Quaternary high-entropy alloy nano-enzyme as well as preparation method and application thereof

Abstract

The invention provides a quaternary high-entropy alloy nano enzyme as well as a preparation method and application thereof, and belongs to the technical field of nano biological materials and biological medicines. The quaternary high-entropy alloy nano-enzyme comprises a single-phase alloy and polyvinylpyrrolidone, the single-phase alloy is composed of four metal elements of M, rhodium, iridium and ruthenium, the surface of the single-phase alloy is modified with the polyvinylpyrrolidone, the element M is selected from palladium or platinum, and the particle size of the quaternary high-entropy alloy nano-enzyme is smaller than 10 nm. The quaternary high-entropy alloy nano-enzyme disclosed by the invention can penetrate through a blood brain barrier, is enriched at a cerebral hemorrhage focus, and comprehensively blocks reactive oxygen species (ROS) cascade reaction after cerebral hemorrhage.

Description

Technical Field

[0001] This invention relates to the fields of nanobiomaterials and biomedicine, and in particular to a quaternary high-entropy alloy nanoenzyme, its preparation method, and its application. Background Technology

[0002] Intracerebral hemorrhage (ICH) is a neurological emergency with extremely high mortality and disability rates. Its pathological mechanisms are complex and not yet fully understood. Current research indicates that oxidative stress is one of the core driving factors of early brain tissue damage after ICH. When a cerebral blood vessel ruptures, the damaged brain tissue suffers from ischemia and hypoxia, leading to mitochondrial dysfunction and an abnormal accumulation of large amounts of reactive oxygen species (ROS). These ROS can oxidize lipids, proteins, and DNA, disrupt cell membrane integrity, induce neuronal necrosis or apoptosis, and further activate microglia to polarize into the pro-inflammatory M1 type, releasing large amounts of pro-inflammatory factors (such as IL-1β and TNF-α), forming a vicious cycle of "ROS-inflammation-cell death".

[0003] Meanwhile, excessive ROS induce ferroptosis—an iron-dependent, lipid peroxidation-mediated regulatory necrosis that plays a crucial role in neurological damage following ICH. Ferroptosis is characterized by decreased glutathione peroxidase 4 (GPX4) activity, accumulation of lipid peroxidation products (such as malondialdehyde, MDA), and mitochondrial structural damage. Clinical studies have confirmed that MDA levels are significantly elevated and GPX4 expression is decreased in the brain tissue of ICH patients, and GPX4 levels are positively correlated with the degree of neurological deficit, suggesting that ferroptosis is an important target for ICH treatment.

[0004] However, in the clinical treatment of ICH, the focus is on symptomatic support, such as controlling intracranial pressure and maintaining vital signs, and there is a lack of drugs that specifically target oxidative stress and ferroptosis. Summary of the Invention

[0005] In view of this, in order to at least partially solve the aforementioned technical problems, the present invention provides a quaternary high-entropy alloy nanozyme, its preparation method and application, which can achieve integrated treatment of "penetrating the barrier-targeted enrichment-comprehensive removal of ROS" in the treatment of cerebral hemorrhage.

[0006] According to one aspect of the present invention, a quaternary high-entropy alloy nanozyme is provided, comprising a single-phase alloy composed of four metal elements: M, rhodium, iridium, and ruthenium, and polyvinylpyrrolidone modified on the surface of the single-phase alloy, wherein the M element is selected from palladium or platinum, and the particle size of the quaternary high-entropy alloy nanozyme is less than 10 nm.

[0007] According to another aspect of the present invention, a method for preparing a quaternary high-entropy alloy nanozyme is provided, comprising: mixing and dissolving the salts corresponding to four metal elements M, rhodium, iridium and ruthenium in a solvent to obtain a mixed solution; and adding polyvinylpyrrolidone and a reducing agent to the mixed solution, carrying out a reduction reaction at 25°C to 35°C, and obtaining the quaternary high-entropy alloy nanozyme by separation.

[0008] According to another aspect of the present invention, the above-described quaternary high-entropy alloy nanozyme is provided in the preparation of a drug for treating cerebral hemorrhage.

[0009] The quaternary high-entropy alloy nanozyme provided by this invention can comprehensively scavenge multiple ROS. Specifically, the quaternary high-entropy alloy nanozyme significantly enhances the activities of superoxide dismutase (SOD)-like enzymes, catalase (CAT)-like enzymes, and DPPH-like free radical scavenging activities through a "cocktail effect," and its SOD-like activity scavenges superoxide anions (•O2). — It can decompose hydrogen peroxide (H2O2) with CAT-like active decomposition and DPPH-like free radical scavenging active catalysis to generate harmless products from H2O2, thus completely blocking the ROS cascade reaction after ICH.

[0010] The quaternary high-entropy alloy nanozyme provided by the present invention can penetrate the blood-brain barrier. Specifically, by controlling the particle size of the quaternary high-entropy alloy nanozyme to be less than 10 nm, the particle size of less than 10 nm precisely matches the physiological pore size of the blood-brain barrier (approximately 8 nm), enabling the quaternary high-entropy alloy nanozyme to penetrate the blood-brain barrier.

[0011] The quaternary high-entropy alloy nanozyme provided by this invention can target and enrich lesions. Specifically, PVP modification reduces non-specific uptake while prolonging circulation time, and achieves passive targeting by recognizing the highly inflammatory microenvironment (such as TNF-α) in ICH lesion areas. In vivo experiments have confirmed that, for example, PtRhIr / Ru SAN high-entropy alloy nanozymes show significant enrichment at cerebral hemorrhage lesions in ICH model mice.

[0012] The quaternary high-entropy alloy nanozyme provided by this invention can inhibit ferroptosis and inflammation. Specifically, the quaternary high-entropy alloy nanozyme reduces inflammatory stimulation by scavenging ROS, directly protecting nerve cells by inhibiting ferroptosis; simultaneously, it directly regulates the phenotypic transformation of microglia, promoting the polarization of M1 cells to the anti-inflammatory M2 cells, reducing the release of pro-inflammatory factors and increasing the secretion of anti-inflammatory factors (such as IL-10 and TGF-β), thereby alleviating neuroinflammation. Thus, an integrated therapeutic system of "penetrating the barrier - targeting the lesion - clearing ROS - regulating inflammation" is formed.

[0013] The quaternary high-entropy alloy nanozyme provided by the present invention exhibits excellent biocompatibility. Specifically, the surface of the quaternary high-entropy alloy nanozyme is coated with PVP, which can reduce the dissolution of metal ions. Animal experiments show that liver and kidney function indicators are normal after tail vein injection. Attached Figure Description

[0014] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings of the embodiments will be briefly described below. Obviously, the drawings described below only relate to some embodiments of the present invention and are not intended to limit the present invention.

[0015] Figure 1 This is a TEM image of a quaternary high-entropy alloy nanozyme provided in Embodiment 1 of the present invention;

[0016] Figure 2 The image shown is an HRTEM image of a quaternary high-entropy alloy nanozyme provided in Example 1 of this invention.

[0017] Figure 3 The XRD patterns of the PtRhIr nanozyme of Comparative Example 2 and the quaternary high-entropy alloy nanozyme provided in Example 1 of this invention are shown.

[0018] Figure 4 Figures A-D in the figure are the XPS full spectrum and high-resolution spectrum of the quaternary high-entropy alloy nanozyme provided in Example 1 of the present invention;

[0019] Figures 5A-5E HAADF-STEM and EDS elemental mapping of the quaternary high-entropy alloy nanoenzyme provided in Example 1 of this invention;

[0020] Figure 6 The above are the SOD-like activity detection results of the quaternary high-entropy alloy nanozyme provided in Example 1 of the present invention;

[0021] Figure 7 The oxygen generation kinetics curve of the CAT-like activity of the quaternary high-entropy alloy nanoenzyme provided in Example 1 of the present invention;

[0022] Figure 8 The results show the detection of DPPH radical scavenging activity of the quaternary high-entropy alloy nanozyme provided in Example 1 of this invention.

[0023] Figure 9 The results of SOD-like activity assays for different concentrations of PtRhIr nanozymes in Comparative Example 2 are shown.

