Nano enzyme as well as preparation method and application thereof

Abstract

The invention relates to the field of biological medicine, in particular to a nano-enzyme and a preparation method and application thereof.The nano-enzyme comprises a Mo2C substrate, and ruthenium atoms and cobalt atoms are anchored to the Mo2C substrate; the nano enzyme can directly catalyze NADH to be regenerated into NAD +, and a simple and effective treatment strategy is provided for ischemia / reperfusion (I / R) treatment.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a nanozyme, its preparation method, and its application. Background Technology

[0002] Myocardial infarction remains a leading cause of death worldwide. Reperfusion therapy is currently the standard treatment for myocardial infarction, but this process can lead to cardiomyocyte dysfunction, known as ischemia-reperfusion (I / R) injury. During ischemia, myocardial hypoxia impairs mitochondrial aerobic respiration, leading to reduced ATP synthesis and enhanced anaerobic glycolysis, which in turn causes mitochondrial dysfunction. After reperfusion, mitochondrial dysfunction results in incomplete reduction reactions during reoxygenation, triggering a surge in reactive oxygen species (ROS) production, ultimately leading to a "cytokine storm." Over time, ROS and inflammatory mediators exacerbate mitochondrial damage, disrupting its bioenergy metabolism and ultimately causing cell death. Current pharmacological interventions for ischemia-reperfusion injury primarily focus on alleviating oxidative stress and inflammatory responses. However, these strategies cannot prevent ischemia-reperfusion injury because they cannot restore mitochondrial bioenergy metabolism or redox homeostasis.

[0003] Nicotinamide adenine dinucleotide (NAD) + As a key metabolic coenzyme, NADH maintains mitochondrial bioenergy metabolism through electron transport mechanisms in the tricarboxylic acid cycle (TCA) and the mitochondrial electron transport chain (ETC), while also activating the SIRT1 protein to maintain mitochondrial structural integrity. During myocardial ischemia, ETC dysfunction hinders NADH oxidation, leading to NAD+ depletion. + It is depleted due to enhanced glycolysis and PARP-1-mediated consumption, thereby destroying NAD. + NADH homeostasis and influences ATP production. + NADH imbalance disrupts energy metabolism: on the one hand, it inhibits mitochondrial oxidative phosphorylation (leading to reduced ATP production), and on the other hand, it forces cells to shift to inefficient glycolysis. Simultaneously, this imbalance also disrupts redox balance through excessive ROS accumulation—NADH-driven electron chain leakage exacerbates oxidative stress, while NAD... + Decreased levels weaken the antioxidant defense system. Reperfusion exacerbates NAD through reactive oxygen species (ROS)-induced oxidation. + This leads to exhaustion, while simultaneously inhibiting SIRT1-dependent repair mechanisms and triggering an inflammatory cascade, ultimately creating a vicious cycle of oxidative stress, mitochondrial breakdown, and cardiomyocyte death.

[0004] To address the above issues, numerous studies have explored exogenous NAD. + Complementary strategies, including intravenous NAD+ +Or its precursors (such as nicotinamide mononucleotide NMN and nicotinamide ribonucleoside NR), via exogenous NAD + Supplement, alleviate NAD + / NADH imbalance. However, due to NAD + Due to poor membrane permeability and low intracellular transport efficiency, direct delivery methods have low bioavailability. Furthermore, the application of NMN and NR is limited by their NAD+ content. + Situations such as low supplementation efficiency, concerns about potential side effects, and the need for high-dose administration. Summary of the Invention

[0005] This invention aims to provide a nanozyme, its preparation method, and its application for the direct catalytic regeneration of NADH into NAD. + This replenishes NAD + And restore normal NAD + NADH balance reduces apoptosis, inhibits the release of inflammatory cytokines, and reactivates the NAMPT-SIRT1 axis, providing a simple and effective treatment strategy for ischemia / reperfusion (I / R) therapy.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: a nanozyme comprising a Mo2C substrate, wherein ruthenium atoms and cobalt atoms are anchored on the Mo2C substrate.

[0007] Preferably, as an improvement, the nanozyme is modified with an M2 macrophage membrane. By modifying the nanozyme with an M2 macrophage membrane, the nanozyme acquires a certain degree of targeting, enabling it to precisely accumulate in ischemic myocardial tissue.

[0008] The principle and advantages of the above scheme are as follows: This application provides a dual single-atom nanozyme RC (RC is a nanozyme without M2 macrophage membrane), which promotes in situ NAD by mimicking NOX activity. + Biotransformation, while simultaneously exhibiting activities similar to SOD and CAT to efficiently scavenge reactive oxygen species, thus overcoming the limitations of traditional NAD. + Limitations of prodrug replacement therapy. Its M2 membrane coating structure allows the nanozyme to precisely aggregate in ischemic myocardial tissue. The nanozyme of this application can restore NAD+. + NADH homeostasis is restored by enhancing ATP metabolism and oxidative phosphorylation to rebuild mitochondrial energy metabolism, thereby inhibiting cardiomyocyte apoptosis and alleviating inflammatory responses. From a mechanistic perspective, RC / M@M (RC / M@M is a nanozyme containing the M2 macrophage membrane) achieves synergistic regulation of metabolic-inflammatory pathways by activating the NAMPT-SIRT1 axis and inhibiting the TNF / NF-κB pathway. Therefore, the nanozyme of this application, through precise targeting, restoration of redox balance, and multimodal regulation of mitochondrial function and inflammatory responses, opens up a novel therapeutic strategy for the treatment of myocardial ischemia-reperfusion injury.

