Preparation method and application of heterostructure supercluster enzyme drug

Abstract

The invention discloses a preparation method and application of a heterostructure supercluster enzyme drug, and belongs to the technical field of atomic cluster materials and biological medicines. The surface of the heterostructure supercluster enzyme is modified with a ligand group (a functional group or a zwitterionic compound) and coupled with a special drug and / or a specific target, so that the supercluster enzyme drug with an anti-tumor effect is obtained. The size of the heterostructure supercluster is 0.1 nm to 2 nm, and the number of atoms is 2 to 300. The composition, the hierarchical structure, the physical and chemical properties, the biocompatibility and the like of the material are regulated and controlled on the atomic scale, and the biocompatibility of the material and cancer cells can be improved through the ultra-small size structure of the material.

Description

Technical Field

[0001] The present invention belongs to the field of atomic cluster materials and biomedicine technology, and specifically relates to a method for preparing a heterogeneous structure super-atomic cluster enzyme drug, and the application of coupling special drugs and / or specific targets to prepare targeted and autonomously targeted anti-tumor cell drugs, belonging to the field of anti-cancer drug technology. Background Art

[0002] Studies by WHO and others show that about one-fifth of people will develop cancer in their lifetime. With population growth, aging and increased risk factors (such as nuclear sewage), the global cancer burden will further increase. It is estimated that there will be 35 million new cases and 19.4 million deaths in 2050, an increase of 77% and 100% respectively from 2022. Even in the United States, where medical technology is the most developed, more than 2 million new cases and 610,000 deaths are expected in 2024. In 2024, my country will have 4.82 million newly diagnosed cancer cases, accounting for 24.1% of global cases; 2.57 million cancer deaths, accounting for 26.5% of global cases. Statistics on cancer in my country from 2005 to 2020 found that the total number of deaths increased by 21.6%, with lung cancer, liver cancer and gastric cancer being the top three. Liver cancer patients in my country account for about 50% of the world's total; the sum of deaths from liver cancer and pancreatic cancer accounts for 13% of the total mortality rate. China has become a veritable "cancer power".

[0003] The progress of basic disciplines and applied technologies has continuously promoted the cross-innovation of materials science and medicine, and has spawned many innovative drugs and therapies, such as the birth of nanozymes featuring ferroptosis, copper death and enzyme-like cancer cell apoptosis or pyroptosis. The single-atom enzymes developed in recent years have high atomic utilization and metal ligand synergistic efficacy, showing better enzyme-like activity, selectivity and lower toxic side effects. However, most of the life processes related to tumors are cascade or disproportionate reactions involving multiple electrons or multiple centers, with multiple transition states, and require the joint participation of multiple atoms or even multiple heteroatoms and ligands to enhance the reactions related to enzyme-like cancer cell apoptosis. Single-atom enzymes (such as VN4, RhN4) generally require carriers (such as ZIF-8, MOF) to anchor to improve their stability, so the biocompatibility and active site density are not high (such as Pt1-Ptn / MoC: (2wt%), which restricts the overall efficacy. Therefore, it is necessary to develop super atoms constructed by multiple / heteroatoms, freely dispersed, with high biocompatibility and active site density.

[0004] Superatoms, also called atomic groups or clusters, are single-layer to dozens of-layer atomic aggregates or clusters (core size is about 10 ... ), in a broad sense, also includes single-atom site aggregates (clusters), which can have more than 90% surface atoms; they not only show the characteristics of elemental atoms, but also can couple with ligands to produce unique spatial topological conformations and quantum effects. The electronic structure has a transition effect from energy bands to molecular orbitals, and has molecular-like characteristics and molecular electronic energy level structures as a whole. The electrons in the clusters tend to concentrate on each atomic group, showing more significant atom-ligand-submicro-region coupling synergistic effects and multi-scale synergistic electronic structure regulation capabilities. The materials constructed by them often show peculiar physical and chemical properties and biomedical functions. At present, people have conducted research in the field of the relationship between superatomic microstructures and physical and chemical properties such as magnetism, light, and electricity, as well as their medical functions, and have found that: by optimizing the spatial topological structure of the central atom with multi-metals and multi-organic ligands and self-assembling into multi-center clusters, its enzyme-like medical functions can be greatly improved. Recently, Xie et al. reported that fructose-coated silver superatom enzyme-catalyzed drugs have significant anti-pancreatic cancer and lung cancer efficacy without obvious toxicity to healthy tissues (Wang, Z.-X. et al. Angstrom-Scale Silver Particles as a Promising Agent for Low-Toxicity Broad-Spectrum Potent Anticancer Therapy. Adv. Funct. Mater. 2019, 1808556); the ZIF-8 loaded single-atom RhN4 and VN4 enzymes developed by Zhang et al. have POD / CAT / GP-like x Enzyme catalytic activity, its catalytic reaction affinity exceeds that of natural enzymes by 4 to 20 times (Zhang, S. et al. Single-atom nanozymes catalytically surpassing naturally occurring enzymes as sustained stitching for brain trauma. Nature communications. 2022, 13, 4744). Recently, Gao et al. reported that iRGD-modified bovine serum albumin-modified Pt cluster enzyme catalytic drugs also have higher efficacy and safety than the first-line anticancer drug "carboplatin" (Zhang, C. et al. Ultrasmall platinum nanoclusters: A potent chemotherapeutic drug for cancer-specific DNA damage with optimizing therapeutic efficacy while minimizing systemic toxicity. Nano Today 2024, 55, 102195).