[0024] Figure 10 A comparison chart of SOD-like activity detection results between the PtRhIr nanozyme provided in Comparative Example 2 and the quaternary high-entropy alloy nanozyme provided in Example 1.

[0025] Figure 11 Oxygen generation kinetics curves for CAT-like activity of PtRhIr nanozymes at different concentrations in Comparative Example 2.

[0026] Figure 12 A comparison of the oxygen generation kinetics curves of the CAT-like activity of the PtRhIr nanozyme provided in Comparative Example 2 and the quaternary high-entropy alloy nanozyme provided in Example 1.

[0027] Figure 13 The results show the DPPH radical scavenging activity of different concentrations of PtRhIr nanozymes in Comparative Example 2.

[0028] Figure 14 This is a comparison chart showing the detection results of DPPH radical scavenging activity of the PtRhIr nanozyme of Comparative Example 2 and the quaternary high-entropy alloy nanozyme provided in Example 1.

[0029] Figure 15 Image showing the HE staining results of brain tissue from different groups of ICH model mice;

[0030] Figure 16 Image showing the Nissl staining results of brain tissue from different groups of ICH model mice;

[0031] Figure 17 The image shows the ROS content detection results in the brain tissue of ICH model mice from different groups; and

[0032] Figure 18 The image shows the results of GPX4 content detection in the brain tissue of ICH model mice from different groups. Detailed Implementation

[0033] Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. However, it should be understood that these descriptions are exemplary only and are not intended to limit the scope of the invention. In the following detailed description, numerous specific details are set forth to provide a thorough understanding of the embodiments of the invention for ease of explanation. However, it will be apparent that one or more embodiments may be practiced without these specific details. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concept of the invention.

[0034] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "comprising" as used herein indicates the presence of features, steps, or operations, but does not exclude the presence or addition of one or more other features.

[0035] When using expressions such as "at least one of A, B and C", they should generally be interpreted in accordance with the meaning that is commonly understood by those skilled in the art (e.g., "a system having at least one of A, B and C" should include, but is not limited to, a system having A alone, a system having B alone, a system having C alone, a system having A and B, a system having A and C, a system having B and C, and / or a system having A, B and C, etc.).

[0036] In related technologies, excessive ROS can disrupt cell membrane integrity by oxidizing lipids, proteins, and DNA, leading to neuronal necrosis or apoptosis. It further activates microglia to polarize into the pro-inflammatory M1 form, releasing large amounts of pro-inflammatory factors and creating a vicious cycle of ROS-inflammation-cell death. On the other hand, it induces ferroptosis—an iron-dependent, lipid peroxidation-mediated regulatory necrosis pathway that plays a crucial role in neurological damage following ICH. Clinical studies have confirmed that MDA levels are significantly elevated and GPX4 expression is decreased in the brain tissue of ICH patients, and its level is positively correlated with the degree of neurological deficit, suggesting that ferroptosis is an important target for ICH treatment.

[0037] Currently, clinical treatment for ICH remains primarily supportive and symptomatic, including controlling intracranial pressure and maintaining vital signs, lacking drugs specifically targeting oxidative stress and ferroptosis. Traditional antioxidants such as vitamin C and edaravone face three major challenges:

[0038] 1. Lack of targeting: It cannot actively identify inflammatory signals or damage microenvironment in ICH lesion areas, and is easily taken up non-specifically by tissues throughout the body, resulting in insufficient effective concentration in lesion areas;

[0039] 2. Weak blood-brain barrier penetration: Small molecule drugs are metabolized quickly and are easily rejected by blood-brain barrier transport proteins, while nanomedicines are often too large (>10nm) or have strong hydrophobic surfaces, making it difficult to pass through the physiological pore size of the blood-brain barrier (about 8nm) and reach the lesions in the brain.

[0040] 3. Incomplete ROS clearance and difficulty in regulating neuroinflammation: It can only target specific types of ROS (such as •OH or H2O2) and cannot block •O2. — The cascade reaction of →H2O2→•OH cannot reverse the amplification of inflammation caused by excessive polarization of microglia, and it is difficult to inhibit the vicious cycle of "oxidative stress-inflammation-ferroptosis".

[0041] Therefore, nanozymes, as a class of nanomaterials with enzyme-mimicking activity, have become a research hotspot for the treatment of ROS-related diseases due to their advantages such as high catalytic efficiency, strong stability, and multifunctional design. Single-metal nanozymes (such as Pt and Ir) have been proven to have antioxidant activity, but their catalytic activity is limited by the electronic structure of a single element, and they are prone to aggregation or oxidative inactivation in complex physiological environments. Bimetallic alloy nanozymes (such as PtIr) can enhance activity through elemental synergistic effects, but they still cannot meet the clearance requirements of multiple ROS after ICH, and they cannot effectively regulate the polarization state of microglia. Similarly, they have not solved the core problems of targeting and blood-brain barrier penetration.

[0042] The emergence of high-entropy alloy nanozymes offers a new approach to solving the aforementioned problems. High-entropy alloys are typically composed of multiple metallic elements and exhibit superior catalytic activity, stability, and multifunctionality compared to single-metal or low-entropy alloys due to characteristics such as the "cocktail effect" and "lattice distortion effect." Furthermore, the synergistic effect of multiple elements in high-entropy alloys can reduce the toxicity of individual metals; for example, alloying Pt with Ir can reduce Pt dissolution, ensuring its stability in complex physiological environments.

[0043] However, research on high-entropy alloy nanozymes has primarily focused on non-neurological diseases, with reports on their application in ICH treatment remaining scarce. More importantly, even existing high-entropy alloy nanozymes have not specifically addressed the core challenges of ICH: most high-entropy alloys are too large to fit the blood-brain barrier pore size (approximately 8 nm); their surfaces lack specific modification, preventing active accumulation in the hemorrhage area; and some alloy enzymes exhibit limited activity, failing to effectively cover the •O2 after ICH. — The cascade clearance requirements of H2O2 and •OH are not met, and microglia polarization cannot be regulated to alleviate neuroinflammation.

[0044] In view of this, and in response to the three major limitations of drugs in the treatment of cerebral hemorrhage—namely, the inability to precisely target the lesion (easily taken up non-specifically by the whole body), difficulty in penetrating the blood-brain barrier (size or surface properties mismatch), and inability to comprehensively eliminate multiple ROS (only targeting a single type of ROS)—this invention provides a quaternary high-entropy alloy nanozyme, its preparation method, and its application. This quaternary high-entropy alloy nanozyme can achieve integrated treatment of cerebral hemorrhage by "penetrating the barrier-targeted enrichment-comprehensive elimination of ROS".

[0045] According to an exemplary embodiment of the present invention, the present invention provides a quaternary high-entropy alloy nanozyme, comprising a single-phase alloy composed of four metal elements: M, rhodium, iridium and ruthenium, and polyvinylpyrrolidone (PVP) modified on the surface of the single-phase alloy, wherein the M element is selected from palladium or platinum, and the nanozyme has a particle size of less than 10 nm, for example, it can be 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, etc.

[0046] In embodiments of the present invention, the cocktail effect of quaternary alloys is used to enhance the activity of antioxidant enzymes such as SOD, CAT, and DPPH free radical scavengers, thereby blocking the ROS cascade reaction and thus covering the complex oxidative stress reaction after ICH. Specifically, Ru and Rh enhance the activity of •O2. — The disproportionation ability of M and Ir is enhanced, the decomposition efficiency of H2O2 is improved, the electronic structures of the four elements are complementary, and the reaction energy barrier is reduced by regulating the d-band center, so as to achieve complete blocking of ROS cascade reaction.

[0047] At the same time, by controlling the particle size to ensure that the drug can effectively cross the blood-brain barrier, and with the hydrophilic polyvinylpyrrolidone coating structure, non-specific adsorption to cerebral vascular endothelial cells can be reduced, circulation time can be prolonged, and specific enrichment in the cerebral hemorrhage area can be achieved by recognizing the high-inflammatory microenvironment. This forms an integrated treatment system of "penetrating the barrier - targeting the lesion - clearing ROS - regulating inflammation", which can not only comprehensively clear ROS, but also regulate the polarization state of microglia to reduce neuroinflammation.

