Multi-enzyme activity high-entropy alloy material as well as preparation method and application thereof

Abstract

The invention belongs to the field of catalytic materials, and particularly relates to a multi-enzyme activity high-entropy alloy material and a preparation method and application thereof. The multi-enzyme activity high-entropy alloy material comprises the following components: Ga, Pd, Ru, Ir and Mn, wherein the crystal phase structure of the high-entropy alloy material is a single-phase solid solution structure. The multi-enzyme activity high-entropy alloy material provided by the invention has pH-adaptive multi-enzyme activity, and not only can effectively treat tumors, but also can protect damage caused by radiotherapy.

Description

Technical Field

[0001] The present invention belongs to the field of catalytic materials, and in particular relates to a multi-enzyme active high-entropy alloy material and a preparation method and application thereof. Background Art

[0002] Radioactive materials and the ionizing radiation they produce have been widely used in fields such as nuclear energy utilization, medical imaging, and tumor radiotherapy, profoundly impacting social progress and development. However, ionizing radiation can damage surrounding healthy tissues and cause various side effects. In radiotherapy for abdominal / pelvic solid tumors (such as pancreatic cancer, prostate cancer, and colorectal cancer), the small intestine, with its high radiosensitivity and large size, is a common site of radiation-induced injury. Intestinal injury caused by enhanced radiotherapy can lead to gastrointestinal dysfunction such as nausea, diarrhea, vomiting, bleeding, infection, perforation, and even death. Radiation-induced intestinal injury is a common complication of tumor radiotherapy with a poor prognosis, limiting the application and therapeutic efficacy of abdominal and pelvic radiotherapy. Therefore, research on radiation-induced injury protection has important social value and practical significance. Currently, although many drugs and natural ingredients have shown potential for radiation protection, their oral administration faces numerous challenges, making it difficult to effectively protect the intestine and the entire body from radiation-induced injury. Therefore, the development of drugs that can effectively treat tumors and protect the intestine after radiotherapy is of particular urgency and importance.

[0003] In tumor catalytic therapy, enzymes serve as essential initiators of catalytic reactions and determine the therapeutic effect. Although much research has been conducted on the use of natural enzymes (such as glucose oxidase, catalase, and lactate oxidase) in tumor catalytic therapy, these natural enzymes still face certain foreseeable challenges, such as poor selectivity, stability, reusability, and strict physiological conditions. This has prompted researchers to continuously strive to develop superior enzyme-like materials.

[0004] High-entropy alloys (HEAs) are an emerging class of alloy materials characterized by the uniform distribution of five or more primary elements in equimolar or equiatomic proportions. They have attracted considerable attention due to their wide compositional tunability and potentially unlimited biological applications. Recent studies have revealed that the interactions between the multiple elements in HEAs promote the formation of stable single-phase solid solutions, rather than fragile intermetallic compounds. This structural stability allows HEAs to significantly tune the electronic and geometric structures of the alloys, leading to the design of catalysts with superior catalytic properties. Furthermore, HEAs, through their inherent synergistic effects, overcome inherent elemental incompatibility, thereby optimizing catalytic performance. Compared to conventional (non-high-entropy) MXenes, HEAs exhibit superior biocatalytic performance. Summary of the Invention

[0005] The purpose of the present invention is to provide a multi-enzyme active high entropy alloy material, namely a GaPdRuIrMn high entropy alloy material, which has pH-adaptive multi-enzyme activity and can effectively treat tumors and protect damage caused by radiotherapy.

[0006] Specifically, in one aspect, the present invention provides a multi-enzyme active high entropy alloy material comprising the following components: Ga, Pd, Ru, Ir and Mn.

[0007] Furthermore, the crystal phase structure of the high entropy alloy material is a single-phase solid solution structure.

[0008] Furthermore, the high entropy alloy material contains equimolar amounts of Ga, Pd, Ru, Ir and Mn.

[0009] Furthermore, the multi-enzyme active high entropy alloy material has POD enzyme-mimicking, SOD enzyme-mimicking, CAT enzyme-mimicking and / or XOR enzyme-mimicking activities.

[0010] Furthermore, the multi-enzyme active high entropy alloy material has pH-adaptive POD enzyme-mimicking, SOD enzyme-mimicking, CAT enzyme-mimicking and / or XOR enzyme-mimicking activities.

[0011] As used herein, pH adaptability means that the multi-enzyme-active high entropy alloy material exhibits different enzyme catalytic activities at different pH values.

[0012] Furthermore, the multi-enzyme active high entropy alloy material can exhibit POD enzyme-mimicking activity at acidic pH, such as in a tumor microenvironment; and can exhibit SOD enzyme-mimicking, CAT enzyme-mimicking and / or XOR enzyme-mimicking activity at neutral or alkaline pH, such as in an intestinal environment.

[0013] In other aspects, the present invention also provides a method for preparing a multi-enzyme active high entropy alloy material as described herein, which comprises the following steps: subjecting Ga salt, Pd salt, Ru salt, Ir salt and Mn salt to a solvothermal reaction in a solvent to obtain a multi-enzyme active high entropy alloy material.

