High-entropy sub-nano enzyme as well as preparation method and application thereof

Abstract

The invention discloses a high-entropy sub-nano enzyme and a preparation method and application thereof.The preparation method comprises the steps that ruthenium acetylacetonate, rhodium acetylacetonate, platinum acetylacetonate, iridium acetylacetonate and molybdenum acetylacetonate serve as raw materials, and the high-entropy sub-nano enzyme is generated in a high-boiling-point organic solvent through a high-temperature and high-pressure reaction; and then the high-entropy sub-nano enzyme with the tumor tissue targeting function is obtained by using the post-modified fluorescence-labeled cyclic peptide. The high-entropy sub-nano enzyme disclosed by the invention has excellent peroxidase-like activity and functions of active tumor tissue targeting, intracellular imaging and the like, and can be used for treating tumors.

Description

Technical Field

[0001] The present invention belongs to the technical field of metal functional materials, and particularly relates to a high-entropy sub-nanozyme and a preparation method and application thereof. Background Art

[0002] Nanozymes are a class of artificial mimetic enzymes that have both the unique physical and chemical properties of nanomaterials and the catalytic functions of bioenzymes. Nanozymes have physical and chemical properties such as being designable, macroscopically preparable, low-cost, easy to modify, stable to heat, acid, and alkali, and capable of regulating enzyme-like catalytic activity through external stimuli. Therefore, the research and development of new nanozymes have important value.

[0003] Sub-nanomaterials are nanomaterials with a diameter less than 1 nm, and their difference from nanomaterials lies in that they are closer to the atomic and molecular levels. Due to their special physical and chemical properties in terms of size, morphology, surface composition, and structure.

[0004] High-entropy alloys refer to alloys formed by five or more equal or approximately equal amounts of metals. The latest research shows that the concentration of each element is between 5 and 35 atomic percentages. In recent years, high-entropy alloys have attracted great attention in the academic community. So far, there has been no report on the development of high-entropy alloy materials with sub-nanoscale 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 improve the value of nanozymes, the present invention provides the following technical solutions.

[0006] In the first aspect, the present invention provides a preparation method of a high-entropy sub-nanozyme, and the preparation method includes the following steps:

[0007] S1: Weigh glucose and a surfactant, place them in a reaction vessel, and then add an organic solvent. After mixing and dissolving, a first solution is obtained;

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

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

[0010] S4: Centrifuge the fourth solution, wash the precipitate, and freeze-dry to obtain a high-entropy sub-nanozyme;

[0011] S5: Weigh polyvinylpyrrolidone and the high-entropy sub-nanozyme, add them to cyclohexane, mix and dissolve, then add a fluorescently labeled cyclic peptide, and mix evenly to obtain a fifth solution;

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

[0013] Preferably, the mass ratio of glucose to the 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 cetyltrimethylammonium chloride, cetyltrimethylammonium bromide, sodium dodecylbenzenesulfonate, sodium octadecyl sulfate, or sodium dioctyl sulfosuccinate, and further preferably cetyltrimethylammonium chloride.

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

[0016] Preferably, the mass ratio of ruthenium acetylacetonate, rhodium acetylacetonate, platinum acetylacetonate, iridium acetylacetonate to the second solution in S2 is 0.01-0.001:0.01-0.001:0.01-0.001:0.01-0.001:1, 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 to the second solution in S3 is 0.01-0.001:1, 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, 3-5 g of polyvinylpyrrolidone is added to every 100 mL of cyclohexane in S5, for example: 3 g, 3.5 g, 4 g, 4.5 g, 5 g.

[0019] Preferably, 1-5 mg of the high-entropy sub-nanozyme is added to every 100 mL of cyclohexane in S5, for example: 1 mg, 2 mg, 3 mg, 4 mg, 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, 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 200-300 W and a time of 20-60 min.

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

[0024] Preferably, the diameter of the high-entropy sub-nanozyme is 0.5-1.0 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 sub-nanozyme is 30-100 nm, for example: 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm.