[0009] To achieve the above objectives, the present invention also adopts the following technical solution: a method for preparing nanozymes, comprising the following steps:

[0010] S1. Disperse Mo2C powder in deionized water and stir to form a homogeneous suspension;

[0011] S2. Subsequently, H2O2 solution is added dropwise to induce surface oxidation;

[0012] S3. After centrifugation to remove unreacted residues, collect the supernatant containing oxidation products;

[0013] S4. Disperse the oxidation product in deionized water, add polyvinylpyrrolidone, RuCl3 and CoCl2·6H2O, sonicate the mixture, and then heat it to 90-98℃ and keep it for 25-35 minutes.

[0014] S5. Add NaBH4 aqueous solution and continue the reaction at 90-98℃ for 50-70 minutes; then collect the product RC by centrifugation.

[0015] Preferably, as an improvement, the process further includes washing S6 and RC with deionized water, mixing them with the M2 macrophage membrane, sonicating the mixture, and preparing RC / M@M using a liposome extruder.

[0016] Preferably, as an improvement, in S6, the mixture is ultrasonically treated and then sequentially passed through polycarbonate membranes with pore sizes of 400 nm, 200 nm, and 100 nm.

[0017] Furthermore, based on the properties of the nanozyme of this application, it can also be used in the preparation of some drugs, specifically:

[0018] Application of a nanozyme in the preparation of drugs that can reconstruct mitochondrial energy metabolism.

[0019] Application of a nanozyme in the preparation of drugs that inhibit cardiomyocyte apoptosis and alleviate inflammatory responses.

[0020] A nanozyme for preparing NAD+ recovery + / Application of drugs for NADH homeostasis.

[0021] Application of a nanozyme in the preparation of drugs for treating myocardial ischemia-reperfusion injury. Attached Figure Description

[0022] Figure 1 TEM and EDS elemental distribution maps of RC (scale bar = 25 nm).

[0023] Figure 2 The image shows the Ru 3p XPS spectrum of RC.

[0024] Figure 3 The image shows the Co 2p XPS spectrum of RC.

[0025] Figure 4 AC-HAADF-STEM image of RC (white dots, red circles).

[0026] Figure 5 The NOx-like activity of Mo2C and RC is illustrated.

[0027] Figure 6 The diagram illustrates the change in NOx-like activity of Mo2C and RC over time.

[0028] Figure 7 This is a colorimetric concentration-dependent assessment of the NOx-like activity and NADH scavenging efficiency of RC.

[0029] Figure 8 This illustrates the conversion of Mo2C and RC to NAD via NADH oxidation. + The rate.

[0030] Figure 9 Illustration of NAD in Mo2C and RC + LC-MS analysis of the transformation.

[0031] Figure 10 The Michaelis-Menten kinetics of NADH oxidation under different concentrations of Mo2C and RC are illustrated.

[0032] Figure 11 , Figure 12 The diagrams illustrate the removal of Mo2C and RC·O. 2- The efficiency of ·OH.

[0033] Figure 13 The diagram illustrates the removal of O2 by Mo2C and RC. - ESR spectra of ·OH and ·OH.

[0034] Figure 14 The diagram illustrates the CAT-like activity of Mo2C and RC as detected by a dissolved oxygen analyzer.

[0035] Figure 15 , Figure 16 The relative levels of MDA and ATP after ischemia-reperfusion injury in each group are shown separately (n=3).

[0036] Figure 17 The diagram illustrates the total NAD after ischemia-reperfusion in each group. + Levels and NAD + / NADH ratio (n=3).

[0037] Figure 18Representative immunofluorescence staining images of NAMPT, SIRT1, PGC-1α, and TFAM in H9C2 cells after ischemia-reperfusion (scale bar = 40 μm; n = 3).

[0038] Figure 19 Representative Western blot results of NAMPT, SIRT1, PGC-1α and TFAM in H9C2 cells after ischemia-reperfusion (n=3).

[0039] Figure 20 Images of reactive oxygen species fluorescence in RAW264.7 and H9C2 cells.

[0040] Figure 21 The relative fluorescence ratios of RAW264.7 and H9C2 cell viability are illustrated.

[0041] Figure 22 Representative JC-1 flow cytometry images of RAW264.7 cells (n=3).

[0042] Figure 23 Representative JC-1 flow cytometry images of H9C2 cells (n=3).

[0043] Figure 24 Representative JC-1 fluorescence images and relative fluorescence ratios of RAW264.7 cells (scale bar = 20 μm; n = 3).

[0044] Figure 25 Representative JC-1 fluorescence images and relative fluorescence ratios of H9C2 cells (scale bar = 20 μm; n = 3).

[0045] Figure 26 The diagram illustrates representative flow cytometry images of apoptosis in RAW264.7 cells and the quantification of relative apoptosis rate (n=3).

[0046] Figure 27 The image shows a representative flow cytometry image of H9C2 cells undergoing apoptosis and the quantification of the relative apoptosis rate (n=3).

[0047] Figure 28 This image shows representative Western blot results of Bax and Bcl-2 in H9C2 cells after ischemia-reperfusion (n=3).

[0048] Figure 29 The image shows representative Western blot results of IL-1β, IL-6, iNOS and TNF in RAW264.7 cells stimulated with LPS / H2O2 (n=3).

[0049] Figure 30The results show the in vivo imaging of DiR-labeled RC and RC / M@M in ischemia-reperfusion mice at 4, 8, 12 and 24 hours after tail vein injection (n=4).

[0050] Figure 31 The diagram illustrates the distribution of nanoparticles in an ex vivo organ after 24 hours (n=4).

[0051] Figure 32 Representative M-mode echocardiogram images for each group.

[0052] Figure 33 The echocardiographic measurements of EF, FS, DS and DD are shown 3 days after ischemia-reperfusion (n>5).

[0053] Figure 34 This diagram illustrates the levels of MDA, ATP, and total NAD in myocardial homogenate three days after ischemia-reperfusion. + and NAD + The relative level of the / NADH ratio (n=3).

[0054] Figure 35 Representative TTC staining images (n=3) one day after ischemia-reperfusion.