[0014] Further, as used herein, the metal salt is at least one of the metal acetate, acetylacetonate, chloride and nitrate.

[0015] Furthermore, the solvent is a mixture of one or more of methanol, ethanol and acetone, or a mixture of one or more of methanol, ethanol and acetone and water.

[0016] Preferably, the solvent is a mixed solvent of ethanol and acetone, wherein the volume ratio of ethanol to acetone can be 1:1.

[0017] Furthermore, the solvent thermal reaction comprises reacting at 150-240°C for 1-3 hours.

[0018] Furthermore, the preparation method includes dissolving Ga salt, Pd salt, Ru salt, Ir salt and Mn salt in a solvent, ultrasonically mixing, performing a solvent thermal reaction, collecting the product by centrifugation, washing and drying to obtain a GaPdRuIrMn high entropy alloy catalyst.

[0019] In other aspects, the present invention also provides the use of the multi-enzyme active high entropy alloy material as described herein for preparing a multi-enzyme active catalyst with pH adaptability.

[0020] Furthermore, the multi-enzyme activity includes POD enzyme-mimicking, SOD enzyme-mimicking, CAT enzyme-mimicking and / or XOR enzyme-mimicking activities.

[0021] Furthermore, pH adaptability refers to the ability to exhibit different enzyme catalytic activities at different pH levels. Specifically, at acidic pH levels, such as in a tumor microenvironment, POD-like enzyme activity may be exhibited; and at neutral or alkaline pH levels, such as in the intestinal tract, SOD-like, CAT-like, and / or XOR-like enzyme activity may be exhibited.

[0022] In other aspects, the present invention also provides the use of the multi-enzyme active high entropy alloy material as described herein in the preparation of drugs for radiosensitization.

[0023] Furthermore, the radiosensitization includes enhancing the effect of radiotherapy for tumors.

[0024] In other aspects, the present invention also provides the use of the multi-enzyme active high entropy alloy material as described herein in the preparation of drugs for protecting against radiation damage.

[0025] Furthermore, the radiation injury includes radiation-induced intestinal injury.

[0026] In other aspects, the present invention also provides the use of the multi-enzyme active high entropy alloy material as described herein in the preparation of drugs for radiosensitization and protection against radiation damage.

[0027] Advantageous Effects of the Invention

[0028] The present invention has systematically studied and shown that GaPdRuIrMn high entropy alloy materials exhibit strong POD-like enzyme activity in acidic environments and strong SOD and CAT-like enzyme activity in neutral or alkaline environments. Schematic diagram of the anti-tumor sensitization and intestinal radiotherapy protection mediated by GaPdRuIrMn under different pH environments is shown in the figure below. Figure 1 In the acidic tumor microenvironment, GaPdRuIrMn catalyzes the decomposition of H2O2 and produces superoxide anions (·O2 - ) and singlet oxygen ( 1 O2) mediates tumor cell apoptosis. However, in the neutral or alkaline environment of the intestine, thanks to the catalytic activity brought by the diverse components and rich defects of GaPdRuIrMn, GaPdRuIrMn has a strong affinity for H2O2, ·O2 - The GaPdRuIrMn nanoparticles exhibited excellent ROS scavenging capabilities, effectively preventing radiotherapy damage to intestinal epithelial cells. Furthermore, compared to single Pd metal particles, GaPdRuIrMn exhibited higher catalytic activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram showing the radiosensitization anti-tumor and intestinal radiotherapy protection effects mediated by GaPdRuIrMn under different pH environments.

[0030] Figure 2 Shown is a schematic diagram of the synthesis and structure of GaPdRuIrMn.

[0031] Figure 3 Shown are the XRD patterns of GaPdRuIrMn and Pd particles.

[0032] Figure 4 Shows the XRD patterns of GaPdRuIrMn prepared by different methods.

[0033] Figure 5 Shown are (a), (b) SEM spectra of GaPdRuIrMn, (c), (d) SEM spectra of Pd, (e) TEM image of GaPdRuIrMn, (f) HRTEM of GaPdRuIrMn, (g) lattice spacing of GaPdRuIrMn, and (h) related element mapping image of GaPdRuIrMn.

[0034] Figure 6 The spherical aberration characterization of GaPdRuIrMn is shown, where Figures b and c are partial enlargements of Figure a.

[0035] Figure 7 XPS spectra of GaPdRuIrMn are shown: high-resolution XPS spectra of (a) Ga 2p, (b) Pd 3p, (c) Ru 3p, (d) Ir 4f, and (e) Mn 2p.

[0036] Figure 8 The results of ROS production activity evaluation are shown: (a) The color reaction of H2O2 catalyzed by GaPdRuIrMn and Pd particles to produce ROS. (b) and (c) The radiosensitization reaction of GaPdRuIrMn 1 O2 and O2 - effect.

[0037] Figure 9 Shown are (a) SOD enzyme activity of different catalysts; (b) DPPH· scavenging rate of GaPdRuIrMn catalyst; (c) H2O2 scavenging rate of different catalysts; (d) O2 concentration produced by different catalysts measured by dissolved oxygen meter in the presence of H2O2; (e), (f) xanthine oxidation performance of different catalysts.