[0026] In a third aspect, the present invention provides the use of the high-entropy sub-nanozyme described in the second aspect in the preparation of anti-tumor drugs.

[0027] Advantages of the present invention:

[0028] (1) The high-entropy sub-nanozyme prepared by the present invention has a size of only 0.5-1 nm, which is significantly smaller than existing nano-scale materials. Therefore, it has excellent catalase-like activity and a more excellent tumor-targeted therapy function.

[0029] (2) The present invention uses ruthenium acetylacetonate, rhodium acetylacetonate, platinum acetylacetonate, iridium acetylacetonate and molybdenum acetylacetonate in combination to obtain a small-sized high-entropy sub-nanozyme with a specific structure.

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

[0031] (4) The present invention expands the application range of nanozymes, improves the value of nanozymes, and provides a new method for the development of sub-nanozymes. Description of the Drawings

[0032] Figure 1 Shown are the electron microscopy images of the high-entropy sub-nanozyme of Example 1. (A) is the TEM image, and (B) is the HAADF-STEM image;

[0033] Figure 2 Shown is the TEM image of the high-entropy sub-nanozyme of Example 1;

[0034] Figure 3 Shown is the TEM image of the high-entropy nanomaterial of Comparative Example 1;

[0035] Figure 4 Shown is the TEM image of the high-entropy nanomaterial of Comparative Example 2;

[0036] Figure 5 Shown is the TEM image of the high-entropy nanomaterial of Comparative Example 3;

[0037] Figure 6 Shown is the TEM image of the high-entropy nanomaterial of Comparative Example 4;

[0038] Figure 7 Shown is the TEM image of the high-entropy nanomaterial of Comparative Example 5;

[0039] Figure 8 Shown is the TEM image of the high-entropy nanomaterial of Comparative Example 6;

[0040] Figure 9 Shown is the TEM image of the high-entropy nanomaterial of Comparative Example 7;

[0041] Figure 10 Shown is the TEM image of the high-entropy nanomaterial of Comparative Example 8;

[0042] Figure 11 Shown is the TEM image of the high-entropy nanomaterial of Comparative Example 9;

[0043] Figure 12 Shown are the detection results of the peroxidase activity of the high-entropy sub-nanozyme;

[0044] Figure 13 Shown is the effect diagram of the high-entropy sub-nanozyme scavenging reactive oxygen species in cells;

[0045] Figure 14 Shown is the effect diagram of the high-entropy sub-nanozyme targeting and imaging in cells;

[0046] Figure 15 Shown is the weight change of the mice treated with the high-entropy sub-nanozyme;

[0047] Figure 16 Shown is the in vivo imaging diagram of the tumor in the mice treated with the high-entropy sub-nanozyme;

[0048] Figure 17The therapeutic effect of the high-entropy sub-nanozyme on mouse tumors is shown. (A) shows the size of the mouse tumors, and (B) shows the graph of the change in the tumor volume of the mouse at different parts over time;

[0049] Figure 18 The fluorescence staining results of the mouse tumor tissues are shown. (i) is H&E staining, (ii) is DAPI staining, (iii) is Tunel staining, (iv) is the combination of DAPI and Tunel staining, (v) is Ki67 staining, (A) is the control group, (B) is the laser group, (C) is the nanozyme group, and (D) is the nanozyme + laser group. Specific Embodiments

[0050] The technical solution of the present invention will be further described below in conjunction with the embodiments and the accompanying drawings. The advantages and features of the present invention will become clearer as the description progresses. However, it should be understood that the embodiments are merely exemplary and do not limit the scope of the present invention.

[0051] Example 1: Preparation of High-Entropy Sub-Nanozyme Sample 1

[0052] (1) Weigh 10 mg of glucose and 30 mg of cetyltrimethylammonium chloride and place them in a high-pressure reaction flask. Then transfer 4 mL of ethylene glycol to the above reaction flask. Ultrasonically treat the mixed solution with a power of 250 W for 30 min. After the compounds in the reaction solution are fully dissolved, a first solution is obtained.

