS at the site of myocardial injury. In contrast, the DHE fluorescence signal on myocardial sections from the snowflake-shaped RuMn BNSP drug group was significantly weakened, indicating that the nanosheet drug can clear ROS generated during myocardial ischemia-reperfusion, thereby protecting myocardial tissue. Furthermore, paraffin sections of some mouse hearts were prepared and immunofluorescence staining was performed. Neutrophils in the infarcted area were identified using anti-Ly6G primary antibody, followed by detection of the anti-Ly6G primary antibody using FITC-labeled secondary antibody. Specifically... Figure 10 As described above, the immunofluorescence imaging of mouse myocardium confirmed that the number of neutrophils in the reperfusion area of ​​the heart was significantly reduced in the RuMn BNSP treatment group with snowflake-like morphology compared with the saline treatment group, indicating that RuMn BNSP can inhibit the invasion of neutrophils into myocardial tissue, thereby reducing myocardial damage in the reperfusion area.

[0054] Example 11

[0055] Eighteen healthy male ICR mice were randomly divided into three groups: a control group, a low-dose group, and a high-dose group (n=6 per group). Then, different doses of snowflake-shaped RuMn BNSP agent (the modified ruthenium manganese nanosheets of Example 1) (0, 10, 20 μg g) were injected via the tail vein. -1After a 14-day observation period, whole blood was collected from all mice. Serum was separated for blood biochemical analysis, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea, and creatinine. Whole blood was used for routine blood tests, including white blood cell (WBC), lymphocytes, granulocytes, red blood cells (RBC), and platelets (PLT). The mice were then euthanized, and their hearts, livers, spleens, lungs, and kidneys were harvested to prepare paraffin sections of myocardial tissue, which were then stained with hematoxylin and eosin (H&E). (Details follow...) Figure 11 As shown in a-j, no significant abnormalities were observed in the blood biochemical indicators and routine blood tests of the mice in each group. H&E staining showed no significant pathological changes such as inflammation, necrosis, congestion, or hemorrhage in the major organs (heart, liver, spleen, lungs, and kidneys) of the mice in each group. In summary, the snowflake-shaped RuMn BNSP agent exhibits good biocompatibility in animals.

Claims

1. The application of an improved ruthenium-manganese nanosheet in the preparation of a drug for treating myocardial ischemia-reperfusion injury, wherein the improved ruthenium-manganese nanosheet is peripherally coated with phospholipids; the preparation method of the improved ruthenium-manganese nanosheet includes the following steps: S1) Mix dodecyltriruthenium carbonyl, decacarbonyldimanganese and salicylic acid solid powders, dissolve in oleylamine, heat under a protective atmosphere to obtain ruthenium manganese nanosheets with initial snowflake morphology, cool to room temperature, wash with a mixture of cyclohexane and anhydrous ethanol, and collect the product by centrifugation; The mass ratio of dodecacarbonyltriruthenium, decacarbonyldimanganese, and salicylic acid is 8:(1~3):10, and the purity of the oleylamine is 70%~90%; the amount of oleylamine added is such that the concentration of salicylic acid is maintained at 1 mg / mL. -1 ~ 4 mg / mL -1 ; The protective atmosphere is provided by argon or nitrogen, the volume ratio of the cyclohexane and anhydrous ethanol mixture is 1:(1~3), the washing is performed 2~4 times, the centrifugation speed is 8000 g~12000 g, the time is 5 min~15 min, and the temperature is set to 24 ℃~26 ℃. S2) The product obtained in S1) is dispersed with phospholipids in an organic solvent, then the organic solvent is removed, ultrapure water is added, ultrasonic oscillation is performed, and the product is washed with an isotonic liquid. The final product is collected by centrifugation to obtain phospholipid-coated ruthenium manganese nanomaterials. The phospholipid is any one or a combination of two or more of dipalmitoylphosphatidylcholine, distearylphosphatidylcholine, lecithin, distearylphosphatidylethanolamine-methoxy polyethylene glycol, distearylphosphatidylethanolamine-amino polyethylene glycol, and distearylphosphatidylethanolamine-carboxylated polyethylene glycol. The polyethylene glycol has a molecular weight of 1000 Da to 5000 Da. The organic solvent is chloroform or dichloromethane, and the amount of added organic solvent is such that the mass percentage concentration ratio of ruthenium manganese nanosheets to phospholipids in the organic solvent is 1:(2~4). The ultrasonic oscillation is continuous ultrasonic oscillation with a power of 60 W to 180 W and a time of 10 min to 20 min. The isotonic liquid is physiological saline or PBS solution. The washing is performed 2 to 4 times. The centrifugation collection requires a centrifugation speed of 8000 g to 12000 g for 5 min to 15 min, and the temperature is set to 24 ℃. 26 ℃.

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