[0038] Figure 10 The enzymatic activity evaluation of the metal-deficient control group is shown: (a) POD enzyme activity of the catalyst; (b) CAT enzyme activity of the catalyst; (c) SOD enzyme activity of the catalyst.

[0039] Figure 11 The in vitro experimental results of anti-tumor sensitization are shown: representative ROS images (a) and quantitative statistics (e) of CT26 cells co-incubated with GaPdRuIrMn and 24 hours after radiotherapy; representative Calcein-AM / PI fluorescence staining (b) and quantitative statistics (f); representative γ-H2AX fluorescence staining (c) and quantitative statistics (g); representative flow cytometry detection of cell apoptosis images (d) and quantitative statistics (h).

[0040] Figure 12 The in vivo experimental results of anti-tumor sensitization are shown: in vivo imaging of abdominal tumors in MC38-luc tumor-bearing mice on days 0, 4, and 7.

[0041] Figure 13The in vitro experimental results of intestinal radiotherapy protection are shown: representative ROS images (a) and quantitative statistics (e) of NCM460 cells co-incubated with GaPdRuIrMn and 24 hours after radiotherapy; representative Calcein-AM / PI fluorescence staining (b) and quantitative statistics (f); representative γ-H2AX fluorescence staining (c) and quantitative statistics (g); representative flow cytometry detection of cell apoptosis images (d) and quantitative statistics (h).

[0042] Figure 14 The in vivo experimental results of intestinal radiotherapy protection are shown: representative images of the anus of mice in different treatment groups on the 7th day of radiation enteritis BALB / C mouse model (a), PDS staining image (b), Ki67 immunohistochemistry image (c), and γ-H2AX immunohistochemistry image (d). DETAILED DESCRIPTION

[0043] The present invention is further described below with reference to specific examples, which, however, are not intended to limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the art.

[0044] Example: Preparation of GaPdRuIrMn high entropy alloy catalyst

[0045] The synthesis process of GaPdRuIrMn high entropy alloy catalyst is as follows Figure 2 As shown, a GaPdRuIrMn high-entropy alloy catalyst was synthesized by a solvothermal method at 200°C using gallium acetylacetonate, palladium acetylacetonate, ruthenium acetylacetonate, iridium acetylacetonate, and manganese acetylacetonate in an ethanol / acetone mixture. Specifically, equimolar amounts of metal salts (0.01 mmol of each metal, corresponding to the following masses: 36.7 mg of gallium acetylacetonate, 30.5 mg of palladium acetylacetonate, 39.8 mg of ruthenium acetylacetonate, 49 mg of iridium acetylacetonate, and 35.2 mg of manganese acetylacetonate) were added to a hydrothermal autoclave. 15 ml of an ethanol / acetone mixture (volume ratio of 1:1) was then added and sonicated for 1 hour to achieve uniform mixing. The solution was then transferred to a hydrothermal autoclave jacket, tightened, and reacted in a 200°C oven for 77 minutes. The product was washed by centrifugation, washed three times with acetone, and once with ethanol, and then dried overnight in a 60°C vacuum oven to obtain the GaPdRuIrMn high-entropy alloy catalyst.

[0046] Comparative Example: Preparation of Single Pd Particles and Preparation of Multi-metal Composite Materials Using Different Methods

[0047] A single Pd particle was prepared as a control by a solvothermal method using only palladium acetylacetonate as a raw material in the same manner as in the example, in order to confirm the advantages of the multi-component GaPdRuIrMn high entropy alloy catalyst in exerting catalytic activity.

[0048] A multimetallic composite material with the same elemental composition as the GaPdRuIrMn high-entropy alloy catalyst but prepared using a different synthesis method (H2 reduction) was also used as a control. This synthesis method incorporates liquid metal Ga as a catalyst to promote elemental fusion, and is synthesized under high-temperature, H2-reducing conditions. The detailed synthesis method involves weighing equimolar amounts of metal salts (i.e., palladium acetylacetonate, ruthenium acetylacetonate, iridium acetylacetonate, and manganese acetylacetonate) and mixing them with liquid metal Ga particles. The mixture is encapsulated in a small porcelain boat and placed in a tube furnace for reaction. The tube furnace is programmed to heat the temperature from room temperature to 650°C over 30 minutes and maintain this temperature for 6 hours until the reaction is complete. During the reaction, argon is passed through the tube furnace for protection during the heating and cooling phases, and hydrogen is introduced during the holding phase to reduce the metal salts. As verified by XRD in the test examples below, this method does not produce a single-phase high-entropy alloy, while the method of the present invention produces a single-phase solid solution high-entropy alloy.

