High-entropy sub-nanose and preparation method and application thereof

Abstract

The application discloses high-entropy sub-nanometer enzymes, a preparation method and application thereof, and is characterized in that acetylacetone ruthenium, acetylacetone rhodium, acetylacetone platinum, acetylacetone iridium and acetylacetone molybdenum are used as raw materials, high-entropy sub-nanometer enzymes are generated through high-temperature and high-pressure reaction in high-boiling-point organic solvents, and then the high-entropy sub-nanometer enzymes with tumor tissue targeting function are obtained by using post-modification fluorescently labeled cyclic peptides. The high-entropy sub-nanometer enzymes have excellent peroxidase-like activity, active tumor tissue targeting and intracellular imaging functions, and can be used for tumor treatment.

Description

Technical Field

[0001] This invention belongs to the field of functional metal materials technology, specifically relating to a high-entropy subnanozyme, its preparation method, and its application. Background Technology

[0002] Nanozymes are a class of artificial enzymes that combine the unique physicochemical properties of nanomaterials with the catalytic functions of biological enzymes. Nanozymes possess several key physicochemical properties, including designability, large-scale production capability, low cost, ease of modification, stability to heat, acids, and bases, and the ability to modulate enzyme-like catalytic activity through external stimuli. Therefore, the research and development of novel nanozymes is of significant value.

[0003] Subnanomaterials are nanomaterials with a diameter of less than 1 nanometer. They differ from nanomaterials in that they are closer to the atomic and molecular level. They possess unique physicochemical properties in terms of size, morphology, surface composition, and structure.

[0004] High-entropy alloys are alloys formed from five or more metals in equal or approximately equal amounts. Recent studies indicate that the concentration of each element ranges from 5% to 35% atomically. In recent years, high-entropy alloys have attracted considerable attention in the academic community. To date, however, there have been no reports on the development of high-entropy alloy materials with sub-nanometer dimensions and the study of their enzyme-like catalytic activity. Summary of the Invention

[0005] In order to expand the application scope of nanozymes and enhance their value, the present invention provides the following technical solutions.

[0006] In a first aspect, the present invention provides a method for preparing a high-entropy subnanozyme, the method comprising the following steps:

[0007] S1: Weigh out glucose and surfactant, place them in a reaction vessel, add organic solvent, mix and dissolve to obtain the first solution;

[0008] S2: Weigh out ruthenium acetylacetone, rhodium acetylacetone, platinum acetylacetone, and iridium acetylacetone, add them to the first solution, mix and dissolve to obtain the second solution;

[0009] S3: Weigh molybdenum acetylacetonate, add it to the second solution, mix and dissolve to obtain the third solution, and then react at 190-220℃ for 60-240 minutes to obtain the fourth solution;

[0010] S4: Centrifuge the fourth solution, wash the precipitate, freeze-dry it, and obtain the high-entropy subnanozyme;

[0011] S5: Weigh polyvinylpyrrolidone and the high-entropy subnanozyme, add them to cyclohexane, mix and dissolve them, then add fluorescently labeled cyclic peptides, mix well and obtain the fifth solution.

[0012] S6: Centrifuge the fifth solution, wash the precipitate, freeze-dry it, and obtain a high-entropy subnanozyme with tumor tissue targeting function.

[0013] Preferably, the mass ratio of glucose to surfactant in S1 is 1-5:3-10, for example: 1:3, 1:5, 1:8, 1:10, 2:3, 2:5, 2:8, 2:10, 3:3, 3:5, 3:8, 3:10, 5:3, 5:5, 5:8, 5:10.

[0014] Preferably, the surfactant in S1 is selected from any one or a combination of two or more of hexadecyltrimethylammonium chloride, hexadecyltrimethylammonium bromide, sodium dodecylbenzenesulfonate, sodium octadecyl sulfate, or sodium dioctyl succinate sulfonate, and more preferably hexadecyltrimethylammonium chloride.

[0015] Preferably, the organic solvent in S1 is selected from any one or a combination of two or more of ethylene glycol, isopropanol, n-butanol, oleylamine or octadecylamine, and more preferably ethylene glycol or oleylamine.