[0053] (2) Mix ruthenium acetylacetonate, rhodium acetylacetonate, platinum acetylacetonate, and iridium acetylacetonate in a certain proportion and transfer them to the first solution. The mass ratio of this mixed solution is First solution: Ruthenium acetylacetonate: Rhodium acetylacetonate: Platinum acetylacetonate: Iridium acetylacetonate = 1:0.01:0.01:0.01:0.01. Ultrasonically treat the mixed solution with a power of 250 W for 30 min. After the compounds in the reaction solution are fully dissolved, a second solution is obtained.

[0054] (3) Transfer molybdenum acetylacetonate to the second solution in a certain proportion. The mass ratio of this mixed solution is Second solution: Molybdenum acetylacetonate = 1:0.01. Ultrasonically treat the mixed solution with a power of 250 W for 60 min. After the compounds in the reaction solution are fully dissolved, a third solution is obtained.

[0055] (4) Heat the third solution in an oil bath to 190 °C, heat and react for 60 min, with a stirring speed of 600 rpm, then stop the reaction to obtain a black reaction solution, i.e., the fourth solution. Centrifuge the fourth solution at 10000 rpm for 5 min, wash the precipitate obtained by centrifugation with a mixed solution of cyclohexane and absolute ethanol in an equal volume ratio, centrifuge the washing solution, repeat this step 2 - 3 times, and freeze-dry the finally obtained centrifuged precipitate to obtain the high-entropy sub-nanozyme.

[0056] (5) Add a certain amount of polyvinylpyrrolidone to 20 mL of cyclohexane solution to obtain a solution with a mass percentage of 3%, then add 1 mg of high-entropy sub-nanozyme, and ultrasonically treat the mixed solution with a power of 250 W for 60 min. After the compounds in the reaction solution are 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, then stop the reaction to obtain the fifth solution. Centrifuge the fifth solution, wash the precipitate obtained by centrifugation with absolute ethanol, centrifuge the washing solution, repeat centrifugation and washing 2 - 3 times, freeze-dry the finally obtained precipitate to obtain the high-entropy sub-nanozyme sample 1 with tumor tissue targeting function, and then observe this sample by electron microscopy scanning.

[0057] As Figure 1 and Figure 2 shown, the length of the high-entropy sub-nanozyme sample 1 is about 30 nm, and the diameter is about 1.0 nm.

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

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

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

[0061] In step (2), the first solution: ruthenium acetylacetonate: rhodium acetylacetonate: platinum acetylacetonate: iridium acetylacetonate = 1:0.001:0.001:0.001:0.001.

[0062] In step (3), the second solution: molybdenum acetylacetonate = 1:0.001.

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

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

[0065] Example 3: Preparation of High-Entropy Sub-Nanozyme Sample 3

[0066] The preparation process is the same as that of Example 1, with the differences as follows:

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

[0068] In step (2), the first solution: ruthenium acetylacetonate: rhodium acetylacetonate: platinum acetylacetonate: iridium acetylacetonate = 1:0.005:0.005:0.005:0.005.

[0069] In step (3), the second solution: molybdenum acetylacetonate = 1:0.005.

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

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

[0072] Comparative Example 1:

[0073] The preparation process is the same as that of Example 1, with the differences as follows:

[0074] The ethylene glycol in step (1) is replaced with oleylamine.

[0075] The molybdenum acetylacetonate in step (3) is replaced with iron acetylacetonate.

[0076] As Figure 3 shown, the high-entropy nanomaterial prepared in step (4) is not formed, and no high-entropy sub-nanozyme is obtained.

[0077] Comparative Example 2:

[0078] The preparation process is the same as that of Example 1, with the differences as follows:

[0079] The molybdenum acetylacetonate in step (3) is replaced with copper acetylacetonate.

[0080] As Figure 4 shown, the high-entropy nanomaterial prepared in step (4) is not formed, and no high-entropy sub-nanozyme is obtained.

[0081] Comparative Example 3:

[0082] The preparation process is the same as that of Example 1, with the differences as follows:

[0083] The molybdenum acetylacetonate in step (3) is replaced with cobalt acetylacetonate.