[0048] In embodiments of the present invention, the particle size of the quaternary high-entropy alloy nanozyme is 3nm to 5nm, for example, 3nm, 3.5nm, 4nm, 4.5nm, 5nm, but is not limited to the values ​​mentioned.

[0049] According to embodiments of the present invention, by precisely controlling the particle size of the quaternary high-entropy alloy nanozyme to be between 3nm and 5nm, it is possible to better adapt to the pore size of the blood-brain barrier, ensuring that the quaternary high-entropy alloy nanozyme penetrates the blood-brain barrier and achieves targeted enrichment of the quaternary high-entropy alloy nanozyme in the lesion.

[0050] In embodiments of the present invention, the quaternary high-entropy alloy nanozyme is in the form of spherical particles. It has good structural stability, but is not limited to this, and can also be particles of other shapes.

[0051] In embodiments of the present invention, the single-phase alloy composed of four metallic elements—M, rhodium, iridium, and ruthenium—exhibits a face-centered cubic (FCC) structure. The thermodynamic stability of the FCC structure can prevent phase transitions or aggregation of the quaternary high-entropy alloy nanozymes in the in vivo environment, which is beneficial for extending the catalytic activity cycle of the quaternary high-entropy alloy nanozymes.

[0052] In embodiments of the present invention, ruthenium exists primarily in monatomic form, for example, more than 50% of ruthenium may exist in monatomic form, such as more than 60%, 70%, or 80% of ruthenium may exist in monatomic form. The highly dispersed and exposed active sites of ruthenium in monatomic form are beneficial for exerting its enzymatic catalytic activity.

[0053] In embodiments of the present invention, PVP modified on the surface of a single-phase alloy stabilizes the metal surface through coordination, which helps reduce ion dissolution. For example, PVP can be selected from PVP K10, PVP K30, PVP K40, etc., depending on its molecular weight.

[0054] In embodiments of the present invention, the surface of the quaternary high-entropy alloy nanozyme can also be coated with a composite layer of a biomimetic cell membrane and polyethylene glycol, wherein the biomimetic cell membrane is selected from neutrophil membranes or microglia membranes. This composite layer, as a biomimetic membrane coating layer (or simply biomimetic membrane), helps reduce the immunogenicity of the nanozyme, while the polyethylene glycol layer further inhibits non-specific protein adsorption, jointly avoiding excessive immune clearance responses in the body. This gives the nanozyme system both excellent biosafety and structural stability, laying a solid foundation for clinical translational applications.

[0055] According to an exemplary embodiment of the present invention, the present invention also provides a method for preparing the above-mentioned quaternary high-entropy alloy nanozyme, comprising: operation S1 to operation S2.

[0056] In step S1, the salts corresponding to the four metal elements M, rhodium, iridium and ruthenium are mixed and dissolved in a solvent to obtain a mixed solution.

[0057] In embodiments of the present invention, the molar ratio of the four metallic elements M, rhodium, iridium, and ruthenium is (0.5~2):(0.5~2):(0.5~2):1; the molar ratio is, for example, 0.5:0.5:0.5:1, 0.5:1:1:1, 1:1:1:1, 2:0.5:0.5:1, 2:2:2:1, but is not limited to the values ​​listed. By controlling the molar ratio of the four metallic elements within a suitable range, a high-entropy alloy can be formed with equal or approximately equal amounts of the four metals.

[0058] In embodiments of the present invention, the solvent may be water (e.g., ultrapure water) and / or ethylene glycol, but is not limited thereto, as long as it enables the four metal salts to dissolve separately and facilitates the subsequent reduction reaction.

[0059] In step S2, polyvinylpyrrolidone (e.g., PVP K30) and a reducing agent are added to the mixed solution, and a reduction reaction is carried out at 25℃~35℃. The quaternary high-entropy alloy nanozyme is obtained by separation.

[0060] In embodiments of the present invention, the amount of polyvinylpyrrolidone (PVP) used is a mol, and the total amount of the four metal elements added is b mol, wherein a / b = 1.5:1 to 2.5:1, for example, a / b = 1.5:1, 2:1, 2.5:1, but not limited to the values ​​mentioned. If the value of a / b is too small, PVP will not be able to completely coat the four metals, which will easily lead to the dissolution of metal ions and reduce the activity of the quaternary high-entropy alloy nanozyme; if the value of a / b is too large, it will easily lead to unnecessary waste of consumables.

[0061] In the embodiments of the present invention, polyvinylpyrrolidone is used as a stabilizer. Compared with the case where no polyvinylpyrrolidone is added, it not only helps to reduce the particle size of nanozymes, but also effectively avoids the precipitation of nanozymes during use, thereby improving the stability of nanozymes.

[0062] In embodiments of the present invention, the amount of reducing agent used is c mol, and the total amount of the four metal elements added is b mol, wherein c / b is greater than or equal to 4, for example, it can be 4, 5, 6, 8, 10, etc., but is not limited to the values ​​listed above. Preferably, c / b is 4 to 8. If the value of c / b is too small, it is difficult to fully and quickly reduce the metal; if the value of c / b is too large, it leads to unnecessary waste of consumables.

[0063] According to embodiments of the present invention, the amount of raw materials such as the four metal elements, polyvinylpyrrolidone, and reducing agent is related to the particle size of the quaternary high-entropy alloy nanozyme. Controlling the appropriate ratio is more conducive to preparing quaternary high-entropy alloy nanozymes with a particle size of 3-5 nm.

[0064] In embodiments of the present invention, the reducing agent is selected from sodium borohydride and ascorbic acid, with sodium borohydride being the preferred reducing agent.

[0065] In embodiments of the present invention, the reduction reaction time is 1 to 3 hours, for example, 1 hour, 2 hours, or 3 hours, but is not limited to the values ​​mentioned.

[0066] In the embodiments of this invention, sodium borohydride has strong reducing properties, which is beneficial for preparing nanozymes with a particle size of 3-5 nm at room temperature, with a particle size deviation of less than or equal to 0.5 nm. The reaction time is short (preparation can be completed in only 6 hours), the raw material conversion rate can reach more than 90%, the production efficiency is increased by 3 times compared with traditional methods, and no expensive equipment is required, which significantly reduces the industrialization cost and provides a feasibility guarantee for clinical translation.

[0067] According to embodiments of the present invention, the present invention is based on a one-step co-reduction method, which uses a reducing agent to reduce at room temperature to synthesize quaternary high-entropy alloy nanozymes without the need for complex equipment or multi-step modification. This method is beneficial for preparing small-size nanozyme particles, with small particle size deviation and good reproducibility of the product.

[0068] According to an embodiment of the present invention, the preparation method may further include operation S3: coating the surface of the quaternary high-entropy alloy nanoenzyme with a composite layer of biomimetic cell membrane and polyethylene glycol, the composite layer serving as a biomimetic membrane coating layer (or simply biomimetic membrane), the biomimetic membrane coating layer being prepared by ultrasonic-assisted extrusion, the process being simple to operate and having high coating efficiency (>90%), and being able to achieve uniform and stable coating.

[0069] According to an embodiment of the present invention, the specific operation process of coating a biomimetic film on the surface of a quaternary high-entropy alloy nanoenzyme may include sub-steps 31 and 32:

[0070] In sub-step 31, the composite dispersion of biomimetic cell membrane and polyethylene glycol (PEG) is mixed with quaternary high-entropy alloy nanozyme to ensure full contact between the biomimetic cell membrane and the quaternary high-entropy alloy nanozyme, thereby obtaining a mixture.

[0071] For example, fresh mouse microglia can be washed with buffer and then lysed in Tris-HCl lysis buffer containing EDTA on ice. The cell membrane precipitate can be collected by centrifugation and resuspended in buffer containing polyethylene glycol, such as PEG 2000. The mixture can be stirred to prepare a microglia cell membrane-PEG composite dispersion and stored for later use. Then, nanozymes can be added to the above composite dispersion at a certain mass ratio, such as 1:2 to 1:4 (which can be 1:2, 1:3, 1:4, etc.) and gently stirred for 1 hour.