[0049] Test Example 1: Structural Characterization of GaPdRuIrMn High Entropy Alloy Catalyst

[0050] The crystal structures of GaPdRuIrMn high entropy alloy catalysts and materials prepared in comparative example were characterized by powder X-ray diffraction (XRD). Figure 3 The XRD spectra of the GaPdRuIrMn high-entropy alloy catalyst and Pd particles are shown. The peaks at 39.06°, 45.41°, and 66.17° are attributed to the (111), (200), and (220) crystal planes of Pd (PDF#87-0637), respectively. Compared to the Pd particles, the XRD peaks of GaPdRuIrMn exhibit a certain shift, with the peaks at 39.4°, 45.66°, and 66.95° being attributed to the (111), (200), and (220) crystal planes of GaPdRuIrMn, respectively. This is because the difference in atomic radius causes lattice distortion, i.e., the incorporation of heteroatoms with a smaller atomic radius than the main atoms results in a smaller lattice constant. Figure 4 The XRD spectra of the GaPdRuIrMn high entropy alloy catalyst and the multi-metal composite material prepared by the H2 reduction method are shown. It can be seen that the H2 reduction method cannot obtain a high entropy alloy with a single-phase structure, while the method of the present invention obtains a high entropy alloy with a single-phase solid solution structure.

[0051] Scanning electron microscopy (SEM) was used to characterize the catalyst morphology. Figure 5 As shown in ab, GaPdRuIrMn exhibits a uniform spherical flower-like structure, and the magnified result shows that GaPdRuIrMn presents a rough, uneven surface. Figure 5As shown in Figures cd, the Pd particles are spherical particles with a morphology similar to that of GaPdRuIrMn, but with a smoother surface. The uneven surface can increase the contact area with the reaction system, which is more conducive to improving the catalytic activity. The morphology of GaPdRuIrMn was further observed by transmission electron microscopy (TEM). Figure 5 As shown in e, GaPdRuIrMn The morphology is a small granular structure with a size of about 1 μm. Further HRTEM shows the grain size and lattice spacing of the material. Figure 5 fg). Energy dispersive X-ray spectroscopy (EDS) mapping images show ( Figure 5 h) The catalyst contains Ga, Pd, Ru, Ir, and Mn elements, and the elements are relatively evenly distributed throughout the high-entropy alloy structure.

[0052] In order to further study the atomic structure of GaPdRuIrMn, aberration-corrected high-angle annular dark-field scanning transmission electron microscopy (AC-HAADF-STEM) was used for characterization. Figure 6 ac clearly shows the defects in the material structure, including dislocations (blue T), insertions of different atoms (blue and yellow circles), and distortions of the lattice. The abundance of defects will significantly enhance the catalytic activity of the material.

[0053] Furthermore, X-ray photoelectron spectroscopy (XPS) was used to analyze the surface chemical composition and electronic structure of the catalyst. Figure 7 As shown in Figures ae and c, the XPS spectrum of Ga 2p indicates that Ga in the catalyst is in a zero-valent state, the XPS spectrum of Pd 3d indicates that Pd in ​​the catalyst is in a mixture of zero-valent and oxidized states, the XPS spectrum of Ru 3p indicates that Ru in the catalyst is in a zero-valent state, the XPS spectrum of Ir 4f indicates that Ru in the catalyst is in a zero-valent state, and the XPS spectrum of Mn 2p indicates that Mn in the catalyst is in an oxidized state. These results indicate that the elemental valence states of the GaPdRuIrMn high-entropy alloy are a mixture of zero-valent and oxidized states. The presence of oxidized states indicates that the metals on the material surface have undergone some degree of oxidation.

[0054] The test equipment and parameters used in this test example are as follows:

[0055] 1. Scanning electron microscopy (SEM) images were acquired using an Apreo S HiVoc (Thermo Fisher Scientific, FEI).

[0056] 2. X-ray diffraction (XRD) patterns were obtained using a DX-2700BH multi-purpose X-ray diffractometer (Haoyuan Instruments) using Cu radiation at 40 kV. The samples were scanned over the 2θ range of 5° to 80°.

[0057] 3. Transmission electron microscopy (TEM) and energy dispersive spectroscopy (EDS) imaging were performed using a 300 kV FEI Tecnai G2 F30 microscope (available from FEI, Inc., USA). Aberration-corrected high-angle annular dark-field scanning TEM (AC HAADF-STEM) was performed using a 200 kV Titan Themis 60-300 microscope (available from FEI, Inc., USA).

[0058] 4. X-ray photoelectron spectroscopy (XPS) was performed using a K-Alpha™+ X-ray photoelectron spectrometer (ThermoScientific), equipped with a 180° dual-focus analyzer and a 128-channel detector. The prepared powder sample was pressed onto a carbon tap and then attached to a sample holder for measurement. Data were collected using an X-ray spot size of 400 μm and 20 scans.

[0059] Test Example 2: Performance Test of GaPdRuIrMn High Entropy Alloy Catalyst

[0060] After successfully characterizing the morphology and electronic structure of the GaPdRuIrMn and Pd particle biocatalysts, the present inventors further tested their performance data. Thanks to the catalytic activity brought about by the diverse components and abundant defects of GaPdRuIrMn, GaPdRuIrMn exhibits pH-adaptive multienzyme activity. Specifically, it exhibits strong POD-like enzyme activity in acidic environments, as well as strong SOD and CAT-like enzyme activities in neutral or slightly alkaline environments.