[0055] Figure 36 The immunofluorescence staining results of TUNEL and phalloidin in frozen sections of the heart 3 days after ischemia-reperfusion are shown (scale bar: 100 μm; n=5).

[0056] Figure 37 The image shows representative Western blot results of Bax and Bcl-2 in the infarct boundary region 3 days after ischemia-reperfusion (n=3).

[0057] Figure 38 This illustrates the qPCR analysis of pro-inflammatory cytokines in whole heart samples 3 days after ischemia-reperfusion (n=5).

[0058] Figure 39 The immunofluorescence staining results of Ly6G and phalloidin in frozen sections of the heart are shown (scale bar: 20 μm; n=3).

[0059] Figure 40 The immunofluorescence staining results of CD68 and CD86 in frozen sections of the heart are shown, revealing the distribution of pro-inflammatory macrophages (scale bar: 50 μm; n = 3).

[0060] Figure 41 Representative flow cytometry atlas of pro-inflammatory cells in cardiac tissue 3 days after ischemia-reperfusion (n>5).

[0061] Figure 42The results show representative Western blots of NAMPT, SIRT1, PGC-1α and TFAM in cardiac tissue 3 days after ischemia-reperfusion (n=3).

[0062] Figure 43 Immunohistochemical staining of NAMPT and SIRT1 in frozen sections of the heart 3 days after myocardial infarction reperfusion is shown (n=3). Scale bar = 200 μm.

[0063] Figure 44 This study illustrates representative Western blot analysis of NAMPT, SIRT1, PGC-1α, and TFAM in the infarct border region 3 days after myocardial infarction reperfusion (n=3). Detailed Implementation

[0064] The following detailed description illustrates the specific implementation method:

[0065] The basic implementation examples are as follows: Figures 1-44 As shown.

[0066] This embodiment discloses a nanozyme comprising a Mo2C substrate anchored with ruthenium and cobalt atoms. The nanozyme in this embodiment is also modified with an M2 macrophage membrane. The M2 macrophage membrane in this embodiment was isolated from M2-polarized RAW264.7 cells.

[0067] This embodiment also discloses a method for preparing nanozymes, including the following steps:

[0068] S1. Disperse 2g of Mo2C powder in 15ml of deionized water and magnetically stir at 800 rpm for 30 minutes to form a homogeneous suspension.

[0069] S2. Then add 2 ml of 30% (w / v) H2O2 solution and react for 12 h to induce surface oxidation.

[0070] S3. After centrifugation to remove unreacted residues, collect the supernatant containing oxidation products;

[0071] S4. Disperse 50 mg of the oxidation product in 50 mL of deionized water, add 15 mg of polyvinylpyrrolidone, 20 mg of RuCl3 and 20 mg of CoCl2·6H2O, sonicate the mixture (40 kHz, 10 min), then transfer it to a three-necked flask and heat to 90-98 °C and maintain for 25-35 min, specifically heat to 95 °C and maintain for 30 min.

[0072] S5. Add NaBH4 aqueous solution (100mM, 1 mL) and continue the reaction at 90-98℃ for 50-70 minutes (specifically, continue the reaction at 95℃ for 60 minutes); then collect the product RC (nanozyme without M2 macrophage membrane, the nanozyme includes a Mo2C substrate, the Mo2C substrate is anchored with ruthenium and cobalt atoms) by centrifugation at 8000 rpm.

[0073] S6 and RC were washed three times with deionized water and then mixed with M2 macrophage membrane at a mass ratio of 10:1. The mixture was sonicated for 5 minutes and then passed through polycarbonate membranes with pore sizes of 400 nm, 200 nm and 100 nm eight times each. RC / M@M (nanozymes containing M2 macrophage membranes) was prepared using a liposome extruder.

[0074] experiment

[0075] The performance and effects of the nanozyme prepared above will be verified through a series of experiments.

[0076] I. Nanozyme Characterization Experiment

[0077] Combination Figure 1 As shown, the morphology and structural characteristics of the nanozyme RC prepared in this embodiment were characterized by transmission electron microscopy (TEM). During the procedure, the nanozyme powder was ultrasonically dispersed in anhydrous ethanol for 5 minutes. 10 μL of the suspension was then dropped onto a 200-mesh carbon film copper grid, air-dried at room temperature, and observed using TEM. The TEM image showed that the RC exhibited a monodisperse spherical structure with a uniform diameter distribution, approximately 50 nm. Energy-dispersive X-ray spectroscopy (EDS) revealed a uniform distribution of carbon, molybdenum, ruthenium, and cobalt elements within the RC nanozyme.

[0078] The oxidation states of ruthenium and cobalt in RC were further investigated using X-ray photoelectron spectroscopy (XPS) (using a Thermo Fisher Nexsa instrument equipped with a monochromatic Al Kα X-ray source (1486.6 eV, 150 W)). The oxidation states of Ru 3p were analyzed by XPS deconvolution. Figure 2 ) and Co 2p ( Figure 3 It was discovered that metallic Ru 0 / Co 0 With oxidation state (Ru) 4+ and Co 2+ / Co 3+ ) coexist. Furthermore, in aberration-corrected high-angle annular dark-field scanning transmission electron microscopy (AC-HAADF-STEM) images, clearly dispersed individual Ru and Co atoms are visible. Figure 4 (marked with a yellow circle in the middle).

[0079] Transmission electron microscopy (TEM) revealed a continuous membrane coating structure on the RC / M@M surface. Dynamic light scattering (DLS) analysis showed that the hydrodynamic diameter of RC / M@M was 177.4 ± 3.4 nm, significantly larger than that of uncoated RC (60.76 ± 2.43 nm), and the zeta potential increased from -31 ± 1.39 mV to -43.32 ± 1.75 mV after coating. These findings confirm that the RC nanozyme successfully achieved membrane coating of M2 macrophages.