[0005] In recent years, studies on the physiological functions of related natural enzymes have shown that they can not only activate specific biomolecules and efficiently catalyze key biomolecule pathways (such as the STING-mediated type I interferon signaling pathway), but also catalyze reaction products (such as the products of CD-NTase) or even directly interact with immune cells (such as activating T cells), activate or regulate the immune system or induce enzyme-linked immunosuppression effects and regulate downstream effector protein effects to reshape the tumor immune microenvironment or enhance the innate immune response. Inspired by this, people have carried out research on nanozymes or single-atom enzymes with immunological effects, and found that they can activate systemic anti-tumor immunity or reverse the immunosuppression of cancer cells and prevent tumor recurrence and metastasis by regulating the tumor microenvironment. At the same time, they have the function of enzyme-like cell ferroptosis and targeting the organelles closely related to immunity-mitochondria. Therefore, with superatom cluster enzymes as the core, targets and special drugs can be conjugated and self-assembled with them into enzyme-like molecular microstructures to construct multi-targeted and multi-modal anticancer drugs to solve the toxic and side effects of traditional drugs and the cytokine storm of inflammatory factors and their drug resistance and immune resistance. At present, there are very few research reports in this area. Summary of the invention

[0006] In order to overcome the shortcomings of single-atom enzymes such as low biocompatibility and active site density, as well as to improve the toxic and side effects of traditional drugs, cellular inflammatory factor storms, and their drug resistance and immune resistance, the present invention proposes a heterogeneous structure superatom cluster enzyme drug and a preparation method thereof, by modifying the surface of the heterogeneous structure superatom cluster enzyme with ligand groups (functional groups or zwitterionic compounds) and coupling special drugs and / or specific targets, thereby obtaining a class of superatom cluster enzyme drugs with anti-tumor effects.

[0007] The heterogeneous structure superatom cluster enzyme is a heterogeneous structure superatom cluster enzyme composed of metal or metal atom cluster (such as a polyatomic cluster constructed by one or more elements of Au, Ag, Pt, Cu, Ru, Fe, Co, Ni, Gd, Bi, Sn, Mn, Ac series, Sc, Y, or La series elements), ceramic atom cluster enzyme (such as Al2O3, ZrO2, HfO2, SiN, SiC and other inorganic non-metallic materials ceramics, metal oxide materials ceramics one or more polyatomic clusters) and / or metal compound atom cluster enzyme (such as metal sulfide / nitride / boride / phosphide, etc., excluding metal oxides) as units. The heterogeneous structure superatom cluster enzyme at least includes metal atom clusters, such as a heterojunction of polymetallic atom cluster aggregates, a core-shell structure heterojunction with metal atom clusters as the core and ceramics or metal compound clusters as the shell, or a gradient structure heterojunction with a gradual transition from metal clusters to metal compound clusters.

[0008] Heterostructure superatom cluster enzyme drugs are constructed by coupling heterostructure superatom cluster enzymes with drugs or / and specific target molecules through surface-modified ligand groups. Superatom cluster enzyme drugs have specific spatial stereostructures: with the surface atoms of the heterostructure superatom cluster enzyme as the center or vertex, a topological structure similar to the spatial stereostructure of organic metal compounds is formed, such as tetrahedron, octahedron, icosahedron, pyramid rhombus, half pyramid, planar quadrilateral, pentagon and hexagon, etc. (such as Figure 7 shown).

[0009] The size of the heterostructure superatom cluster is (i.e. 0.1 nm to 2 nm), more preferably (i.e. 0.5nm-1.5nm), the number of atoms is 2-300; the optimal number is 2-150, or has 0-3 shells;

[0010] The surface modification ligand group of the superatom cluster enzyme is one or more of functional groups or zwitterionic compounds, wherein the functional groups are selected from hydroxyl, carboxyl, thiol, amine and amide groups, etc.; the zwitterionic compounds are selected from alkaloids (such as betaine), cystine, selenoic acid, selenocysteine ​​and 20 essential amino acids for the human body, etc., preferably amino, hydroxyl, carboxyl, cysteine ​​and selenoic acid.