[0061] 1) POD-like enzyme activity in acidic environment

[0062] In the acidic tumor microenvironment (TME), H2O2 is higher than that in normal cells, and the excess H2O2 is converted into more cytotoxic •O2 − and 1 O2 deoxidation damage to tumor cells is an effective treatment method. To this end, the catalytic performance of simulated peroxidase (POD) was tested. The difference in absorbance of the system with different materials shows that GaPdRuIrMn has a higher POD activity ( Figure 8 a). By controlling the radiation conditions of the system and observing the intensity of ROS production, it can be seen that GaPdRuIrMn has a significant radiosensitization effect on ROS production ( Figure 8 b and c).

[0063] 2) SOD and CAT-like enzyme activities in neutral or alkaline environments

[0064] Radiotherapy may induce oxidative stress in intestinal epithelial cells. The SOD and CAT-like enzyme activities in the neutral or alkaline environment of the intestine can alleviate this side effect. Since GaPdRuIrMn has pH-adaptive multienzyme activity, its effect on ·O2 - , DPPH· and H2O2 scavenging effects.

[0065] GaPdRuIrMn to superoxide radicals (·O2 - ) clearance rate reached over 80% (simulating superoxide dismutase activity (SOD)), which was better than that of Pd particles ( Figure 9 a).

[0066] To further verify the antioxidant properties of the samples, a free radical scavenging experiment was conducted using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method. After DPPH was paired with a free radical scavenger, the color of DPPH changed from purple to yellow, and the absorbance at a wavelength of 519 nm decreased. The absorbance at 519 nm was measured and the DPPH scavenging efficiency was calculated, as shown in Figure 2. Figure 9 As shown in (b), the material exhibits a concentration-dependent DPPH· scavenging effect. When the concentration in the system is 100 ng / ml, the DPPH· scavenging rate can reach more than 80%.

[0067] From the time-varying CAT enzyme activity curve, it can be seen that the H2O2 concentration of GaPdRuIrMn is significantly lower than that of Pd particles ( Figure 9 c) It takes about 10 minutes to completely remove all H2O2 in the system, which fully demonstrates the performance advantages of high entropy alloys. The O2 concentration produced by different catalysts is measured using a dissolved oxygen meter, such as Figure 9 As shown in Figure d, the rate of producing O2 by catalytic decomposition of H2O2 over GaPdRuIrMn is the fastest and the O2 concentration is the highest, reaching above 40 mg / L at 180 s.

[0068] In addition, the XOR performance of the material was tested, and GaPdRuIrMn could effectively convert xanthine into uric acid ( Figure 9 e), and outperformed single Pd particles ( Figure 9 f).

[0069] Taken together, these results demonstrate that GaPdRuIrMn possesses diverse enzyme-mimetic properties due to its unique material structure.

[0070] The performance test method used in this test case is as follows:

[0071] 1. Peroxidase (POD) catalytic activity assay: The test catalyst decomposes hydrogen peroxide (H2O2) into reactive oxygen species (ROS), which then oxidize the colorless chromogenic agent 3,3',5,5'-tetramethylbenzidine (TMB) to produce blue oxidized 3,3',5,5'-tetramethylbenzidine (oxTMB). The ROS generation capacity was evaluated as follows: a final concentration of 1.25 mM H2O2 (Aladdin, Shanghai, China), 0.12 mg / M lde1TMB, and 50 μg mL -1 The catalyst was mixed in acetic acid / sodium acetate buffer (pH = 4.5) to a volume of 2 mL. After 10 minutes of reaction, 200 μL of the solution was added to a 96-well plate, and the absorbance of the solution at 652 nm was measured to assess the extent of ROS generation.

[0072] 2. Experimental verification of radiosensitization ROS production: Electron paramagnetic resonance (EPR) technology was used to detect the types of ROS generated during the POD process. Since the lifespan of free radicals is very short, spin traps are required to capture and stabilize the free radicals for a period of time. DMPO and TEMP were used to capture superoxide anions (·O2 - ) and singlet oxygen ( 1 O2), the specific implementation method is: ①·O2 - Capture: Add 10 μL of 10 mg / ml catalyst solution, 10 μL of DMPO, and 10 μL of 30% hydrogen peroxide to 0.5 ml of DMSO, and use a capillary to draw a small amount of the reaction solution for testing. ② 1 O2 capture: Add 10 μL of a 10 mg / mL catalyst solution, 20 μL of TEMO, and 10 μL of 30% hydrogen peroxide to 0.5 mL of acetic acid / sodium acetate buffer. A small amount of the reaction solution is pipetted using a capillary tube for testing. To investigate the radiosensitization effect, the experimental steps are the same as above, except that the reaction solution must be irradiated.

[0073] 3. Superoxide dismutase (SOD)-like activity: 1 mg KO2 (Sigma-Aldrich, MO, USA) was dissolved in 1 ml 18-crown-6-dimethylsulfoxide (DMSO) solution (Aladdin, Shanghai, China) (3 mg mL -1 ). Then, the catalyst with a final concentration of 50 μg / mL and the above 60 μL KO2-crown ether-DMSO solution were added to the crown ether-DMSO (3 mg / mL) solution to make the volume 990 μL. After reacting for 5 min, 10 μL nitro blue tetrazolium (NBT)-DMSO solution (10 mg / mL) was added. -1NBT, Aladdin, Shanghai, China) to detect the remaining O2 - The absorbance of the solution at 680 nm was then measured to quantitatively assess its ability to remove O2 - ability.