[0080] The bioactivity of macrophage membrane-coated nanoparticles depends on the retention of surface membrane proteins. Therefore, the proteomic profiles of whole-cell lysates of M2 macrophages, isolated M2 membranes, and RC / M@M were analyzed and compared. Coomassie brilliant blue staining showed a high degree of similarity between the proteomic profiles of RC / M@M and M2 membranes, confirming the retention of the biological properties of the RC / M@M membrane. Western blotting experiments further verified the preservation status of key membrane proteins in RC / M@M (including integrins α4 and β1, which mediate inflammation targeting). The experimental results demonstrate that the RC material successfully achieved surface functionalization through M2 macrophage membrane coating technology.

[0081] II. The effect of dual single-atom catalysis on NAD + Experiment on the effects of supplementation and ROS scavenging

[0082] Myocardial ischemia leads to intracellular NADH and NAD accumulation through mitochondrial electron transport chain dysfunction. + This depletion leads to an explosion of oxidative stress and an excessive inflammatory response during reperfusion. Therefore, promoting in situ NAD through NADH elimination is crucial. + Biotransformation plays a crucial therapeutic role in alleviating myocardial ischemia / reperfusion injury.

[0083] Based on this, the inventors first evaluated the NADH oxidase activity of RC. NADH has a characteristic absorption peak at 340 nm, which can quantify its scavenging ability. The inventors first evaluated the NADH scavenging ability of Mo2C and RC. Mo2C and RC were incubated with NADH for 10 minutes, followed by 280-400 nm spectral absorption analysis. Specifically, equal amounts of RC and control group Mo2C were co-incubated with NADH at 37°C in the dark for 10 minutes (during incubation, Mo2C, RC, and NADH were each 100 μg / mL, and the total system volume was 200 μL). NADH consumption was monitored by measuring the absorbance at 340 nm or by collecting the full-band UV-Vis spectrum from 280-500 nm (during measurement, the final concentration of each material was 1-5 μg / mL). The results showed that both materials could scavenge NADH, with RC showing significantly better scavenging efficiency than Mo2C. Figure 5 Time-dependent experiments further confirmed that RC's NADH scavenging ability was superior to Mo2C. Figure 6The residual NADH level after catalysis was visually observed through the colorimetric reaction of WST-8 with NADH. Specifically, after centrifugation, the supernatant (190 μL) of the incubated sample was reacted with WST-8 in the dark at 37°C for 10-20 minutes. An orange-yellow formazan was formed. The reaction progress was quantified by measuring absorbance at 450 nm or visually observing the color development; a deeper color indicated a higher NADH concentration. If the color was lighter, the incubation time could be extended to 30-60 minutes. Visually, the colorimetric change showed that the NADH scavenging efficiency of RC increased with increasing concentration. Figure 7 ). To quantify NAD + Biotransformation was performed by adding an ethanol-ethanol dehydrogenase system to the WST-8 reaction system (specifically, after centrifugation of the incubated sample, 190 μL of the supernatant was reacted with 10 μL of ethanol-ethanol dehydrogenase (37°C for 10 minutes in the dark), followed by the addition of WST-8 reaction (37°C for 10-20 minutes), and the absorbance at 450 nm was measured. If the color development was too light, the incubation time could be extended to 30-60 minutes). This enzymatic conversion of NAD3 facilitated the transformation of NAD3. + The NADH level is used to calculate the indirect efficiency based on the regenerated NADH level. The results show that the RC's NAD... + The conversion efficiency is significantly higher than that of Mo2C ( Figure 8 Liquid chromatography-mass spectrometry (LC-MS) analysis confirmed that RC can catalyze the conversion of NADH to NAD. + ( Figure 9 ).

[0084] In addition, combined Figure 10 As shown, enzyme kinetic analysis indicates that the RC-catalyzed NADH oxidation reaction follows typical Michaelis-Menten kinetics, with a Michaelis constant (Km) of 47.57 μM and a maximum reaction rate (Vmax) of 33.18 μM·min. -1 In contrast, the zymosomal protein (Mo2C) has a Km value of 65.87 μM and a Vmax of 15.39 μM·min. -1 This indicates that NADH has a higher substrate affinity and catalytic efficiency for RC. Overall, RC exhibits better NADH scavenging ability and NAD+ catalytic efficiency. + In terms of regeneration efficiency, it is significantly superior to that of zymosomal protein (Mo2C).

[0085] In addition, the inventors evaluated the reactive oxygen species (ROS) scavenging capabilities of Mo2C and RC using various free radical models. Superoxide dismutase-like activity assays (the assay method involved using a riboflavin-methionine-nitroblue tetrazolium (NBT) superoxide anion generation system to detect the SOD-like activity of RC; riboflavin (10 μM), methionine (10 mM), and NBT (75 μM) were mixed with different concentrations of RC (or Mo2C), irradiated under ultraviolet light for 1 minute, and the absorbance at 560 nm was measured) showed that superoxide anion (·O2) activity... - The scavenging ability increases with increasing concentration, with RC showing significantly better scavenging activity than Mo2C. Figure 11 The scavenging effect of hydroxyl radicals (·OH) was evaluated using a Fenton reaction system monitored by methylene blue (MB) degradation kinetics (100 μL FFeSO4 (5 mg / mL) was reacted with 10 μL DMPO, 10 μL 30% H2O2 and 80 μL deionized water (control group) or sample solution (test group, concentration 1-5 μg / mL) at 25 °C for 5 min). The absorption peak intensity at 652 nm of RC and Mo2C decreased with increasing concentration, indicating that the ·OH scavenging efficiency of RC was significantly higher than that of Mo2C. Figure 12 Furthermore, electron spin resonance (ESR) spectroscopy confirmed the RC pair with ·O. 2- Both ·OH radical signal suppression effects are superior to those of Mo2C ( Figure 13 The CAT-like activity of RC was verified by measuring the characteristic superoxide dismutase (CAT) absorption peak at 240 nm (CAT-like activity was assessed by measuring oxygen produced by the decomposition of H2O2 using a dissolved oxygen meter; after calibration with standard solutions, H2O2 (100 μM) was co-incubated with 10-50 μg / mL RC (or Mo2C), and the dissolved oxygen concentration (mg / L) was recorded in a time-dependent manner; in addition, since H2O2 has a characteristic absorption peak at 240 nm, its absorbance at 240 nm was measured after co-incubation with RC). This indicated that RC's ability to scavenge superoxide was significantly higher than that of Mo2C. Dissolved oxygen measurements further quantified this activity, showing that RC produced significantly more superoxide anions than Mo2C. Figure 14Peroxidase-like activity was assessed using a peroxidase (POD) oxidation assay (RC) using 3,3',5,5'-tetramethylbenzidine (TMB) and H2O2 as substrates. The POD-like activity of RC was evaluated in PBS buffer (pH 7.4) at room temperature with TMB (0.5 mM), H2O2 (0.1 mM), and 10–50 μg / mL RC (or Mo2C). The formation of oxidized TMB (oxTMB) resulted in a blue color, and quantification was performed by measuring absorbance at 652 nm). oxTMB produced a significant absorption peak at 652 nm, and the POD-like activity of RC was significantly higher than that of Mo2C. Overall, these results strongly demonstrate that RC possesses superior NAD+ activity. + Conversion efficiency and multi-target antioxidant capacity.