[0072] In sub-step 32, the mixture is repeatedly extruded through a porous polycarbonate filter membrane based on an extrusion method to coat the surface of the quaternary high-entropy alloy nanoenzyme with a composite layer of the biomimetic cell membrane and polyethylene glycol as a biomimetic membrane.

[0073] For example, the mixture can be passed through a polycarbonate filter membrane with a pore size of, for example, 400 nm, and repeatedly squeezed at a pressure of 0.2 to 0.4 MPa, for example, 0.3 MPa; then the squeezed sample is centrifuged to discard the supernatant, resuspended in buffer and washed, and the precipitate is collected to obtain a biomimetic membrane-coated nanozyme.

[0074] According to an exemplary embodiment of the present invention, the present invention provides the application of the above-mentioned quaternary high-entropy alloy nanozyme in the preparation of a drug for treating cerebral hemorrhage.

[0075] In an embodiment of the present invention, the application of quaternary high-entropy alloy nanozymes in the preparation of drugs for treating cerebral hemorrhage includes treating cerebral hemorrhage by scavenging reactive oxygen species, inhibiting ferroptosis, and / or inhibiting neuroinflammation.

[0076] In an embodiment of the present invention, the application of quaternary high-entropy alloy nanozymes in the preparation of drugs for treating cerebral hemorrhage includes reactive oxygen species including superoxide anions, hydrogen peroxide, and hydroxyl radicals.

[0077] According to an embodiment of the present invention, four elements, Rh, M, Ir, and Ru, form a face-centered cubic (FCC) single-phase alloy. Through the "cocktail effect" and lattice distortion optimization of the electronic structure, the quaternary high-entropy alloy nanozyme simultaneously possesses SOD-like activity, CAT-like activity, and DPPH-like free radical scavenging activity, enabling comprehensive and efficient removal of •O2. — This invention targets various ROS such as H2O2 and •OH, blocking the oxidative stress cascade reaction. Specifically, related single-metal nanozymes (such as Pt and Ir) are limited by the electronic configuration of a single element and can only scavenge specific ROS; bimetallic alloys (such as PtIr) have a synergistic effect, but still cannot cover the complex ROS spectrum after cerebral hemorrhage. The Ru and Rh enhancements of the quaternary high-entropy alloy nanozyme of this invention target •O2. — The disproportionation ability of M and Ir is enhanced, the decomposition efficiency of H2O2 is improved, the electronic structures of the four elements are complementary, and the reaction energy barrier is reduced by regulating the d-band center, so as to achieve complete blocking of ROS cascade reaction.

[0078] According to embodiments of the present invention, by precisely controlling the particle size of the quaternary high-entropy alloy nanozyme to be between 3 nm and 5 nm, and coating the surface of the quaternary high-entropy alloy nanozyme with hydrophilic PVP K30, the quaternary high-entropy alloy nanozyme can efficiently penetrate the blood-brain barrier and target and accumulate in lesions. Specifically, although the blood-brain barrier is damaged after cerebral hemorrhage, it still has structural limitations (physiological pore size of about 8 nm), and nanoparticles larger than 10 nm are difficult to penetrate; moreover, exposed metal nanozymes are easily cleared by the reticuloendothelial system and cannot accumulate in lesions. In this invention, the small particle size of 3 nm to 5 nm can better match the pore size of the blood-brain barrier, the hydrophilic shell of PVP K30 reduces non-specific adsorption to cerebral vascular endothelial cells, prolongs circulation time, and achieves targeted accumulation by recognizing the highly inflammatory microenvironment of the lesion area.

[0079] According to embodiments of the present invention, Transwell experiments verified that the quaternary high-entropy alloy nanozyme of the present invention can effectively cross the blood-brain barrier. In vivo experiments further demonstrated that, after tail vein injection, the quaternary high-entropy alloy nanozyme not only efficiently penetrates the blood-brain barrier but also specifically targets and accumulates in the cerebral hemorrhage area. This significantly increases the local drug concentration in the brain, solving the problems of weak penetration and poor targeting of traditional drugs.

[0080] According to the quaternary high-entropy alloy nanozyme provided in the above embodiments of the present invention, the quaternary high-entropy alloy nanozyme can increase the survival rate of microglia and neurons in the ICH model by 1.45 times and 6.67 times, respectively. It not only directly protects nerve cells by clearing ROS and inhibiting ferroptosis (reducing MDA level and maintaining GPX4 activity), but also effectively alleviates neuroinflammation.

[0081] Specifically, this quaternary high-entropy alloy nanozyme significantly reduced lipid peroxidation (MDA decreased by 55.68%) by maintaining GPX4 activity (increased by 6.32 times) and effectively inhibited ferroptosis. The reduction in neuroinflammation was manifested in reducing microglia polarization towards the pro-inflammatory M1 type (M1 marker CD86 expression decreased by 74.13%) and promoting their conversion to the anti-inflammatory M2 type (M2 marker CD206 expression increased by 623%), thereby downregulating the pro-inflammatory factor IL-6 (decreased by 69.32%) and upregulating the anti-inflammatory factor IL-10 (increased by 1.29 times), thus alleviating nerve damage through a dual pathway of ferroptosis inhibition and inflammation regulation.

[0082] The following exemplifies the design of a quaternary high-entropy alloy nanozyme, its preparation method, and its applications. It should be noted that this exemplification is merely a specific embodiment of the present invention and does not limit the scope of protection of the present invention.

[0083] Example 1

[0084] A quaternary high-entropy alloy nanozyme was prepared. Specifically, K₂RhCl₆ (0.2 mmol), K₂PtCl₄ (0.2 mmol), IrCl₃ (0.2 mmol), and RuCl₃ (0.2 mmol) were weighed and dissolved in 20 mL of ultrapure water. 100 mg of PVP K₃₀ was added, and the mixture was magnetically stirred for 30 minutes. 4 mL of freshly prepared 10 mM sodium borohydride solution was added, and the reaction was carried out at 30 °C for 2 hours. The product was prepared by ultrafiltration at 6000 rpm for 30 minutes, followed by re-washing with 15 mL of ultrapure water. This process was repeated three times to obtain the PtRhIr / Ru SAN (RRPI) nanozyme.

[0085] Structural characterization and testing:

[0086] (1) Structural characterization

[0087] Figure 1 This is a TEM image of a quaternary high-entropy alloy nanozyme provided in Example 1 of the present invention.

[0088] refer to Figure 1 As shown in the TEM image, the prepared quaternary high-entropy alloy nanozyme is a spherical particle with a particle size of 2.2 nm ± 0.3 nm.

[0089] Figure 2 The image is an HRTEM image of a quaternary high-entropy alloy nanozyme provided in Example 1 of this invention.

[0090] refer to Figure 2 As shown, HRTEM images revealed clear lattice fringes on the quaternary high-entropy alloy nanozyme, indicating that the material has a good crystal structure and has formed a single-phase alloy.

[0091] Figure 3The XRD patterns are of the PtRhIr nanozyme of Comparative Example 2 and the quaternary high-entropy alloy nanozyme provided in Example 1 of this invention.

[0092] refer to Figure 3 As shown, the XRD pattern of the PtRhIr nanozyme has changed compared to that of the PtRhIr nanozyme, which is considered to be due to the more dispersed elements. Furthermore, no obvious 44° corresponding Ru diffraction peak appears in the pattern, which verifies that no crystals with specific crystal planes have been formed or that the morphology is mainly single-atom.

[0093] Figure 4 Figures A through D in the figure show the XPS full spectrum and high-resolution spectrum of the quaternary high-entropy alloy nanozyme provided in Example 1 of this invention.

[0094] refer to Figure 4 As shown in Figures A through D, the XPS full spectrum indicates that four elements exist in the quaternary high-entropy alloy nanozyme, with Ru mainly existing as single atoms.

[0095] Figures 5A-5E HAADF-STEM and EDS elemental mapping of the quaternary high-entropy alloy nanoenzyme provided in the embodiments of the present invention.

[0096] refer to Figures 5B-5E As shown, the EDS mapping reveals a uniform distribution of the four elements.

[0097] (2) Superoxide dismutase (SOD) activity test

[0098] The superoxide dismutase (SOD) activity of the quaternary high-entropy alloy nanozyme prepared in Example 1 was detected by the WST-8 colorimetric method.