[0016] Preferably, the mass ratio of ruthenium acetylacetone, rhodium acetylacetone, platinum acetylacetone, iridium acetylacetone, and the second solution in S2 is 0.01–0.001:0.01–0.001:0.01–0.001:1, for example: 0.01:0.01:0.01:0.01:1, 0.008:0.008:0.008:0.008:1, 0.005:0.005:0.005:0.005:1, 0.003:0.003:0.003:0.003:1, 0.001:0.001:0.001:0.001:1.

[0017] Preferably, the mass ratio of molybdenum acetylacetonate in S3 to the second solution is 0.01 to 0.001:1, for example: 0.01:1, 0.008:1, 0.006:1, 0.005:1, 0.003:1, 0.002:1, 0.001:1.

[0018] Preferably, 3-5g of the polyvinylpyrrolidone is added to every 100mL of cyclohexane in S5, for example: 3g, 3.5g, 4g, 4.5g, or 5g.

[0019] Preferably, 1 to 5 mg of the high-entropy subnanozyme is added to every 100 mL of cyclohexane in S5, for example: 1 mg, 2 mg, 3 mg, 4 mg, or 5 mg.

[0020] Preferably, 1-5 mg of the cyclic peptide is added to every 100 mL of cyclohexane in S5, for example: 1 mg, 2 mg, 3 mg, 4 mg, or 5 mg.

[0021] Preferably, the cyclic peptide in S5 is selected from any one or a combination of two or more of cRGD-PEG2000-Cy5, cRGD-PEG2000-Cy7, or cRGD-PEG2000-IR825.

[0022] Preferably, the mixing method in S1-S5 is ultrasonic treatment, with an ultrasonic power of 200-300W and a time of 20-60min.

[0023] In a second aspect, the present invention provides a high-entropy subnanozyme, which is prepared according to the preparation method described in the first aspect.

[0024] Preferably, the diameter of the high-entropy subnanozyme is 0.5 to 1.0 nm, for example: 0.5 nm, 0.6 nm, 0.7 nm, 0.8 nm, 0.9 nm, 1.0 nm.

[0025] Preferably, the length of the high-entropy subnanozyme is 30-100 nm, for example: 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm.

[0026] Thirdly, the present invention provides the application of the high-entropy subnanozyme described in the second aspect in the preparation of antitumor drugs.

[0027] The beneficial effects of this invention are:

[0028] (1) The high-entropy subnanozyme prepared in this invention has a size of only 0.5-1 nm, which is significantly smaller than existing nanoscale materials. Therefore, it has excellent catalase-like activity and superior tumor-targeting therapy function.

[0029] (2) The present invention uses ruthenium acetylacetonate, rhodium acetylacetonate, platinum acetylacetonate, iridium acetylacetonate and molybdenum acetylacetonate to obtain small-sized high-entropy subnanozymes with specific structures.

[0030] (3) The present invention optimizes specific processing conditions, especially oil bath temperature and oil bath time, thereby achieving the effect of further optimizing the microstructure of high-entropy sub-nanozymes, which is conducive to further reducing their size and improving their catalase activity and tumor-targeted therapy function.

[0031] (4) This invention expands the application scope of nanozymes, increases the value of nanozymes, and provides a new method for the development of sub-nanozymes. Attached Figure Description

[0032] Figure 1 The image shown is an electron microscope image of the high-entropy subnanozyme of Example 1. (A) is a TEM image, and (B) is a HAADF-STEM image.

[0033] Figure 2 The image shown is a TEM image of the high-entropy subnanozyme from Example 1.

[0034] Figure 3 The image shown is a TEM image of the high-entropy nanomaterial in Comparative Example 1.

[0035] Figure 4 The image shown is a TEM image of the high-entropy nanomaterial in Comparative Example 2.

[0036] Figure 5 The image shown is a TEM image of the high-entropy nanomaterial in Comparative Example 3.

[0037] Figure 6 The image shown is a TEM image of the high-entropy nanomaterial in Comparative Example 4.