[0086] III. Experiments demonstrating the targeting effect of RC / M@M

[0087] To assess the cellular uptake efficiency of RC / M@M, researchers co-incubated DiL-labeled RC / M@M and RC cells with RAW264.7 macrophages and H9C2 cardiomyocytes (37°C, CO2 concentration (approximately 5%), humidity (approximately 95%)). Fluorescence intensity was quantitatively analyzed at 0, 2, 4, and 6 hours after incubation using confocal fluorescence microscopy. Compared to RC, RC / M@M exhibited higher uptake efficiency in RAW264.7 cells at all time points. Consistent results were obtained in H9C2 cell experiments, with RC / M@M consistently showing higher fluorescence signals. These results confirm that macrophage membrane coating enhances cell affinity and promotes targeted internalization.

[0088] IV. Biocompatibility Experiment of RC / M@M

[0089] Treatment of H9C2 rat cardiomyocytes with different concentrations of RC / M@M showed that even at concentrations as high as 200 μg / mL, cell viability remained above 80%. In particular, the inventors discovered that RC / M@M effectively rescued H9C2 cardiomyocytes under hypoxia / reoxygenation (H / R) conditions. In this embodiment, the H / R model is summarized as follows: when H9C2 cardiomyocytes reached 90% confluence, they were cultured in a hypoxic chamber under hypoxic conditions (0.1% O2) in serum-free, glucose-free DMEM medium. After 10 hours of hypoxia treatment, the medium was replaced with complete DMEM medium containing RC / M@M (200 μg / mL), and the cells were then placed back in a normoxic incubator for another 14 hours.

[0090] V. RC / M@M and its NAD + Experiment on the key role of biotransformation and antioxidant capacity in cardiomyocyte protection

[0091] H9C2 rat cardiomyocytes were co-cultured after being treated with PBS, Mo2C, and RC / M@M, respectively (when the cell density reached 70%-80%, H9C2 cells were exposed to 0.1% oxygen in serum-free and glucose-free DMEM at 37°C for 10 hours of oxygen-glucose deprivation. After 10 hours of incubation, the cells were placed in a complete culture medium with the appropriate material concentration of 100 μg / ml and then placed in an atmospheric pressure incubator for 14 hours to recover). The inventors tested oxidative stress and NAD. + Metabolic biomarkers were detected using the following methods: For MDA: 0.1 mL of lysis buffer or homogenate per million cells. After homogenization or lysis, centrifuge at 10,000-12,000 g for 10 minutes and collect the supernatant for subsequent assays (refer to Beyotime S0131S); For NAD... + Approximately 1 × 10⁶ cells (roughly equivalent to the number of cells that can grow to a full volume in one well of a 6-well plate). Aspirate the culture medium and add 200 μl of NADPH extract per million cells using a pipette. + The NADH extract was gently pipetted to promote cell lysis, followed by centrifugation at 12,000×g, 4°C for 5-10 minutes. The supernatant was collected as the sample for testing (refer to Beyotime S0175). Following myocardial ischemia-reperfusion injury, H9C2 cardiomyocytes treated with RC / M@M showed restoration of bioenergy homeostasis and an increase in ATP levels. Figure 15 , Figure 15 The first group (light blue) represents the untreated group, the second group (dark blue) represents the H / R group, the third group (red) represents the Mo2C group, and the fourth group (orange) represents the RC / M@M group. Other groups in the attached figures follow the same pattern. Lipid peroxidation (MDA) was also reduced. Figure 16 Notably, RC / M@M significantly increased intracellular NAD. + Level and enhance NAD + / NADH ratio ( Figure 17 These results highlight the RC / M@M and its NAD. + The crucial role of biotransformation and antioxidant capacity in cardiomyocyte protection.

[0092] VI. NAD + Supplemental experiments on the correlation with mitochondrial repair

[0093] To verify NAD + To further investigate the connection with mitochondrial repair, the inventors used immunofluorescence and Western blotting techniques, referencing previous studies, to verify this connection.

[0094] Previous studies have shown that elevated NAD + Levels enhanced the activity of sirtuin 1 (SIRT1), an NAD-dependent enzyme.+ The deacetylase in NAD+ drives the expression of mitochondrial transcription factor A (TFAM) by enhancing the transcriptional activity of peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α), thereby restoring mitochondrial biogenesis and further restoring mitochondrial function. Elevated NAD+ + Levels of SIRT1 enhance SIRT1 expression, further promoting the activation of nicotinamide phosphoribosyltransferase (NAMPT), thereby reinforcing the NAMPT-NAD pathway. + The "-SIRT1" positive feedback loop produces an "amplification effect" in mitochondrial metabolism.