[0084] As Figure 5 shown, the high-entropy nanomaterial obtained in step (4) is flaky with a diameter of 5 nm, and no high-entropy sub-nanozyme is obtained.

[0085] Comparative Example 4:

[0086] The preparation process is the same as that of Example 1, and the differences are as follows:

[0087] Molybdenum acetylacetonate in step (3) is replaced with nickel acetylacetonate.

[0088] As Figure 6 shown, the high-entropy nanomaterial obtained in step (4) is flaky with a diameter of 5-10 nm, and no high-entropy sub-nanozyme is obtained.

[0089] Comparative Example 5:

[0090] The preparation process is the same as that of Example 1, and the differences are as follows:

[0091] Molybdenum acetylacetonate in step (3) is replaced with manganese acetylacetonate.

[0092] As Figure 7 shown, the high-entropy nanomaterial obtained in step (4) is flaky with a diameter of 5-10 nm, and no high-entropy sub-nanozyme is obtained.

[0093] Comparative Example 6:

[0094] The preparation process is the same as that of Example 1, and the differences are as follows:

[0095] Molybdenum acetylacetonate in step (3) is replaced with palladium acetylacetonate.

[0096] As Figure 8 shown, the high-entropy nanomaterial obtained in step (4) is not formed, and no high-entropy sub-nanozyme is obtained.

[0097] Comparative Example 7:

[0098] The preparation process is the same as that of Example 1, and the differences are as follows:

[0099] Ruthenium acetylacetonate in step (2) is replaced with palladium acetylacetonate.

[0100] As Figure 9 shown, the high-entropy nanomaterial obtained in step (4) is not formed, and no high-entropy sub-nanozyme is obtained..

[0101] Comparative Example 8:

[0102] The preparation process is the same as that of Example 1, and the differences are as follows:

[0103] The oil bath reaction temperature in step (4) is 170 °C.

[0104] As Figure 10 shown, the high-entropy nanomaterial prepared in step (4) is rod-shaped with a diameter of 3-5 nm, and no high-entropy sub-nanozyme is obtained.

[0105] Comparative Example 9:

[0106] The preparation process is the same as that of Example 1, and the differences are as follows:

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

[0108] As Figure 11 shown, the high-entropy nanomaterial prepared in step (4) is rod-shaped with a diameter of 3-5 nm, and no high-entropy sub-nanozyme is obtained.

[0109] Comparative Example 10:

[0110] The preparation process is the same as that of Example 1, and the differences are as follows:

[0111] In step (4), the oil bath reaction time is 30 min, and the prepared high-entropy nanomaterial is not formed, and no high-entropy sub-nanozyme is obtained.

[0112] Comparative Example 11:

[0113] The preparation process is the same as that of Example 1, and the differences are as follows:

[0114] In step (4), the oil bath reaction time is 250 min, and the prepared high-entropy nanomaterial is not formed, and no high-entropy sub-nanozyme is obtained.

[0115] After statistically analyzing the electron microscope observation results of the products prepared in Example 1 and Comparative Examples 1-9, Table 1 is obtained. 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 a diameter reaching the sub-nanometer level can be prepared under specific processing conditions, and their morphologies are uniform and regular, and are expected to have good functional activities.

[0116] Table 1

[0117]

[0118] Performance Test Example

[0119] Performance Test Experiment 1 Detection of Ultraviolet Absorption of High-Entropy Sub-Nanozyme

[0120] (1) Disperse the high-entropy sub-nanozyme prepared in Example 1 in ultrapure water to make the mass-volume concentration of the high-entropy sub-nanozyme 0.01 mg / mL, and perform ultrasonic treatment for 10 min with an ultrasonic power of 250 W to obtain the sixth solution.

[0121] (2) Add 20 μL of TMB solution with a mass concentration of 5 mg / mL to the HAc-NaAc buffer solution with a pH value of 4.5, then add 5 μL of the sixth solution, and use a UV spectrophotometer to measure the change in UV absorption of the reaction solution. The detection results are shown in Figure 12 .