[0038] Figure 7 The image shown is a TEM image of the high-entropy nanomaterial in Comparative Example 5.

[0039] Figure 8 The image shown is a TEM image of the high-entropy nanomaterial in Comparative Example 6.

[0040] Figure 9 The image shown is a TEM image of the high-entropy nanomaterial in Comparative Example 7.

[0041] Figure 10 The image shown is a TEM image of the high-entropy nanomaterial in Comparative Example 8.

[0042] Figure 11 The image shown is a TEM image of the high-entropy nanomaterial compared to Example 9.

[0043] Figure 12 The results of peroxidase activity detection for high-entropy subnanozymes are shown.

[0044] Figure 13 The image shows the effect of high-entropy subnanozymes in scavenging reactive oxygen species within cells.

[0045] Figure 14 The image shows the targeting and imaging effects of high-entropy subnanozymes within cells.

[0046] Figure 15 The figure shows the weight changes in mice treated with high-entropy subnanozymes.

[0047] Figure 16 The image shown is an in vivo imaging diagram of high-entropy subnanozyme therapy for mouse tumors.

[0048] Figure 17The image shows the therapeutic effect of high-entropy subnanozymes on mouse tumors. (A) shows the size of the mouse tumor, and (B) shows the change in tumor volume at different locations in the mouse over time.

[0049] Figure 18 The results of fluorescence staining of mouse tumor tissue are shown in the figures: (i) H&E staining, (ii) DAPI staining, (iii) TUNEL staining, (iv) DAPI and TUNEL staining combined, (v) Ki67 staining, (A) control group, (B) laser group, (C) nanozyme group, and (D) nanozyme + laser group. Detailed Implementation

[0050] The technical solution of the present invention will be further described below with reference to embodiments and accompanying drawings. The advantages and features of the present invention will become clearer as the description unfolds. However, it should be understood that the embodiments are merely exemplary and do not constitute a limitation on the scope of the present invention.

[0051] Example 1: Preparation of high-entropy subnanozyme sample 1

[0052] (1) Weigh 10 mg of glucose and 30 mg of hexadecyltrimethylammonium chloride into a high-pressure reaction flask, and then transfer 4 mL of ethylene glycol into the flask. Sonicate the mixture at 250 W for 30 min until the compounds in the reaction solution are fully dissolved to obtain the first solution.

[0053] (2) Ruthenium acetylacetone, rhodium acetylacetone, platinum acetylacetone, and iridium acetylacetone were mixed in a certain proportion and transferred to the first solution. The mass ratio of the mixed solution was: first solution:ruthenium acetylacetone:rhodium acetylacetone:platinum acetylacetone:iridium acetylacetone = 1:0.01:0.01:0.01:0.01. The mixed solution was ultrasonically treated with a power of 250W for 30 minutes until the compounds in the reaction solution were fully dissolved, thus obtaining the second solution.

[0054] (3) Transfer molybdenum acetylacetonate to the second solution in a certain proportion, the mass ratio of the mixed solution being second solution:molybdenum acetylacetonate = 1:0.01. Sonicate the mixed solution with a power of 250W for 60 minutes until the compounds in the reaction solution are fully dissolved to obtain the third solution.

[0055] (4) The third solution was heated to 190°C in an oil bath for 60 min with a stirring speed of 600 rpm. The reaction was then stopped to obtain a black reaction solution, i.e., the fourth solution. The fourth solution was centrifuged at 10,000 rpm for 5 min. The precipitate obtained by centrifugation was washed with a mixture of cyclohexane and anhydrous ethanol in equal volume ratio. The washing solution was centrifuged again. This step was repeated 2 to 3 times. The final centrifuged precipitate was freeze-dried to obtain the high-entropy subnanozyme.