[0099] Specifically, the CheKine™ Superoxide Dismutase (SOD) Activity Assay Kit purchased from AbbKine was used to prepare the reaction working solution. Specifically, sample solution, enzyme working solution, enzyme diluent, and substrate reaction solution were added to a 96-well plate according to Table 1 below. The sample was the supernatant obtained by homogenizing and centrifuging microglia. The enzyme working solution was xanthine oxidase (XOD) solution, and the substrate reaction solution was WST-8 solution. The substrate reaction solutions for the sample groups contained nanozymes at concentrations of 0 μg / mL, 3 μg / mL, 6 μg / mL, 12 μg / mL, 24 μg / mL, and 48 μg / mL, respectively, with three replicates per group. No sample solution was added to the blank group. After incubating the working solution in each well at 37°C for 10 minutes, a full-spectrum scan was performed using a microplate reader at wavelengths from 360 nm to 525 nm to measure the absorbance value A at each well. The values ​​were calculated as follows: ΔAsample = Asample - Asample control, and ΔAblank = Ablank - Ablank control. The absorbance calculated based on ΔAsample - ΔAblank can be used to characterize nanozyme activity. A decrease in absorbance compared to the state without nanozyme indicates the generation of •O2. — The amount decreased, WST-8 in •O2 — The amount of orange-yellow formazan reduced by the action decreases, indicating the presence of SOD-like activity.

[0100] Table 1

[0101]

[0102] Figure 6 The results of SOD-like activity detection for the quaternary high-entropy alloy nanozyme provided in Example 1 of this invention.

[0103] refer to Figure 6 As shown in the full spectrum curve, when the amount of quaternary high-entropy alloy nanozyme added is 0, i.e., without nanozyme, •O2 — The WST-8 was not removed and was fully reduced, forming the highest absorption peak (absorbance of about 1.37au) at 450nm~460nm, with a complete peak shape covering the entire range of 360nm~525nm.

[0104] The WST-8 colorimetric method was used to scan and detect the entire spectrum from 360 nm to 525 nm. The results showed that superoxide anion (•O2) —The nanozyme can reduce WST-8 to an orange-yellow formazan product, forming a characteristic absorption peak at 450 nm–460 nm. With increasing concentration of the PtRhIr / RuSAN quaternary high-entropy alloy nanozyme (0→48 μg / mL), the absorbance across the entire spectrum decreases in a concentration-dependent manner. Specifically, at a concentration of 48 μg / mL, the absorbance at the characteristic peak is reduced by 70.07% compared to the concentration without nanozyme (P<0.001), directly confirming that the nanozyme can efficiently scavenge •O2. — It exhibits significant SOD-like activity.

[0105] (3) Catalase-like activity test

[0106] The CAT-like activity of the quaternary high-entropy alloy nanozyme prepared in Example 1 was detected.

[0107] Specifically, 10 mL of 0.0003 wt% H₂O₂ substrate solution was added to a 20 mL glass reaction flask, and the dissolved oxygen electrode of a Leici JPSJ-606T dissolved oxygen analyzer was inserted. The initial dissolved oxygen concentration (O₂) was measured and recorded under stirring conditions. 0 Six groups of glass reaction flasks were filled with 100 μL of nanozyme solutions at concentrations of 0 μg / mL, 3 μg / mL, 6 μg / mL, 12 μg / mL, 24 μg / mL, and 48 μg / mL, respectively. Dissolved oxygen concentration was recorded immediately and every 5 seconds for 5 minutes to obtain dissolved oxygen release curves. The amount of dissolved oxygen released was used to characterize nanozyme activity. Compared to the reaction without nanozyme, an increase in dissolved oxygen concentration indicated an increase in oxygen produced by the nanozyme-catalyzed decomposition of H₂O₂, suggesting the presence of CAT-like activity.

[0108] Figure 7 The oxygen generation kinetics curve of the CAT-like activity of the quaternary high-entropy alloy nanoenzyme provided in Example 1 of the present invention.

[0109] refer to Figure 7 As shown, the dissolved oxygen meter of Leici JPSJ-606T showed that after adding quaternary high-entropy alloy nanozymes to a 0.0003% H2O2 solution, the dissolved oxygen concentration increased over time, and the rate of increase was significantly dose-dependent on the concentration of the quaternary high-entropy alloy nanozymes.

[0110] The CAT-like activity and dose-dependent properties of PtRhIr / RuSAN quaternary high-entropy alloy nanozymes in a 0.0003% H2O2 system were systematically evaluated using a Leici JPSJ-606T dissolved oxygen analyzer. The results showed that the oxygen release rate increased significantly with increasing nanozyme concentration from 0 to 24 μg / mL. At 24 μg / mL, the dissolved oxygen concentration increased by 46.05 times within 5 minutes compared to the system without the quaternary high-entropy alloy nanozyme, fully demonstrating that this nanozyme can efficiently catalyze the decomposition of H2O2 into non-toxic O2 and H2O. This catalytic action directly targets H2O2, a key molecule in ROS. In pathological processes such as cerebral hemorrhage, H2O2 further generates the more toxic •OH through the Fenton reaction, triggering oxidative damage to lipids, proteins, and DNA, forming a vicious cycle of "ROS accumulation - oxidative stress - cell death." Nanozymes, by specifically accelerating the harmless transformation of H2O2, can block the ROS cascade amplification effect and reduce the attack of oxidative stress on nerve cells (such as microglia and neurons), thereby alleviating oxidative stress-mediated tissue damage at its source. This highly efficient CAT-like activity and clear dose-dependency provide direct experimental evidence for its antioxidant protective effect in ROS-related diseases such as cerebral hemorrhage, highlighting the potential application value of nanozymes in alleviating pathological damage by scavenging H2O2 and disrupting the oxidative stress chain.

[0111] (4) Test of free radical scavenging activity of 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH).

[0112] The DPPH-like free radical scavenging activity of the quaternary high-entropy alloy nanozyme prepared in Example 1 was tested.

[0113] Specifically, the Abbkine CheKine™ DPPH Radical Scavenging Kit (catalog number: KTB1092) was used for detection, and the reaction working solution was prepared. As shown in Table 2 below, sample groups, a DPPH-free control group, and a nanozyme-free positive control group were set up. In the sample group, 10 μL of nanozyme sample and 190 μL of substrate reaction solution with concentrations of 0 μg / mL, 3 μg / mL, 6 μg / mL, 12 μg / mL, 24 μg / mL, and 48 μg / mL were added to each well of a 96-well plate or microplate. The substrate reaction solution was an anhydrous ethanol solution of DPPH. In the DPPH-free control group, the 190 μL of substrate reaction solution was replaced with an equal volume of anhydrous ethanol. In the nanozyme-free positive control group, the nanozyme was replaced with an equal volume of vitamin C solution. After mixing the reaction working solutions of each group, incubate at room temperature in the dark for 10 min. Use an ELISA reader to perform a full-spectrum scan at wavelengths of 450 nm to 750 nm. With the absorbance at 515 nm as the core indicator, measure the absorbance value A at each well and calculate ΔA_sample = A_sample - A_control to evaluate nanozyme activity. If the absorbance value decreases compared to when no nanozyme was added, it indicates that DPPH free radicals have been cleared and DPPH free radical scavenging activity is present. At the same time, vitamin C solution is used as a positive control to evaluate the strength of nanozyme activity.

[0114] Table 2

[0115]

[0116] Figure 8 The results show the detection of the DPPH radical scavenging activity of the quaternary high-entropy alloy nanozyme provided in Example 1 of this invention.

[0117] refer to Figure 8 As shown, a full-spectral scan from 450 nm to 750 nm reveals that the DPPH free radical alcohol solution exhibits a characteristic absorption peak in the range of 510 nm to 520 nm. With the concentration of the PtRhIr / RuSAN quaternary high-entropy alloy nanozyme increasing from 0 to 48 μg / mL, the absorbance of the full spectrum decreases significantly in a concentration-dependent manner.