[0095] Immunofluorescence analysis in this experiment showed that the above RC / M@M treatment significantly enhanced the fluorescence intensity of NAMPT, SIRT1, PGC1α, and TFAM in H / R H9C2 cells. Figure 18 The results showed that the primary antibodies used for staining differed among the groups, namely NAMPT, SIRT1, PGC1α, and TFAM, while other antibodies were consistent. Western blot analysis revealed that H / R downregulated NAMPT, SIRT1, PGC1α, and TFAM, while RC / M@M treatment reversed these changes. Figure 19 These findings suggest that RC / M@M regulates NAD + It promotes metabolic and mitochondrial repair, and protects macrophages and cardiomyocytes from harmful damage caused by hydrogen peroxide and H / R stress.

[0096] VII. Experiments on how RC / M@M can alleviate oxidative stress and restore mitochondrial function

[0097] RC / M@M protects cardiomyocytes from apoptosis by reducing intracellular ROS production through its ability to restore mitochondrial function and scavenge reactive oxygen species (ROS). Given the complexity of the in vivo therapeutic microenvironment, the inventors subsequently assessed intracellular ROS levels in RAW264.7 and H9C2 cells (specifically, cells (Raw264.7 stimulated with hydrogen peroxide (1 mM H2O2) and H9C2 under H / R conditions) were incubated with DCFH-DA solution for 30 minutes, followed by three washes with PBS. ROS production was finally detected using a Zeiss LSM900 laser scanning confocal microscope and a FACSCanto flow cytometer). Figure 20 and Figure 21As shown, RC / M@M treatment significantly reduced intracellular ROS levels in both cell types, as evidenced by a decrease in green DCF fluorescence intensity. These results confirm the significant ability of RC / M@M to alleviate oxidative stress in different cell models. As a key marker of mitochondrial dysfunction, the inventors further investigated the changes in cell membrane potential (ΔΨm) before and after RC / M@M treatment (mitochondrial membrane potential (ΔΨm) was detected using the JC-1 assay kit manufactured by Beyotime Biotechnology. JC-1 in normal mitochondria forms red fluorescent aggregates, while impaired mitochondrial membrane potential exhibits green fluorescent monomers. The change in ΔΨm was quantified by calculating the red / green fluorescence ratio. Cells subjected to ischemia-reperfusion (H / R) or oxidative stress (H2O2) were incubated with 1 mL of JC-1 working solution at 37°C in the dark for 30 minutes. Finally, the fluorescence signal was analyzed using a Zeiss LSM900 laser confocal microscope and flow cytometry (FACSCanto) to confirm NAD). + The positive effects of elevated levels and ROS clearance on mitochondrial function. Flow cytometry analysis showed that RC / M@M restored mitochondrial membrane potential in both cell types. Figure 22 and Figure 23 ), observed by confocal microscopy ( Figure 24-25 This result was further confirmed, indicating that RC / M@M treatment can alleviate oxidative stress and restore mitochondrial function.

[0098] VIII. Experiment on the anti-apoptotic effect of RC / M@M

[0099] Given the close association between oxidative stress, mitochondrial damage, and the apoptosis pathway, the inventors evaluated the anti-apoptotic effect of RC / M@M (specifically, the following procedure was performed: damaged cells were first labeled with Annexin V-FITC (green fluorescent label) and propidium iodide (PI, red fluorescent label) (Raw264.7 was treated with hydrogen peroxide (H2O2 concentration of 1 mM); H9C2 was treated under H / R conditions). After treatment, the cells were prepared into single-cell suspensions and incubated with the staining solution for 20 minutes. Apoptosis was then analyzed using flow cytometry (FACSCanto). In addition, the inventors used the YO-PRO-1 / PI apoptosis and necrosis detection kit from Beyotime Biotechnology and a Zeiss LSM900 microscope to detect apoptotic and necrotic cells after injury). Flow cytometry showed that RC / M@M significantly reduced the apoptosis rate of macrophages and cardiomyocytes after H2O2-treated ischemia-reperfusion (H / R) injury. Figure 26-27 Western blot analysis showed that RC / M@M regulated the expression of apoptosis-related proteins: H / R treatment upregulated Bax expression and inhibited Bcl-2, while RC / M@M treatment reversed these expression changes. Figure 28 ).

[0100] IX. RC / M@M Modulation of Inflammatory Response Experiment

[0101] Given that reactive oxygen species (ROS) can cause cardiomyocyte damage and activate inflammatory pathways, leading to a persistent inflammatory response throughout the disease course, the inventors hypothesized that RC / M@M might regulate these inflammatory responses by scavenging ROS and restoring mitochondrial function. To this end, the inventors used a RAW264.7 macrophage model stimulated with lipopolysaccharide (LPS) and H2O2 (a model chosen to simulate in vivo I / R changes, where LPS (100 ng / ml) represents the inflammatory response and H2O2 (1 mM) represents reactive oxygen species; dual stimulation better reflects in vivo changes. When the cell density reached 90%, RAW264.7 cells were stimulated with the solution prepared at the above concentrations for 12 hours. After stimulation, the appropriate materials and a placebo (both 100 ng / ml) were added for another 12 hours, and the anti-inflammatory effect of RC / M@M was evaluated. The results showed that RC / M@M treatment significantly inhibited the expression of key pro-inflammatory mediators such as interleukin-1β (IL-1β), IL-6, inducible nitric oxide synthase (iNOS), and tumor necrosis factor-α (TNF-α). Figure 29 These findings indicate that RC / M@M can regulate NAD. + It metabolizes and eliminates reactive oxygen species (ROS), thereby preventing cell apoptosis, reducing inflammatory response, and ultimately inhibiting the deterioration of ischemia / reperfusion injury.