[0056] (5) Add a certain amount of polyvinylpyrrolidone to 20 mL of cyclohexane solution to obtain a 3% (w / w) solution. Then add 1 mg of high-entropy subnanozyme and sonicate the mixture for 60 min using 250 W of ultrasound. After the compound in the reaction solution is fully dissolved, add 1 mg of fluorescently labeled cyclic peptide cRGD-PEG2000-Cy5. React at room temperature for 120 min with a stirring speed of 600 rpm, and then stop the reaction, which is the fifth solution. Centrifuge the fifth solution and wash the precipitate obtained by centrifugation with anhydrous ethanol. Centrifuge the washing solution and repeat the centrifugation and washing process 2-3 times. Freeze-dry the final precipitate to obtain high-entropy subnanozyme sample 1 with tumor tissue targeting function. Then, observe this sample by scanning electron microscopy.

[0057] like Figure 1 and Figure 2 As shown, the high-entropy subnanozyme sample 1 is approximately 30 nm in length and 1.0 nm in diameter.

[0058] Example 2: Preparation of high-entropy subnanozyme sample 2

[0059] The preparation process is the same as in Example 1, with the following differences:

[0060] In step (1), there are 50 mg of glucose, 100 mg of hexadecyltrimethylammonium chloride, and 8 mL of ethylene glycol.

[0061] In step (2), the first solution is composed of: Ruthenium acetylacetone: Rhodium acetylacetone: Platinum acetylacetone: Iridium acetylacetone = 1:0.001:0.001:0.001:0.001.

[0062] The second solution in step (3): molybdenum acetylacetonate = 1:0.001.

[0063] In step (4), the oil bath heating temperature is 220℃ and the time is 240min.

[0064] In step (5), the concentration of the polyvinylpyrrolidone-cyclohexane solution is 5%, the amount of high-entropy subnanozyme added is 5 mg, the cyclic peptide is cRGD-PEG2000-Cy7, and the reaction is carried out for 240 min after the cyclic peptide is added.

[0065] Example 3: Preparation of high-entropy subnanozyme sample 3

[0066] The preparation process is the same as in Example 1, with the following differences:

[0067] In step (1), there are 50 mg of glucose, 100 mg of hexadecyltrimethylammonium chloride, and 8 mL of ethylene glycol.

[0068] In step (2), the first solution is composed of: Ruthenium acetylacetone, Rhodium acetylacetone, Platinum acetylacetone, and Iridium acetylacetone = 1:0.005:0.005:0.005:0.005.

[0069] The second solution in step (3): molybdenum acetylacetonate = 1:0.005.

[0070] In step (4), the oil bath heating temperature is 220℃ and the time is 120min.

[0071] In step (5), the concentration of the polyvinylpyrrolidone-cyclohexane solution is 5%, the amount of high-entropy subnanozyme added is 5 mg, the cyclic peptide is cRGD-PEG2000-IR825, and the reaction is carried out for 240 min after the cyclic peptide is added.

[0072] Comparative Example 1:

[0073] The preparation process is the same as in Example 1, with the following differences:

[0074] In step (1), ethylene glycol is replaced with oleylamine.

[0075] In step (3), molybdenum acetylacetonate is replaced with iron acetylacetonate.

[0076] like Figure 3 As shown, the high-entropy nanomaterials prepared in step (4) were not formed, and no high-entropy subnanozymes were obtained.

[0077] Comparative Example 2:

[0078] The preparation process is the same as in Example 1, with the following differences:

[0079] In step (3), molybdenum acetylacetone is replaced with copper acetylacetone.

[0080] like Figure 4 As shown, the high-entropy nanomaterials prepared in step (4) were not formed, and no high-entropy subnanozymes were obtained.

[0081] Comparative Example 3:

[0082] The preparation process is the same as in Example 1, with the following differences:

[0083] In step (3), molybdenum acetylacetonate is replaced with cobalt acetylacetonate.

[0084] like Figure 5 As shown, the high-entropy nanomaterials prepared in step (4) are sheet-like with a diameter of 5 nm, and no high-entropy subnanozymes were obtained.

[0085] Comparative Example 4:

[0086] The preparation process is the same as in Example 1, with the following differences:

[0087] In step (3), molybdenum acetylacetonate is replaced with nickel acetylacetonate.

[0088] like Figure 6 As shown, the high-entropy nanomaterials prepared in step (4) are sheet-like with a diameter of 5-10 nm, and no high-entropy subnanozymes were obtained.