[0118] Based on the above full-spectral changes, it can be seen that this quaternary high-entropy alloy nanozyme can efficiently act on DPPH free radicals. From the dynamic changes in the spectral curve, as the nanozyme concentration gradually increases, the absorbance of the DPPH free radical solution in the characteristic absorption peak region continuously and significantly decreases, showing a clear concentration-dependent increasing trend. This change reflects that the nanozyme can interact with DPPH free radicals and have an effective influence on them. As DPPH free radicals are a typical oxidative stress-related free radical, the high efficiency of the nanozyme in acting on them fully demonstrates its excellent antioxidant activity. In the pathological process of cerebral hemorrhage, oxidative stress is an important damage mechanism; excessive ROS can trigger a series of cellular damage responses. The antioxidant activity of this nanozyme provides direct experimental evidence for blocking ROS-induced oxidative stress damage in the treatment of cerebral hemorrhage, and also lays the foundation for further exploration of its application potential in the treatment of related diseases.

[0119] Comparative Example 1

[0120] Preparation of single-metal Pt nanozymes. Single-metal Pt nanozymes were prepared using the same method as in Example 1, the main difference being that only K₂PtCl₄ was added as the noble metal precursor. Specifically, potassium chloroplatinate (K₂PtCl₄, 0.2 mmol) was weighed and dissolved in 20 mL of ultrapure water, and 100 mg of polyvinylpyrrolidone (PVP, Mw≈58000) was added. The mixture was magnetically stirred for 30 minutes to ensure thorough dispersion. 4 mL of freshly prepared 10 mM sodium borohydride (NaBH₄) solution was added in batches as a reducing agent, and the reaction was continuously stirred at 30°C for 2 hours. After the reaction, the solid product was collected by filtration and washed three times with ultrapure water to obtain the Pt nanozyme.

[0121] Using a method similar to that in Example 1, the SOD-like activity, CAT-like activity, and DPPH free radical scavenging activity of the single metal Pt nanozyme were tested. It can be seen that the single metal Pt nanozyme prepared in Comparative Example 1 can only target a single type of ROS, and the scavenging rate is generally less than 30%, which cannot cover the complex oxidative stress environment after ICH.

[0122] Furthermore, the SOD-like activity, CAT-like activity, and DPPH free radical scavenging activity of the PtRhIr / RuSAN quaternary high-entropy alloy nanozyme are 3.2 times, 2.8 times, and 4.5 times that of the single-metal Pt nanozyme, respectively, solving the problems of single antioxidant action and incomplete ROS scavenging in traditional antioxidants.

[0123] Comparative Example 2

[0124] PtRhIr nanozymes were prepared using the same method as in Example 1. The difference from Example 1 was that RuCl3 was not added to the raw materials. Specifically, K₂RhCl₆ (0.2 mmol), K₂PtCl₄ (0.2 mmol), and IrCl₃ (0.2 mmol) were weighed and dissolved in 20 mL of ultrapure water. 100 mg of PVP K₃₀ was added, and the mixture was magnetically stirred for 30 minutes. 4 mL of freshly prepared 10 mM sodium borohydride solution was added, and the reaction was carried out at 30°C for 2 hours. The product was prepared by ultrafiltration at 6000 rpm for 30 minutes, followed by washing with 15 mL of ultrapure water. This process was repeated three times to obtain the PtRhIr nanozymes.

[0125] The structure was characterized and tested using a method similar to that in Example 1. It was found that characteristic diffraction peaks of Pt, Rh and Ir were present, indicating that aggregates of the three were present, and PtRhIr nanozyme was successfully obtained.

[0126] Figure 9 The results show the SOD-like activity of different concentrations of PtRhIr nanozymes in Comparative Example 2.

[0127] Figure 10 The graph shows a comparison of the SOD-like activity detection results between the PtRhIr nanozyme provided in Comparative Example 2 and the quaternary high-entropy alloy nanozyme provided in Example 1.

[0128] refer to Figure 9 As shown, the absorbance decreased significantly in a concentration-dependent manner with increasing PtRhIr nanozyme dosage; this indicates that with increasing PtRhIr nanozyme dosage, the absorbance for •O2... — The stronger its cleaning ability.

[0129] refer to Figure 10 As shown, compared to PtRhIr nanozymes, the absorbance of the quaternary high-entropy alloy nanozyme (PtRhIr / Ru SAN) from Example 1 of this invention decreased significantly after addition. In other words, compared to PtRhIr nanozymes, PtRhIr / Ru SAN exhibits a greater absorption of •O2. — It has a stronger removal ability. It should be noted that the lower the absorbance, the stronger the removal ability for •O2. — The stronger the scavenging ability, the stronger the SOD-like catalytic activity of the nanozyme.

[0130] Figure 11 Oxygen generation kinetics curves for CAT-like activity of different concentrations of PtRhIr nanozymes in Comparative Example 2.

[0131] Figure 12 The graph shows a comparison of the oxygen generation kinetics curves of the PtRhIr nanozyme provided in Comparative Example 2 and the quaternary high-entropy alloy nanozyme provided in Example 1, which exhibit CAT-like activity.

[0132] refer to Figure 11 As shown, the concentration of dissolved oxygen generated gradually increases with the increase of the amount of PtRhIr nanozyme added.

[0133] refer to Figure 12 As shown, compared to PtRhIr nanozymes, the concentration of dissolved oxygen generated increased after adding PtRhIr / Ru SAN from Example 1 of this invention. In other words, compared to PtRhIr nanozymes, PtRhIr / Ru SAN has a stronger ability to catalyze the generation of H2O and oxygen from H2O2.

[0134] Figure 13 The results show the detection of DPPH radical scavenging activity of different concentrations of PtRhIr nanozymes in Comparative Example 2.

[0135] Figure 14 This is a comparison chart showing the detection results of DPPH radical scavenging activity of the PtRhIr nanozyme of Comparative Example 2 and the quaternary high-entropy alloy nanozyme provided in Example 1.

[0136] refer to Figure 13 As shown, the absorbance gradually increases with the increase of the amount of PtRhIr nanozyme added.

[0137] refer to Figure 14 As shown, compared to the PtRhIr nanozyme, the absorbance decreased significantly after adding the PtRhIr / Ru SAN of Example 1 of this invention. In other words, compared to the PtRhIr nanozyme, PtRhIr / Ru SAN has a stronger •OH scavenging ability. It should be noted that the lower the absorbance, the stronger the •OH scavenging ability, and the stronger the DPPH-like free radical scavenging activity of the nanozyme.

[0138] Example 2: Biomimetic membrane-coated nanoenzymes

[0139] Fresh mouse M1 microglia were pretreated, washed with PBS, and then lysed in a cell lysis buffer containing 1 mM EDTA and 10 mM Tris-HCl (pH 7.4) on ice for 30 minutes. The cell membrane precipitate was collected and resuspended in PBS buffer containing 2 mg / mL 2000 Da PEG and magnetically stirred for 30 minutes to obtain an M1 microglia membrane-PEG composite dispersion with a membrane protein content of 30%, which was then refrigerated at 4°C for later use. Next, 2-4 nm of the PtRhIr / RuSAN nanozyme prepared in Example 1 was added to the composite dispersion at a mass ratio of 1:3, and gently stirred for 1 hour to allow the nanozyme surface to initially adsorb membrane components. The mixture was then sonicated for 15 minutes to promote membrane structure dispersion and full contact with the nanozyme. Finally, a 400 nm pore size polycarbonate filter membrane was used to inject the sonicated mixture and squeeze it through the filter membrane at a uniform speed. This process was repeated 8 times. This process achieves uniform coating of the composite membrane; the extruded sample is then centrifuged, the supernatant is discarded, and the sample is resuspended in PBS and washed twice. The precipitate is collected to obtain the biomimetic membrane-coated nanozyme (PtRhIr / Ru SAN@M).

[0140] Comparative Example 3: Biomimetic Membrane-Coated Nanoenzymes

[0141] Using a similar procedure to Example 2, the PtRhIr nanozyme in Comparative Example 2 was coated with a biomimetic membrane to obtain a biomimetic membrane-coated nanozyme (PtRhIr@M).

[0142] Example 3:

[0143] The therapeutic effects of quaternary high-entropy alloy nanozymes in ICH model mice were tested.