[0074] 4. DPPH scavenging activity. Mix 1990 µL of a 50 μg / mL ethanolic solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH, Aladdin, Shanghai, China) with 10 µL of a 10 mg / mL catalyst. The mixture was then allowed to react in the dark. After 60 minutes, the absorbance of the solution at 519 nm was measured to calculate the DPPH radical scavenging activity.

[0075] 5. Catalytic activity of catalase (CAT): The catalytic activity of catalase (CAT) was evaluated by testing the scavenging of hydrogen peroxide (H2O2) and the generation of oxygen (O2). The scavenging ability of H2O2 was evaluated as follows: H2O2 (Aladdin, Shanghai, China) with a final concentration of 10 mM and 50 μg mL -1 The catalyst was mixed in PBS to a volume of 2 mL. Then, 50 µL of the solution was added to 100 µL of Ti(SO4)2 solution (13.9 mM, Aladdin, Shanghai, China). The absorbance of the solution at 405 nm was measured at 10, 20, 30, and 40 min to assess the concentration of residual H2O2. The catalytic production of O2 by H2O2 was assessed as follows: a total of 100 mM H2O2 and 2.5 µg mL -1 The catalysts were mixed in 20 mL PBS at room temperature, and the dissolved O2 concentration was measured every 5 s using a dissolved oxygen meter (JPSJ-606L DO Meter, China) until the test was terminated at 3 min.

[0076] 6. Xanthine oxidase (XOR)-like activity: 3 mg of catalyst was weighed and added to 300 μL PBS, followed by a 0.2 mM xanthine solution. The reaction was incubated in a 37°C water bath for 3 days. 200 μL of the solution was measured in a 96-well plate to measure the absorbance at 294 nm to evaluate the ability to oxidize xanthine.

[0077] Test Example 3: Evaluation of enzyme-like activity after reducing the metal element composition in the GaPdRuIrMn high-entropy alloy catalyst

[0078] The metal element composition of the GaPdRuIrMn high-entropy alloy catalyst was reduced to prepare a quaternary metal high-entropy alloy (i.e., lacking Mn, Ir, Ru, Pd, and Ga, respectively). The preparation method was the same as in the Example. The original intention of this control experiment was to design the GaPdRuIrMn high-entropy alloy catalyst based on theoretical expectations. The design concept is to introduce Ru, a metal with a high Z value and enzyme-mimicking activity, which has a radiosensitizing effect, and then select other transition metals to co-construct the high-entropy alloy (HEA). Therefore, the quaternary metal control group was designed to verify the Ru as the pre-defined active site.

[0079] The enzyme-mimicking activity of quaternary metal high entropy alloys was tested, such as Figure 10 As shown in Figures ac, the experimental results show that the experimental performance of the control group without Ru did not drop significantly, but the performance of the control group without Mn and Pd dropped, indicating that there is a difference between the preset active sites and the actual results. It is speculated that the special crystal structure of HEA and the interaction between atoms caused such a result.

[0080] Test Example 4: Radiotherapy sensitization, anti-tumor and intestinal radiotherapy protection effects of GaPdRuIrMn high entropy alloy catalyst.

[0081] After successfully verifying that GaPdRuIrMn has multiple enzyme activities, the present invention further studied the radiosensitization anti-tumor and intestinal radiotherapy protection effects mediated by GaPdRuIrMn under different pH environments.

[0082] 1) Radiotherapy sensitization and anti-tumor effect

[0083] In the in vitro experiment, GaPdRuIrMn was co-incubated with CT26 cells (pH = 6.8) and 24 h after radiotherapy, e.g. Figure 11 As shown in a and e, compared with the control group, the X-ray (radiotherapy) group and the GaPdRuIrMn group did not significantly increase ROS generation, while the GaPdRuIrMn+radiotherapy group had significant ROS generation. In addition, the results of Calcein-AM / PI fluorescence staining showed that ( Figure 11 b and f), the proportion of dead cells increased and the number of live cells decreased in the GaPdRuIrMn+radiotherapy group. γ-H2AX is an important marker of DNA damage. A large number of γ-H2AX foci were formed in the GaPdRuIrMn+radiotherapy group ( Figure 11 c and g), indicating that the number of DNA double-strand breaks generated in CT26 cells in the treatment group increased. In order to evaluate the radiosensitization and anti-tumor activity of GaPdRuIrMn, cell apoptosis was detected by flow cytometry, as shown in Figure 5. Figure 11As shown in d and h, the average cell apoptosis rates of the control group, GaPdRuIrMn group, radiotherapy group and GaPdRuIrMn+radiotherapy group were 4.5%, 10.57%, 10.2% and 26.87%, respectively. The apoptosis rate of the GaPdRuIrMn+radiotherapy group was 16.67% higher than that of the radiotherapy group, demonstrating that GaPdRuIrMn has an excellent radiosensitizing effect.