[0102] 10. RC / M@M in vivo experiments

[0103] Based on the excellent cytoprotective effects of RC / M@M demonstrated in in vitro experiments, the inventors systematically evaluated its therapeutic efficacy in a mouse model of myocardial ischemia-reperfusion injury. In this embodiment, the mouse model of myocardial ischemia-reperfusion injury involved anesthetizing mice with 2% isoflurane and then mechanically ventilating them via endotracheal intubation at a rate of 105 breaths per minute to maintain basal respiration. A small retractor was used to open the thoracic cavity to directly expose the heart. The left atrial appendage (LAD) was ligated approximately 2-3 mm below the left atrial appendage using 7-0 sutures and held for 45 minutes. The ligation was released after ischemia to allow reperfusion. The thoracic cavity was then manually closed, and the muscles and skin were sutured with 5-0 sutures. During reperfusion, 100 μL of RC / M@M (2 mg / mL) was injected via the tail vein using an insulin injector. The sham-operated group underwent the same procedure without ligation.

[0104] To track biodistribution in vivo, the inventors intravenously injected mice with coronary artery ligation-induced ischemia-reperfusion injury with an equivalent amount of DiR-labeled RC or RC / M@M. In vivo fluorescence imaging at 4, 8, 12, and 24 hours post-injection showed (…). Figure 30Significant fluorescence enrichment was observed in the abdominal cavities. Notably, compared to RC, the cardiac signal enrichment in mice receiving RC / M@M was significantly enhanced, indicating that M2 macrophage membrane coating significantly improves cardiac targeting specificity. To verify the cardiac targeting properties, the inventors performed in vitro fluorescence imaging on major organs (heart, liver, spleen, lung, and kidney) 24 hours after injection. Consistent with in vivo results, the enrichment of RC / M@M in the heart was significantly higher than that of RC, accompanied by increased uptake in the liver and spleen, while the distribution in the lungs and kidneys was comparable to that of RC / M. Figure 31 ).

[0105] Reperfusion-induced oxidative stress, energy metabolism disorders, and mitochondrial dysfunction are major contributing factors to irreversible myocardial injury. Early therapeutic intervention to salvage reversibly damaged cardiomyocytes and reduce the inflammatory response is crucial for improving prognosis. The inventors established an acute-phase (3 days, when ischemic myocardium is suddenly restored to blood supply (reperfusion), it triggers a very severe inflammatory response. This response is not instantaneous but a process. 1 to 3 days after reperfusion, inflammatory cells (such as neutrophils) accumulate in large numbers in the damaged myocardial area, reaching a peak. This is the most typical and severe manifestation of acute-phase injury) ischemia-reperfusion (I / R) model. Mice were divided into four experimental groups: sham-operated group, I / R group, Mo2C@M group, and RC / M@M group, each group was injected with 2 mg / ml, 100 μl. Three days post-surgery, echocardiography (mice were first anesthetized with 2% isoflurane to maintain a heart rate of 550 ± 50 beats / min; then, long-axis images of the left ventricle were acquired, and cardiac function parameters, including ejection fraction (EF), fractional shortening (FS), left ventricular end-systolic diameter (LVIDs), left ventricular end-diastolic diameter (LVIDd), left ventricular volume (LVV), and left ventricular anterior wall thickness (LVAW), were assessed using B-mode and M-mode echocardiography) revealed that the RC / M@M group showed significant improvement compared to the I / R group. Figure 32 Notably, ejection fraction (EF) and fractional shortening (FS)—key indicators of myocardial contractility—both showed significant increases. Measurements of left ventricular systolic diameter (Ds), diastolic diameter (Dd), ventricular volume (LV VOL), and anterior wall thickness (LVAW) further confirmed the functional recovery in the RC / M@M group mice. Figure 33 (Different colors represent references above; other figures are the same). Three days after reperfusion, the ATP content in the I / R-damaged heart was severely decreased, and the malondialdehyde (MDA) level was significantly increased, indicating severe energy deficiency and oxidative damage. Figure 34 Surprisingly, RC / M@M treatment not only reversed these pathological changes but also significantly restored cardiac NAD. +The bioavailability of NAD+ was increased under I / R stress. + / NADH ratio ( Figure 34 These results indicate that RC / M@M effectively alleviates excessive reactive oxygen species (ROS) production and corresponding metabolic imbalances. Twenty-four hours after reperfusion, 2,3,5-triphenyltetrazolium chloride (TTC) staining confirmed that RC / M@M administration reduced the infarct area. Figure 35 The degree of cardiomyocyte apoptosis induced by ischemia / reperfusion (I / R) is a key factor determining prognosis. TUNEL staining of myocardial tissue showed that RC / M@M significantly alleviated I / R-induced cardiomyocyte apoptosis. Figure 36 Further validation using Western blot analysis showed that RC / M@M treatment could regulate apoptosis-related proteins: compared with untreated I / R mice, the level of the pro-apoptotic protein Bax was decreased, while the expression of the anti-apoptotic protein Bcl-2 was increased. Figure 37 In summary, these findings indicate that RC / M@M has the ability to inhibit cardiomyocyte apoptosis and alleviate myocardial ischemia-reperfusion injury.

[0106] Neutrophils are a major component of early inflammation, exacerbating oxidative damage and promoting cytokine release, while macrophages exhibit functional heterogeneity, characterized by a pro-inflammatory M1 subtype that secretes IL-1β, IL-6, iNOS, and TNF-α, and an anti-inflammatory M2 subtype that promotes tissue repair. Quantitative PCR analysis of myocardial tissue revealed that RC / M@M treatment significantly reduced the mRNA levels of pro-inflammatory mediators (IL-1β, IL-6, iNOS, TNF-α, ICAM-1, VCAM-1) and alleviated the pathological elevation of atrial natriuretic peptide (ANP), a biomarker of cardiac dysfunction. Figure 38 Notably, RC / M@M also restored antioxidant defense mechanisms, as evidenced by significantly increased activities of heme oxygenase-1 (HO-1), superoxide dismutase 2 (SOD2), glutathione reductase (GSR), and glutathione S-transferase subtypes (GSTu, GSTa) compared to the ischemia-reperfusion (I / R) group. These results collectively indicate that RC / M@M can inhibit the inflammatory response in I / R injury and restore redox homeostasis.