[0122] (3) According to the methods of steps (1) and (2), measure the change in UV absorption of the reaction solutions of the high-entropy nanomaterials in Comparative Examples 1-11 at 652 nm. The detection results are shown in Table 2.

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

[0124]

[0125]

[0126] As Figure 12 shown, the high-entropy sub-nanozyme of Example 1 can convert H2O2 into · OH, with excellent peroxidase-like activity, and the detection wavelength is 652 nm.

[0127] It can be seen from Table 2 that the high-entropy sub-nanozyme of Example 1 has the best peroxidase-like activity, and is significantly higher than the high-entropy nanomaterials of Comparative Examples 1-11, indicating that a high-entropy sub-nanozyme product with excellent peroxidase-like activity can be prepared using ruthenium acetylacetonate, rhodium acetylacetonate, platinum acetylacetonate, iridium acetylacetonate combined with molybdenum acetylacetonate as raw materials. When 4 kinds of precious metal acetylacetonates or 1 kind of acetylacetonate are replaced, or the preparation conditions are changed, it is very difficult to prepare sub-nanomaterials, that is, it is impossible to obtain high-entropy sub-nanozymes with similar morphology and performance.

[0128] Performance test experiment 2 Detection of high-entropy sub-nanozyme cell imaging

[0129] (1) Sterilize the high-entropy sub-nanozyme with tumor tissue targeting function of Example 1 and disperse it in RPMl-1640 medium to make the mass-volume concentration of the high-entropy sub-nanozyme 0.1 mg / mL, and perform ultrasonic treatment for 30 min with an ultrasonic power of 250 W to obtain the seventh solution;

[0130] (2) Culture, passage and make 4T1 cells cover the entire 12-well plate, remove the medium and wash, then add 5 mL of the seventh solution, and incubate for 2 hours, and observe the cells using a laser confocal microscope to obtain its cell imaging effect.

[0131] As Figure 13 shown, an increase in green fluorescence can be observed. The fluorescence intensity of 4T1 cells indicates that there are a large number of ·OH. It shows that the high-entropy sub-nanozyme can catalyze the conversion of H2O2 in the cancer cell culture environment into · OH.

[0132] As Figure 14 shown, 4T1 cells emit red fluorescence under the irradiation of 750 nm excitation light, indicating that Cy7 has been successfully grafted onto the surface of the nanozyme and can penetrate the cell membrane into tumor cells, and also indicating the prospect of in vivo imaging of the nanozyme system.

[0133] Performance test experiment 3 Detection of the active targeting performance of high-entropy sub-nanozymes in tumor tissues

[0134] Inject the 4T1 cell suspension into mice to construct a mouse model bearing breast cancer tumors. When the tumor volume of the mice is about 60 mm 3 3, all tumor-bearing mice are randomly divided into four groups, with five in each group, and administered by tail vein injection. Each group is the control group (Control) injected with 50 μL of PBS intratumorally, the laser group (Laser) treated with laser, the nanozyme group (Nanozyme) injected only with 50 μL of the high-entropy sub-nanozyme solution with tumor tissue targeting function of Example 1 (50 μg / mL), and the nanozyme + laser group (Nanozyme+Laser) injected with 50 μL of the high-entropy sub-nanozyme solution with tumor tissue targeting function of Example 1 (50 μg / mL) combined with laser treatment. The laser treatment group is exposed to 808 nm near-infrared laser (1.0 W / cm2, 10 min). Observe and image the four groups of mice with a Fluke Ti400 infrared thermal imager, and quantify with the Ti400examiner software. Measure the tumor size every other day using vernier calipers. Calculate the tumor volume according to the following formula.

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

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

[0137] As Figure 15 shown, the body weights of the four groups of mice did not change significantly during the treatment, indicating that the treatment process had no obvious effect on the daily life of the mice, the mice could metabolize the nanozyme normally, and there were no obvious side effects.

[0138] As Figure 16 shown, through the in vivo imaging system for small animals, red fluorescence can be observed at the tumor sites of the mice, indicating that the nanozyme is enriched in tumor cells.