[0089] Comparative Example 5:

[0090] The preparation process is the same as in Example 1, with the following differences:

[0091] In step (3), molybdenum acetylacetone is replaced with manganese acetylacetone.

[0092] like Figure 7 As shown, the high-entropy nanomaterials prepared in step (4) are sheet-like with a diameter of 5-10 nm, and no high-entropy subnanozymes were obtained.

[0093] Comparative Example 6:

[0094] The preparation process is the same as in Example 1, with the following differences:

[0095] In step (3), molybdenum acetylacetone is replaced with palladium acetylacetone.

[0096] like Figure 8 As shown, the high-entropy nanomaterials prepared in step (4) were not formed, and no high-entropy subnanozymes were obtained.

[0097] Comparative Example 7:

[0098] The preparation process is the same as in Example 1, with the following differences:

[0099] In step (2), ruthenium acetylacetone is replaced with palladium acetylacetone.

[0100] like Figure 9 As shown, the high-entropy nanomaterials prepared in step (4) were not formed, and no high-entropy subnanozyme was obtained.

[0101] Comparative Example 8:

[0102] The preparation process is the same as in Example 1, with the following differences:

[0103] In step (4), the oil bath reaction temperature is 170℃.

[0104] like Figure 10 As shown, the high-entropy nanomaterials prepared in step (4) are rod-shaped with a diameter of 3-5 nm, and no high-entropy subnanozymes were obtained.

[0105] Comparative Example 9:

[0106] The preparation process is the same as in Example 1, with the following differences:

[0107] In step (4), the oil bath reaction temperature is 240℃.

[0108] like Figure 11 As shown, the high-entropy nanomaterials prepared in step (4) are rod-shaped with a diameter of 3-5 nm, and no high-entropy subnanozymes were obtained.

[0109] Comparative Example 10:

[0110] The preparation process is the same as in Example 1, with the following differences:

[0111] In step (4), the oil bath reaction time was 30 min, and the high-entropy nanomaterials were not formed, and no high-entropy subnanozyme was obtained.

[0112] Comparative Example 11:

[0113] The preparation process is the same as in Example 1, with the following differences:

[0114] In step (4), the oil bath reaction time was 250 min, and the high-entropy nanomaterials were not formed, and no high-entropy subnanozyme was obtained.

[0115] Table 1 is a statistical analysis of the electron microscopy observations of the products obtained in Example 1 and Comparative Examples 1-9. It can be seen that, using ruthenium acetylacetonate, rhodium acetylacetonate, platinum acetylacetonate, iridium acetylacetonate combined with molybdenum acetylacetonate as specific raw materials, high-entropy nanomaterials with sub-nanometer diameters can be prepared under specific processing conditions. These materials exhibit uniform and regular morphology and are expected to possess good functional activity.

[0116] Table 1

[0117]

[0118] Performance test examples

[0119] Performance Testing Experiment 1: Detection of Ultraviolet Absorption of High-Entropy Subnanozymes

[0120] (1) The high-entropy subnanozyme prepared in Example 1 was dispersed in ultrapure water to make the mass volume concentration of the high-entropy subnanozyme 0.01 mg / mL. The solution was then subjected to ultrasonic treatment with an ultrasonic power of 250 W for 10 min to obtain the sixth solution.

[0121] (2) Add 20 μL of 5 mg / mL TMB solution to a pH 4.5 HAc-NaAc buffer solution, then add 5 μL of the sixth solution. Measure the change in UV absorption of the reaction solution using a UV spectrophotometer. The results are shown in [Figure 1]. Figure 12 .

[0122] (3) Following the methods in steps (1) and (2), the ultraviolet absorption changes of the high-entropy nanomaterial reaction solutions of Comparative Examples 1 to 11 were measured at 652 nm. The detection results are shown in Table 2.

[0123] Table 2. UV detection results of high-entropy nanomaterials

[0124]

[0125]

[0126] like Figure 12 As shown, the high-entropy subnanozyme in Example 1 can convert H2O2 into · OH exhibits excellent peroxidase-like activity and can be detected at a wavelength of 652 nm.