[0084] To further evaluate the in vivo radiosensitization and anti-tumor activity of GaPdRuIrMn, a MC38-luc transplant tumor model was established using BALB / C mice to evaluate the anti-tumor ability of GaPdRuIrMn in mice. Figure 12 The experimental results showed that compared with the Control group, the ability of different treatment groups to inhibit mouse tumor volume growth was GaPdRuIrMn+radiotherapy group > radiotherapy group > GaPdRuIrMn group, indicating that GaPdRuIrMn combined with radiotherapy had the most obvious inhibitory effect on tumor growth, further confirming the excellent in vivo radiotherapy sensitization and anti-tumor ability of the prepared GaPdRuIrMn.

[0085] 2) Protective effect of intestinal radiotherapy

[0086] In the in vitro experiment, GaPdRuIrMn was co-incubated with intestinal epithelial cells NCM460 (pH=8.0) and radiotherapy for 24 hours. Figure 13 As shown, the GaPdRuIrMn+ radiotherapy group can reduce ROS production and alleviate oxidative stress compared with the radiotherapy group ( Figure 13 a and e), Calcein-AM / PI fluorescence staining showed that the ratio of dead / living cells was significantly reduced ( Figure 13 b and f), γ-H2AX was significantly reduced ( Figure 13 c and g), flow cytometry detection of apoptosis showed that the average apoptosis rates of cells in the control group, GaPdRuIrMn group, radiotherapy group and GaPdRuIrMn+radiotherapy group were 5.2%, 5.9%, 23.46% and 16.76%, respectively. The apoptosis rate in the GaPdRuIrMn+radiotherapy group was significantly lower than that in the radiotherapy group ( Figure 13 d and h). These results indicate that GaPdRuIrMn reduces radiation-induced oxidative stress, apoptosis, and DNA damage in intestinal epithelial cells.

[0087] In the in vivo experiment, a radiation enteritis model was established using BALB / C mice. Figure 14As shown in Figures ad, the radiotherapy group experienced diarrhea, damage to intestinal villus structure, decreased goblet cell number, suppressed cell proliferation (decreased Ki67), and increased DNA damage markers such as γ-H2AX after radiation. GaPdRuIrMn significantly reduced radiation-induced intestinal damage, alleviated diarrhea, improved cell proliferation, and maintained goblet cell number.

[0088] The experimental materials and methods used in this test case are as follows:

[0089] 1. Cell lines: CT26 cells and NCM460 cells were cultured in RPMI-1640 (Gibco) containing 0.5 mg / mL -1 MC38 cells were cultured in DMEM (Gibco) containing penicillin-streptomycin (Gibco) and 10% certified fetal bovine serum (ZATA, Shanghai, China, Cat Z7185FBS-500). MC38 cells were cultured in DMEM (Gibco) containing 0.5 mg mL -1 The culture medium contained penicillin-streptomycin (Gibco) and 10% certified fetal bovine serum (ZATA, Shanghai, China, Cat Z7185FBS-500).

[0090] 2. In vitro anti-tumor and sensitization experiments

[0091] 2.1 Detection of ROS production in cells after irradiation: NCM460 cells were collected during the growth phase, counted using a cell counting plate, and seeded into 6-well plates (1×10 6 Cells were cultured overnight in a PBS-containing solution (10 μg / mL) containing GaPdRuIrMn (10 μg / mL). The GaPdRuIrMn group and the GaPdRuIrMn+radiotherapy group each contained an equal amount of GaPdRuIrMn, while the control group and the radiotherapy group each contained an equal amount of blank PBS. The original cell culture medium was removed from each cell group and incubated with drug-containing medium for 4 hours. The drug-containing medium was then aspirated and replaced with fresh medium. The cells were then irradiated with 20 Gy of X-rays. The cells in the normal control group were sham-irradiated using a lead block as shielding. Following irradiation, the original culture medium was aspirated and serum-free medium containing DCFH-DA (1 mL / well) prepared according to the ROS detection kit instructions was added. The cells were incubated in a dark incubator for 20 minutes. After washing the cells three times with serum-free medium, the ROS green fluorescence of the cells in each group was observed using a fluorescence microscope.

[0092] 2.2 Live / dead staining Calcein (AM) / Propidium iodide (PI) staining: NCM460 cells were collected in the growth phase and seeded in 6-well plates (1×10 6Cells were cultured overnight in 4% paraformaldehyde (PNA) solution (per well). Cell dosing and irradiation procedures were the same as above. Live cells were stained with 1 µl of AM solution in detection buffer, and dead cells were stained with 1 µl of PI solution. The captured cells in each cell group were then observed and counted using a fluorescence microscope.

[0093] 2.3 DNA Damage: NCM460 cells were harvested from the growing phase and seeded into 96-well plates (5,000 cells / well) for overnight culture. Cell dosing and irradiation procedures were the same as above. After fixation with 4% paraformaldehyde, cells were washed three times with washing buffer (5 minutes each). Immunostaining blocking buffer was added for 20 minutes at room temperature. After blocking, y-H2AX rabbit monoclonal antibody was added, and cells were incubated overnight at 4°C. Cells were then washed three times with washing buffer (5 minutes each). Anti-rabbit 488 was added, and cells were incubated in the dark for 1 hour at room temperature. Cell nuclei were counterstained with DAPI at room temperature. Cells were washed with washing buffer and examined under a fluorescence microscope.