[0107] Furthermore, LY6G immunofluorescence staining showed that the I / R group exhibited significant neutrophil infiltration 3 days after reperfusion. Figure 39 RC / M@M treatment effectively reversed this phenomenon, significantly reducing oxidative damage and inflammatory cytokine storm induced by I / R injury. Quantitative analysis of M1 macrophage infiltration in the myocardial infarction area using CD68 and CD86 co-staining revealed that the number of M1 macrophages in the I / R group was significantly higher than that in the RC / M@M group. Figure 40The decreased polarization of M1 cells was associated with the anti-inflammatory activity and potent antioxidant effect of M2 macrophage membranes in RC / M@M cells. Dynamic flow cytometry analysis of inflammatory cells in cardiac tissue 3 days after reperfusion (…) Figure 41 Experiments showed a significant increase in pro-inflammatory macrophages and a decrease in anti-inflammatory macrophages in the ischemia-reperfusion (I / R) model. RC / M@M treatment successfully restored the balance of macrophage subsets, confirming its anti-inflammatory effect. Western blot analysis showed that, compared with the I / R group, the RC / M@M group had significantly lower levels of IL-1β, IL-6, iNOS, and TNF-α proteins. Figure 42 This is consistent with the trend of changes in transcriptional levels. Experimental data indicate that RC / M@M can effectively maintain NAD. + / NADH homeostasis reduces oxidative stress levels and significantly inhibits inflammatory responses and apoptosis in vivo.

[0108] Cardiac cardiomyocytes are among the cell types with the highest metabolic demands, with mitochondria occupying 30% to 40% of their cell volume to maintain a continuous energy supply. NAD + It is not only an indispensable redox coenzyme in the tricarboxylic acid cycle (TCA cycle) and electron transport chain (ETC), but also a key component of NAD+ such as SIRT1. + It is a key substrate for acetyl-dependent deacetylases. This dual function makes it essential for maintaining cardiac energy metabolism homeostasis.

[0109] Therefore, the inventors investigated whether RC / M@M could restore NAD. + It biosynthesizes and reactivates its downstream cellular protection network. Immunohistochemical analysis showed that the expression of NAMPT and SIRT1 was significantly reduced in myocardium injured by ischemia-reperfusion injury. Figure 43 RC / M@M treatment restored NAMPT and SIRT1 levels, suggesting that it may enhance NAD through a salvage pathway. + Biosynthesis. Western blot analysis confirmed that RC / M@M simultaneously upregulated the protein expression of NAMPT, SIRT1, PGC-1α, and TFAM. Figure 44Enhanced SIRT1 activity induces PGC-1α / TFAM expression, significantly improving mitochondrial DNA stability and energy metabolism, thereby restoring redox balance and bioenergy homeostasis. This directly demonstrates that RC / M@M mediates the repair of mitochondrial functional integrity by activating the NAMPT-SIRT1 axis. Given the central role of the NF-κB pathway in oxidative stress and the inflammatory cascade, and existing evidence that SIRT1 can regulate the AMPK and NF-κB signaling pathways, the inventors investigated how RC / M@M regulates this signaling axis. The results show that RC / M@M can restore the NAMPT-mediated activation of SIRT1, which inhibits p65 transcriptional activity and enhances the AMPK signaling pathway, thereby synergistically inhibiting the excessive generation of reactive oxygen species (ROS) and the inflammatory cascade.

[0110] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A nanozyme, characterized in that: It includes a Mo2C substrate, wherein ruthenium atoms and cobalt atoms are anchored on the Mo2C substrate.

2. The nanozyme according to claim 1, characterized in that: The nanozyme was modified with the membrane of an M2 macrophage.

3. The method for preparing a nanozyme according to claim 1, characterized in that: Includes the following steps: S1. Disperse Mo2C powder in deionized water and stir to form a homogeneous suspension; S2. Subsequently, H2O2 solution is added dropwise to induce surface oxidation; S3. After centrifugation to remove unreacted residues, collect the supernatant containing oxidation products; S4. Disperse the oxidation product in deionized water, add polyvinylpyrrolidone, RuCl3 and CoCl2·6H2O, sonicate the mixture, and then heat it to 90-98℃ and keep it for 25-35 minutes. S5. Add NaBH4 aqueous solution and continue the reaction at 90-98℃ for 50-70 minutes; then collect the product RC by centrifugation.

4. The method for preparing a nanozyme according to claim 3, characterized in that: The process also includes washing S6 and RC with deionized water, mixing them with M2 macrophage membranes, sonicating the mixture, and preparing RC / M@M using a liposome extruder.

5. The method for preparing a nanozyme according to claim 4, characterized in that: In S6, the mixture is ultrasonically treated and then passed sequentially through polycarbonate membranes with pore sizes of 400 nm, 200 nm, and 100 nm.

6. The use of the nanozyme according to claim 1 or 2 in the preparation of drugs for treating myocardial ischemia-reperfusion injury.

7. The nanozyme according to claim 1 or 2 in the preparation of NAD+ recovery + / Application of drugs for NADH homeostasis.

8. The application of the nanozyme according to claim 1 or 2 in the preparation of a drug with the function of reconstructing mitochondrial energy metabolism.

9. The application of the nanozyme according to claim 1 or 2 in the preparation of a drug with the function of inhibiting cardiomyocyte apoptosis and alleviating inflammatory response.

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