[0127] As shown in Table 2, the high-entropy subnanozyme of Example 1 exhibits the best peroxidase-like activity, which is significantly higher than that of the high-entropy nanomaterials in Comparative Examples 1-11. This indicates that high-entropy subnanozyme products with excellent peroxidase-like activity can be prepared using ruthenium acetylacetonate, rhodium acetylacetonate, platinum acetylacetonate, iridium acetylacetonate, and molybdenum acetylacetonate as raw materials. However, when the four noble metal acetylacetonate salts or one acetylacetonate salt are replaced, or the preparation conditions are changed, it is difficult to prepare subnanomaterials, i.e., it is impossible to obtain high-entropy subnanozymes with similar morphology and performance.

[0128] Performance Test Experiment 2: Detection of High-Entropy Subnanozyme Cell Imaging

[0129] (1) The high-entropy subnanozyme with tumor tissue targeting function of Example 1 was sterilized and dispersed in RPM1-1640 medium to make the mass volume concentration of high-entropy subnanozyme 0.1 mg / mL. It was then ultrasonicated with an ultrasonic power of 250 W for 30 min to obtain the seventh solution.

[0130] (2) Culture, passage, and spread 4T1 cells to cover the entire 12-well plate. After removing the culture medium, wash the cells, add 5 mL of the seventh solution, and incubate for 2 hours. Observe the cells using a laser confocal microscope to obtain cell imaging results.

[0131] like Figure 13 As shown, enhanced green fluorescence can be observed. The fluorescence intensity of 4T1 cells indicates the presence of a large number of tumor cells. ·OH. This indicates that high-entropy subnanozymes can catalyze the conversion of H2O2 in the cancer cell culture environment into... · OH.

[0132] like Figure 14 As shown, 4T1 cells emitted red fluorescence under 750nm excitation light, indicating that Cy7 was successfully grafted onto the surface of the nanozyme and could penetrate the cell membrane to enter tumor cells, which also shows that the nanozyme system has the potential for in vivo imaging.

[0133] Performance Testing Experiment 3: Active Targeting Performance Detection of High-Entropy Subnanozymes in Tumor Tissue

[0134] A mouse model of breast cancer was established by injecting a suspension of 4T1 cells into mice. The tumor volume in the mice was approximately 60 mm. 3 Subsequently, all tumor-bearing mice were randomly divided into four groups of five mice each, and administered the drug via tail vein injection. The groups were: a control group (50 μL PBS injected intratumorally), a laser group (laser therapy), a nanozyme group (50 μL of high-entropy sub-nanozyme solution with tumor-targeting function from Example 1 injected only), and a nanozyme+laser group (50 μL of high-entropy sub-nanozyme solution with tumor-targeting function from Example 1 combined with laser therapy). The laser therapy group was exposed to 808 nm near-infrared laser (1.0 W / cm², 10 min). The mice in all four groups were observed and imaged using a Fluke Ti400 infrared thermal imager, and quantification was performed using Ti400examiner software. Tumor size was measured every other day using calipers. Tumor volume was calculated using the following formula.

[0135] V = L × W 2 ×0.5 (L: tumor length, W: tumor width).

[0136] Tumor tissues were stained and analyzed using fluorescent staining methods, including hematoxylin and eosin (H&E) staining, Tunel staining, and Ki67 staining.

[0137] like Figure 15 As shown, the body weight of the four groups of mice did not change significantly during the treatment period, indicating that the treatment process did not significantly affect the daily life of the mice, the mice were able to metabolize nanozymes normally, and there were no obvious side effects.

[0138] like Figure 16 As shown, red fluorescence can be observed at the tumor site in mice using a small animal in vivo imaging system, indicating that nanozymes are enriched in tumor cells.