[0144] Specifically, healthy male C57BL / 6 mice aged 6-8 weeks and weighing 20g-22g were randomly divided into four groups of 6 mice each, for a total of 24 mice. Brain tissue sections were collected and fixed 21 days after modeling for immunofluorescence staining experiments. Specific grouping details are as follows:

[0145] The sham surgery group (Sham group) underwent only anesthesia, scalp preparation, and skull drilling, but no collagenase was injected into the skull. The skull holes were then sealed with sterile bone wax, and the scalp wounds were sutured. No postoperative medication was administered, and the animals were fed as usual.

[0146] The model group (ICH group): An intracranial hemorrhage (ICH) model was constructed according to standard procedures. After drilling a hole at the right striatal location point, 2 μL of collagenase VII working solution at a concentration of 0.5 U / μL was slowly injected 3.5 mm subdurally (striatal location). The hole was then sealed with sterile bone wax, and the scalp was sutured. No postoperative drug intervention was administered; the patient was fed according to routine care.

[0147] PtRhIr@M group: The ICH model was constructed using the same method as the model group. One day before modeling and on days 1, 3, 5, 7 and 14 after modeling, the biomimetic membrane-coated nanoenzyme (PtRhIr@M) solution prepared in Comparative Example 3 was injected via the tail vein at a dose of 1.2 mg / kg. The group was then routinely fed until day 21 after modeling.

[0148] PtRhIr / Ru SAN@M group: The ICH model was constructed using the same method as the model group. One day before modeling and on days 1, 3, 5, 7 and 14 after modeling, the biomimetic membrane-coated nanozyme (PtRhIr / Ru SAN@M) nanozyme solution prepared in Example 2 was injected via the tail vein. The animals were then routinely fed until 21 days after modeling.

[0149] Twenty-one days after modeling, brain tissue was collected from mice in the sham-operated group (Sham group), the modeling group (ICH group), the PtRhIr@M group, and the PtRhIr / Ru SAN@M group. Brain tissue used for subsequent HE staining and Nissl staining could be directly stained. Brain tissue used for immunofluorescence experiments was first fixed with 4% paraformaldehyde solution, then soaked in 30% sucrose solution for 3 days until the brain tissue completely sank to the bottom, and then frozen sectioned for use in immunofluorescence experiments.

[0150] Animal experiments have confirmed that 10 hours after tail vein injection, the concentration of nanozymes in the lesion area is greatly enriched, indicating that nanozymes can effectively cross the blood-brain barrier and target the lesion area. This characteristic solves the key bottlenecks of traditional drugs, namely "difficult blood-brain barrier penetration" and "poor lesion targeting," avoiding the toxicity risks caused by non-specific uptake by systemic tissues while ensuring effective drug concentration in the hemorrhage area. This lays the foundation for efficient removal of local ROS, inhibition of ferroptosis, and inflammation.

[0151] (1) Hematoxylin-eosin (HE) staining was performed on the brain tissue of mice in different groups.

[0152] Specifically, frozen sections of brain tissue (approximately 5 μm thick) were dewaxed and cleared in xylene solution for 10 minutes each time, for a total of 3 times. After removing excess liquid from the surface of the sections, they were sequentially immersed in ethanol of varying concentrations (100% → 95% → 85% → 75%) for gradient hydration, with each concentration gradient lasting 3 minutes, repeated 3 times. After rinsing the sections with distilled water, they were immersed in hematoxylin staining solution for 3 minutes, followed by rinsing with distilled water for 5 minutes. Then, the sections were dehydrated using 70% ethanol and 95% ethanol for 10 minutes each time, followed by immersion in eosin staining solution for 2–3 minutes. After clearing with xylene, the sections were removed, air-dried to remove excess liquid, and mounted with neutral resin. The sections were then observed under an optical microscope to examine the pathological morphological changes of the brain tissue and to capture images, observing the cell structure around the hemorrhage and the infiltration of inflammatory cells.

[0153] Figure 15 The images show the results of HE staining of brain tissue from different groups of ICH model mice.

[0154] refer to Figure 15 As shown in the HE staining results, there were significant differences in the amount of cerebral hemorrhage and the degree of cerebral edema among the different groups of mice, and the differences between the groups were statistically significant (P < 0.005). The sham-operated group showed almost no obvious cerebral hemorrhage or cerebral edema; the model group showed a large number of hemorrhage foci, and the cerebral edema was extensive and severe; the amount of cerebral hemorrhage and the degree of cerebral edema in the PtRhIr@M group were significantly reduced compared with the model group, but still more than those in the PtRhIr / Ru SAN@M group; the amount of cerebral hemorrhage and the degree of cerebral edema in the PtRhIr@M group were even less than those in the model group. Overall, the trend of cerebral hemorrhage and cerebral edema was: model group > PtRhIr@M group > PtRhIr / Ru SAN@M group > sham-operated group.

[0155] (2) Nissl staining was performed on the brain tissue of mice in different groups.

[0156] Specifically, the preparation method for Nissl staining sections is the same as that for HE staining. After dewaxing and rehydration, the sections are placed in toluidine blue staining solution and stained at 37°C for 30 min. Then, they are rinsed with running water for 5 min to remove excess staining solution. They are then differentiated with 95% ethanol until the background is clear. Next, they are dehydrated with 100% ethanol, cleared with xylene, mounted with neutral resin, dried, and observed under an optical microscope. At this time, Nissl bodies (basophilic substances in the cytoplasm of neurons) appear blue. This is used to observe the morphology, number, and distribution of Nissl bodies of neurons, and thus assess the degree of neuronal damage.

[0157] Figure 16 The images show the Nissl staining results of brain tissue from different groups of ICH model mice.

[0158] refer to Figure 16 As shown, Nissl staining was used to observe neuronal damage. The results showed that the comparison of the number of dead neurons among the groups was statistically significant (P < 0.005). In the sham-operated group, neurons were morphologically intact, neatly arranged, and rich in Nissl bodies; in the model-induced group, the number of dead neurons was high, cell structure was severely damaged, and Nissl bodies were reduced or even disappeared; the number of dead neurons in the PtRhIr@M group was lower than that in the model-induced group, but some neurons still showed structural damage; the number of dead neurons in the PtRhIr / Ru SAN@M group was lower than that in the PtRhIr@M group, indicating a relatively milder degree of neuronal damage. In other words, the number of dead neurons was: model group > PtRhIr@M group > PtRhIr / Ru SAN@M group > sham-operated group.

[0159] (3) The ROS content of brain tissue in mice from different groups was detected.

[0160] Specifically, brain tissue sections (approximately 8 μm thick) that had been fixed, dehydrated with sucrose, and frozen were mounted on anti-detachment glass slides, air-dried at room temperature, and then rinsed three times with phosphate-buffered saline (PBS) for 5 min each time. Subsequently, a DCFH-DA probe with a final concentration of 10 μM was added, and the slides were incubated at 37°C in the dark for 30 min. After incubation, the slides were washed three times with PBS for 5 min each time to remove unbound probes. The slides were then placed under a laser confocal microscope, and the excitation wavelength was selected at 488 nm and the emission wavelength at 525 nm for observation, and fluorescence images were acquired. Simultaneously, image analysis software (e.g., ImageJ) was used to quantitatively analyze the fluorescence images, calculating the average fluorescence intensity to reflect the ROS content level in the brain tissue sections, thereby assessing the degree of oxidative stress in the brain tissue of different groups of mice.

[0161] Figure 17 The image shows the ROS content detection results in the brain tissue of ICH model mice from different groups.

[0162] refer to Figure 17 As shown, the ROS content in brain tissue was detected, and the inter-group comparisons were statistically significant (P < 0.005). The sham-operated group showed extremely low ROS fluorescence intensity, indicating low ROS production; the model-induced group showed significantly enhanced ROS fluorescence intensity, indicating a large amount of ROS production; the PtRhIr@M group showed lower ROS fluorescence intensity than the model-induced group, but higher than the PtRhIr / Ru SAN@M group; the PtRhIr / Ru SAN@M group showed lower ROS fluorescence intensity than the PtRhIr@M group. Overall, the ROS content followed the pattern: model-induced group > PtRhIr@M group > PtRhIr / Ru SAN@M group > sham-operated group.