[0139] As Figure 17As shown, different changes occurred in the tumor sites of mouse models treated under different conditions. For the control group and the laser group, over time, the tumor volumes of all mice grew to nearly 700 mm 3 , while for the nanozyme group, the tumor volumes of the mice were inhibited to some extent compared with the control group, and the tumor volumes were approximately 380 mm 3 , and the inhibition rate was approximately 46%. Under the combined treatment of laser and nanozyme, the tumors of the mice completely disappeared, and the tumor inhibition rate reached 100%

[0140] As Figure 18 shown, the H&E staining results showed that the cell nuclei of the tumor tissues were fragmented and a large number of cells died. The Tunel staining results showed a large amount of red fluorescence, indicating that the ROS generated under the action of the nanozyme induced the apoptosis effect of the tumor tissues. The Ki67 staining results showed that the nanozyme system could effectively inhibit the proliferation activity of tumor cells.

[0141] The above results indicate that a high-entropy sub-nanozyme product prepared from ruthenium acetylacetonate, rhodium acetylacetonate, platinum acetylacetonate, iridium acetylacetonate combined with molybdenum acetylacetonate has high safety, can accurately target tumor tissues, inhibit tumor growth, can be used to prepare anti-tumor drugs, and has good medical application prospects.

[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 solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A preparation method of a high-entropy sub-nanozyme, characterized in that, The preparation method comprises the following steps: S1: Weigh glucose and a surfactant, place them in a reaction vessel, and then add an organic solvent. After mixing and dissolving, a first solution is obtained; S2: Weigh ruthenium acetylacetonate, rhodium acetylacetonate, platinum acetylacetonate, and iridium acetylacetonate, and add them to the first solution. After mixing and dissolving, a second solution is obtained; S3: Weigh molybdenum acetylacetonate, add it to the second solution, mix and dissolve to obtain a third solution, and then react at 190 - 220 °C for 60 - 240 minutes to obtain a fourth solution; S4: Centrifuge the fourth solution, wash the precipitate, and freeze-dry it to obtain a high-entropy sub-nanozyme.

2. The preparation method according to claim 1, wherein It further comprises the following steps: S5: Weigh polyvinylpyrrolidone and the high-entropy sub-nanozyme, add them to cyclohexane, mix and dissolve, then add a fluorescently labeled cyclic peptide, and mix well to obtain a fifth solution; S6: Centrifuge the fifth solution, wash the precipitate, and freeze-dry it to obtain a high-entropy sub-nanozyme with tumor tissue targeting function.

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

4. The preparation method according to claim 1, characterized in that, In S1, the surfactant is selected from any one or a combination of two or more of cetyltrimethylammonium chloride, cetyltrimethylammonium bromide, sodium dodecylbenzenesulfonate, sodium octadecyl sulfate, or sodium dioctyl sulfosuccinate.

5. The preparation method according to claim 1, wherein In S1, the organic solvent is selected from any one or a combination of two or more of ethylene glycol, isopropanol, n-butanol, oleylamine, or octadecylamine.

6. The preparation method according to claim 1, characterized in that, In S2, the mass ratio of ruthenium acetylacetonate, rhodium acetylacetonate, platinum acetylacetonate, iridium acetylacetonate to the second solution is 0.01 - 0.001:0.01 - 0.001:0.01 - 0.001:0.01 - 0.001:

1.

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

1.

8. The preparation method according to claim 2, characterized in that, In S5, 3 - 5 g of polyvinylpyrrolidone, 1 - 5 mg of the high-entropy sub-nanozyme, and 1 - 5 mg of the cyclic peptide are added to every 100 mL of cyclohexane; Preferably, 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.

9. A high-entropy sub-nanozyme, characterized in that, The high-entropy sub-nanozyme is prepared by the preparation method according to any one of claims 1 - 8; Preferably, the diameter of the high-entropy sub-nanozyme is 0.5 - 1.0 nm.

10. Use of the high-entropy sub-nanozyme according to claim 9 in the preparation of an anti-tumor drug.

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