[0139] like Figure 17As shown, different changes occurred in the tumor sites of mouse models treated under different conditions. For both the control and laser groups, the tumor volume in all mice increased to nearly 700 mm over time. 3 For the nanozyme group, the tumor volume in mice was inhibited to a certain extent compared with the control group, with the tumor volume being approximately 380 mm². 3 The inhibition rate was approximately 46%. Under the combined treatment of laser and nanozyme, the tumors in mice completely disappeared, achieving a tumor inhibition rate of 100%.

[0140] like Figure 18 As shown, H&E staining results revealed nucleus fragmentation and extensive cell death in the tumor tissue. Tunel staining showed abundant red fluorescence, indicating that ROS generated by the nanozyme induced apoptosis in the tumor tissue. Ki67 staining results showed that the nanozyme system effectively inhibited the proliferative activity of tumor cells.

[0141] The above results indicate that the high-entropy subnanozyme products prepared from ruthenium acetylacetonate, rhodium acetylacetonate, platinum acetylacetonate, iridium acetylacetonate combined with molybdenum acetylacetonate are highly safe, can accurately target tumor tissues, and inhibit tumor growth. They can be used to prepare anti-tumor drugs and have good prospects for medical applications.

[0142] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A method for preparing a high-entropy subnanozyme, characterized in that, The preparation method includes the following steps: S1: Weigh out glucose and surfactant, place them in a reaction vessel, add organic solvent, mix and dissolve to obtain the first solution; S2: Weigh out ruthenium acetylacetone, rhodium acetylacetone, platinum acetylacetone, and iridium acetylacetone, add them to the first solution, mix and dissolve to obtain the second solution; S3: Weigh molybdenum acetylacetonate, add it to the second solution, mix and dissolve to obtain the third solution, then react at 190~220℃ for 60~240 minutes to obtain the fourth solution; S4: Centrifuge the fourth solution, wash the precipitate, freeze-dry it, and obtain the high-entropy subnanozyme; S5: Weigh polyvinylpyrrolidone and the high-entropy subnanozyme, add them to cyclohexane, mix and dissolve them, then add fluorescently labeled cyclic peptides, mix well and obtain the fifth solution. S6: Centrifuge the fifth solution, wash the precipitate, freeze-dry it, and obtain a high-entropy subnanozyme with tumor tissue targeting function; The cyclic peptide is selected from any one or a combination of two or more of cRGD-PEG2000-Cy5, cRGD-PEG2000-Cy7, or cRGD-PEG2000-IR825.

2. The preparation method according to claim 1, characterized in that, The mass ratio of glucose to surfactant in S1 is 1~5:3~10.

3. The preparation method according to claim 1, characterized in that, The surfactant in S1 is selected from any one or a combination of two or more of hexadecyltrimethylammonium chloride, hexadecyltrimethylammonium bromide, sodium dodecylbenzenesulfonate, sodium octadecyl sulfate, or sodium dioctyl succinate sulfonate.

4. The preparation method according to claim 1, characterized in that, The organic solvent mentioned in S1 is selected from any one or a combination of two or more of ethylene glycol, isopropanol, n-butanol, oleylamine, or octadecylamine.

5. The preparation method according to claim 1, characterized in that, The mass ratio of ruthenium acetylacetone, rhodium acetylacetone, platinum acetylacetone, iridium acetylacetone, and the second solution in S2 is 0.01~0.001: 0.01~0.001: 0.01~0.001: 0.01~0.001:

1.

6. The preparation method according to claim 1, characterized in that, The mass ratio of the non-precious metal acetylacetone salt in S3 to the second solution is 0.01~0.001:

1.

7. The preparation method according to claim 1, characterized in that, In S5, 3-5 g of the polyvinylpyrrolidone, 1-5 mg of the high-entropy subnanozyme, and 1-5 mg of the cyclic peptide are added to every 100 mL of cyclohexane.

8. A high-entropy subnanozyme, characterized in that, The high-entropy subnanozyme was prepared according to any one of the preparation methods described in claims 1-7.

9. The high-entropy nanozyme according to claim 8, characterized in that, The high-entropy subnanozyme has a diameter of 0.5~1.0 nm.

10. The use of the high-entropy subnanozyme according to claim 8 or 9 in the preparation of anti-breast cancer drugs.

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