[0094] 3. In vitro intestinal radiotherapy protection experiment

[0095] 3.1 Detection of ROS production in cells after irradiation: CT26 cells were collected during the growth period, counted using a cell counting plate, and seeded into 6-well plates (1×10 6 Cells were cultured overnight in PBS (10 μg / well). Cell culture medium containing GaPdRuIrMn (5 μg / mL) was prepared. The GaPdRuIrMn group and the GaPdRuIrMn + radiotherapy group each contained an equal amount of GaPdRuIrMn, while the control group and the radiotherapy group each contained an equal amount of blank PBS. The original cell culture medium was removed from the cells in each group and incubated with drug-containing medium for 4 hours. The drug-containing medium was then aspirated and replaced with fresh medium. The cells were then irradiated with 6 Gy X-rays. The normal control group was sham-irradiated using a lead block as a shield. The detection method was the same as above.

[0096] 3.2 Live / dead staining Calcein (AM) / Propidium iodide (PI) staining: CT26 cells were collected in the growth phase and seeded in 6-well plates (1×10 6 Cells were cultured overnight in 4% paraformaldehyde (PNA) / well (μg / ml). Cell dosing and irradiation procedures were the same as above. Detection methods were the same as above.

[0097] 3.3 DNA Damage: CT26 cells were harvested from the growing phase and seeded into 96-well plates (5000 cells / well) for overnight culture. The cell administration and irradiation procedures were the same as above, and the detection method was the same as above.

[0098] 2. Animal Model: Female BALB / c mice (6–8 weeks old) were purchased from GemPharmatech Co., Nanjing, China. All animal experimental procedures were performed in accordance with the Regulations for the Administration of Laboratory Animals. All mice were housed under specific pathogen-free (SPF) conditions (temperature ~22°C, humidity ~50%) with a 12 / 12 h dark / light cycle. Each mouse was intraperitoneally injected with MC38 cancer cells (5 × 10 5 MC38 tumor models were established using GaPdRuIrMn cells. The models were randomly divided into control, GaPdRuIrMn, radiotherapy (12 Gy), and GaPdRuIrMn + radiotherapy (12 Gy) groups. The injection concentration of GaPdRuIrMn and GaPdRuIrMn + radiotherapy (12 Gy) groups was 100 μg mL -1 (100 uL) / mouse. Tumor growth was observed by in vivo imaging every 3 days. On the 7th day, mice were euthanized, and intestinal tissue was collected for examination. The collected intestinal tissue was fixed in 4% paraformaldehyde overnight, paraffin sections were prepared, and staining, section scanning, and image analysis were performed. The sections were stained with PDS to detect the number of goblet cells; Ki67 (proliferation-associated antigen) immunohistochemical staining was performed to show normal intestinal crypt cells with proliferative capacity, and the number of complete crypts in each intestinal cross-section was counted and counted; the sections were immunohistochemically stained with γ-H2AX (DNA double-strand break marker) to show the degree of DNA damage in intestinal cells.

[0099] It should be noted that the preferred embodiments of the present invention are given in the specification and drawings of the present invention. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. These embodiments are not intended to be additional limitations on the content of the present invention. The purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive. In addition, the above-mentioned technical features can be combined with each other to form various embodiments not listed above, which are all considered to be within the scope of the description of the present invention. Furthermore, it is obvious to those skilled in the art that improvements or changes can be made based on the above description, and all such improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A multi-enzyme active high entropy alloy material, characterized in that: The high entropy alloy material comprises the following components in equimolar amounts: Ga, Pd, Ru, Ir and Mn; The crystal phase structure of the high entropy alloy material is a single-phase solid solution structure.

2. A method for preparing a multi-enzyme active high entropy alloy material according to claim 1, characterized in that: The following steps are involved: Ga salt, Pd salt, Ru salt, Ir salt and Mn salt are subjected to solvothermal reaction in a solvent to obtain a multi-enzyme active high entropy alloy material.

3. The preparation method according to claim 2, characterized in that The salt includes at least one of acetate, acetylacetonate, chloride and nitrate.

4. The preparation method according to claim 2, characterized in that The solvent is a mixture of one or more of methanol, ethanol and acetone, or a mixture of one or more of methanol, ethanol and acetone and water.

5. The preparation method according to claim 2, characterized in that The solvothermal reaction comprises reacting at 150-240° C. for 1-3 hours.

6. Use of the multi-enzyme active high entropy alloy material as claimed in claim 1 for preparing a multi-enzyme active catalyst with pH adaptability.

7. The use according to claim 6, characterized in that The multi-enzyme activity includes POD enzyme-mimicking, SOD enzyme-mimicking, CAT enzyme-mimicking and / or XOR enzyme-mimicking activities.

8. Use of the multi-enzyme active high entropy alloy material according to claim 1 in the preparation of drugs for radiosensitization.

9. Use of the multi-enzyme active high entropy alloy material according to claim 1 in the preparation of a drug for protecting against radiation damage.

10. Use of the multi-enzyme active high entropy alloy material according to claim 1 in the preparation of a drug for radiosensitization and simultaneous protection against radiation damage.

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