[0163] Quantitative analysis of the aforementioned ROS measurement results revealed that the ROS level in the brain tissue of ICH model mice in the PtRhIr / Ru SAN@M group was 67% lower than that in the model group, thus solving the problems of "single action" of traditional antioxidants and "incomplete clearance" of single / bimetallic nanozymes.

[0164] (4) GPX4 immunofluorescence detection was performed on the brain tissue of mice in different groups.

[0165] Specifically, brain slices were transferred to glass slides, excess liquid was removed with a pipette, and fixed with 4% paraformaldehyde for 10 minutes at room temperature. Then, the brain slices were permeated and blocked with a solution containing 0.5% Triton X-100 and 3% bovine serum albumin at room temperature for 1 hour. Afterward, diluted GPX4 antibody was added, and the slides were incubated overnight at 4°C. The next day, the slides were incubated with appropriately diluted secondary antibody at room temperature for 1 hour. After rinsing, 4',6-diamidinyl-2-phenylindole (DAPI) staining solution was added, and staining was performed under light-protected conditions. After rinsing again, the slides were mounted with anti-fluorescence quenching mounting medium, and nail polish was used to secure the coverslip to prevent movement. Finally, images were acquired using an inverted fluorescence microscope, and fields of equal size were randomly selected for observation. Six independent samples were selected from each group, and the number of positive cells was calculated using image analysis software (e.g., ImageJ).

[0166] Figure 18 The image shows the results of GPX4 content detection in the brain tissue of ICH model mice from different groups.

[0167] refer to Figure 18 As shown, GPX4, an antioxidant-related protein, exhibited statistically significant differences in immunofluorescence results between groups (P < 0.005). The sham-operated group showed high GPX4 fluorescence intensity and abundant expression; the model-induced group showed significantly weakened GPX4 fluorescence intensity and low expression; the PtRhIr@M group showed higher GPX4 fluorescence intensity than the model-induced group; and the PtRhIr / RuSAN@M group showed higher GPX4 fluorescence intensity than the PtRhIr@M group, exhibiting an expression trend of sham-operated group > PtRhIr / RuSAN@M group > RuRhPt@M group > model-induced group. Based on the above tests, the PtRhIr / RuSAN quaternary alloy nanozyme is superior to PtRhIr in reducing cerebral hemorrhage volume and cerebral edema, reducing neuronal death, lowering ROS levels, and upregulating the expression of the antioxidant protein GPX4, demonstrating a more significant neuroprotective effect. This provides strong experimental evidence for its clinical application in the treatment of cerebral hemorrhage and other neurological diseases.

[0168] Immunohistochemical and immunofluorescence staining analyses can also detect the expression levels of MDA, CD86, and CD206. Compared to the model group, the PtRhIr / Ru SAN@M group showed a 58% decrease in the expression of CD86 (M1 marker of microglia), a 72% increase in the expression of CD206 (M2 marker of microglia), and a 58% decrease in MDA content (a marker of ferroptosis) in the brain tissue.

[0169] (5) Test the inflammatory status of mouse brain tissue.

[0170] Specifically, after homogenizing and lysing mouse brain tissue, the expression of inflammatory factors (IL-6 and IL-10) was tested using an enzyme-linked immunosorbent assay (ELISA). The results showed that, compared to the model group, the serum levels of pro-inflammatory IL-6 in ICH mice in the PtRhIr / RuSAN@M group were reduced by 69%, and the levels of anti-inflammatory IL-10 were increased by 1.3 times. This indicates that the PtRhIr / RuSAN quaternary alloy nanozyme of this invention can reduce and alleviate inflammation.

[0171] In addition, biosafety experiments were conducted on mice in the PtRhIr / RuSAN@M group. The results showed that the liver and kidney function indicators (ALT, BUN) of the mice in the PtRhIr / RuSAN@M group remained within the normal range, with no obvious organ damage; thus solving the problem of "high toxicity risk" of existing nanozymes.

[0172] In summary, the comprehensive therapeutic effect of quaternary high-entropy alloy nanozymes is significantly superior to existing drugs with only single functions, achieving multi-dimensional and precise intervention in the pathological process of cerebral hemorrhage. Furthermore, these quaternary high-entropy alloy nanozymes exhibit excellent biocompatibility and stability, reducing the risk of clinical translation. The core characteristics of the quaternary high-entropy alloy nanozymes work synergistically to form an integrated therapeutic system of "barrier penetration - lesion targeting - ROS clearance - inflammation regulation," which not only comprehensively eliminates ROS but also regulates the polarization state of microglia to alleviate neuroinflammation.

[0173] Specifically, treatment options for ICH often suffer from limited efficacy due to their inability to simultaneously address issues such as incomplete ROS clearance, weak penetration, poor targeting, and insufficient inflammatory regulation. The quaternary high-entropy alloy nanozyme of this invention possesses highly efficient ROS clearance capabilities that comprehensively block oxidative stress; its small particle size and PVP modification ensure effective penetration of the blood-brain barrier and accumulation in the lesion; simultaneously, the accumulated nanozyme reduces inflammatory stimulation by clearing ROS and can directly regulate microglial phenotypic transformation, preventing excessive polarization towards the pro-inflammatory M1 type and promoting conversion towards the anti-inflammatory M2 type, thereby blocking the amplification effect of inflammation. These four elements form a closed loop, specifically addressing the multiple pathological barriers of "oxidative stress-ferroptosis-inflammation" in cerebral hemorrhage.

[0174] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A quaternary high-entropy alloy nanoenzyme, comprising a single-phase alloy composed of four metallic elements: M, rhodium, iridium, and ruthenium, and polyvinylpyrrolidone modified on the surface of the single-phase alloy, wherein, The element M is selected from palladium or platinum, and the nanozyme has a particle size of less than 10 nm.

2. The quaternary high-entropy alloy nanozyme according to claim 1, wherein, The nanozyme has a particle size of 3-5 nm.

3. The quaternary high-entropy alloy nanozyme according to claim 1 or 2, wherein, The nanozyme is in the form of spherical particles; And / or, the single-phase alloy has a face-centered cubic structure; And / or, ruthenium exists primarily in monatomic form.

4. The quaternary high-entropy alloy nanozyme according to claim 1 or 2, wherein, The surface of the quaternary high-entropy alloy nanoenzyme is also coated with a composite layer of biomimetic cell membrane and polyethylene glycol, wherein the biomimetic cell membrane is selected from neutrophil membrane or microglia membrane.

5. A method for preparing a quaternary high-entropy alloy nanozyme as described in any one of claims 1 to 4, comprising: The salts corresponding to the four metallic elements M, rhodium, iridium and ruthenium are mixed and dissolved in a solvent to obtain a mixed solution; Polyvinylpyrrolidone and a reducing agent were added to the mixed solution, and a reduction reaction was carried out at 25~35℃. The quaternary high-entropy alloy nanozyme was obtained by separation.

6. The preparation method according to claim 5, wherein, The reducing agent is selected from sodium borohydride; And / or, the reduction reaction takes 1 to 3 hours.

7. The preparation method according to claim 5, wherein, The molar ratio of the four metallic elements M, rhodium, iridium, and ruthenium is (0.5~2):(0.5~2):(0.5~2):1; And / or, the amount of polyvinylpyrrolidone used is a mol, and the total amount of the four metal elements added is b mol, where a / b = 1.5:1~2.5:

1.

8. The preparation method according to claim 5, wherein, The preparation method further includes: The surface of the quaternary high-entropy alloy nanoenzyme is coated with a composite layer of biomimetic cell membrane and polyethylene glycol.

9. The use of a quaternary high-entropy alloy nanozyme as described in any one of claims 1 to 4 in the preparation of a drug for treating cerebral hemorrhage.

10. The application according to claim 9, wherein, The treatment of cerebral hemorrhage includes treating cerebral hemorrhage by scavenging reactive oxygen species, inhibiting ferroptosis, and / or inhibiting neuroinflammation; The reactive oxygen species include superoxide anions, hydrogen peroxide, and hydroxyl radicals.

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