[0011] The coupled special drug is selected from one or more of squalene, ginsenoside, paclitaxel, sea cucumber saponin, saponin, doxorubicin, ginkgo flavonoids, curcumin, polypeptide drugs, antibodies (such as mesothelin antibodies, V-ATPase antibodies, atezolizumab, insulin-like growth factor 1: IGF1), mRNA (such as mRNA-4157), iRNA (such as siRNA, miRNA), DNA (such as mtDNA), preferably ginsenoside, sea cucumber saponin, saponin, ginkgo flavonoids, PDL1, CD8, mRNA;

[0012] The coupled specific target molecules are selected from: specific target molecules of specific cancers (such as lung cancer, liver cancer, pancreatic cancer, gastric cancer, colorectal cancer, breast cancer, etc.) (such as AKT target, EGFR target, NTRK target, GPC3 target, PD-1 / PD-L1 target, Claudin18.2 target, IGF1R target, MET target, UCK2 target, CD4 / 6 / 39 / 47 / 70 target, FGFR target and DDR1 target), and one or more of the related targeting substances targeting tumor microenvironment factors (such as pH value and temperature) (such as interpenetrating network biomacromolecule alloy substances composed of sodium alginate and modified chitosan, etc.).

[0013] The heterogeneous structure superatom cluster enzyme drug further described is a superatom cluster enzyme drug constructed by coupling a heterogeneous structure superatom cluster enzyme with a special drug through a surface-modified ligand group; or a superatom cluster enzyme drug coupled with a specific target through a surface-modified ligand group or a superatom cluster enzyme drug coupled with a special drug and a specific target through a surface-modified ligand group, that is, a superatom cluster enzyme drug ②;

[0014] The present invention provides a method for preparing the superatom cluster enzyme drug, which specifically comprises the following steps:

[0015] Step 1: Preparation of heterogeneous structure superatom cluster enzyme;

[0016] Step 2: Heterostructure superatom cluster enzyme coupled with special drugs;

[0017] (a) adding the heterogeneous structure superatom cluster enzyme prepared in step 1 immediately to an organic solution containing a surface modifier, stirring at room temperature to completely modify it, centrifuging after the reaction, and washing by centrifugation with a mixed solution of water and ethanol to obtain a surface-modified heterogeneous structure superatom cluster enzyme; at the same time, adding a special drug to an organic solution containing a surface modifier, stirring at room temperature to completely modify it, centrifuging after the reaction, and washing by centrifugation with a mixed solution of water and ethanol to obtain a surface-modified special drug;

[0018] (b) dispersing the surface-modified heterogeneous structure superatom cluster enzyme and the cross-linking agent obtained in step (a) into an organic solvent, stirring and incubating, and then centrifuging and washing with a mixed solution of water and ethanol to obtain an activated heterogeneous structure superatom cluster enzyme for use;

[0019] (c) dispersing or dissolving the activated heterogeneous structure superatomic cluster enzyme obtained in step (b) and the special drug after surface modification in step (a) in an organic solvent, stirring them at room temperature to couple them together, and then centrifuging and washing with a mixed solution of water and ethanol and vacuum drying them to obtain a superatomic cluster enzyme drug of the heterogeneous structure superatomic cluster enzyme coupled with the special drug①.

[0020] The coupling process can be referred to patent application numbers 201810060431.5 and 202110360364.0.

[0021] or / and step 3: conjugation to specific target

[0022] (a) adding the heterogeneous structure superatom cluster enzyme prepared in step (1) or the superatom cluster enzyme drug ① prepared in step (2) to an organic solution containing a surface modifier, stirring at room temperature to completely modify it, centrifuging after the reaction, washing by centrifugation with a mixed solution of water and ethanol, and vacuum drying to obtain a surface-modified heterogeneous structure superatom cluster enzyme or a surface-modified superatom cluster enzyme drug ①;

[0023] (b) dissolving the surface-modified heterogeneous superatom cluster enzyme or the surface-modified superatom cluster enzyme drug ① obtained in step (a) in PBS to prepare a solution A;

[0024] (c) preparing a specific target solution and a condensing agent solution with MES buffer and mixing the two; preparing a crosslinking agent solution with MES buffer and then adding the crosslinking agent solution to the above solution, and the final mixed solution is solution B;

[0025] (d) Mix solution A from step (b) and solution B from step (c) as solution C, and incubate overnight at low temperature (the temperature can be adjusted as needed); then dissolve the terminator in PBS to prepare a low-concentration reaction terminator solution; add the reaction terminator solution to solution C to terminate the reaction to obtain the final product, and after centrifugal washing and vacuum drying, obtain a heterogeneous structure superatomic cluster enzyme-coupled superatomic cluster enzyme drug with a specific target or a heterogeneous structure superatomic cluster enzyme-coupled special drug and a superatomic cluster enzyme drug with a specific target, namely, superatomic cluster enzyme drug②.

[0026] According to the method for preparing superatom cluster enzyme drugs of the present invention, either step 2 or step 3 can be used alone to couple the special drug or the specific target; or they can be used simultaneously, and the obtained product can be coupled with the special drug and the specific target at the same time.

[0027] Further step 1: preparing the heterostructure superatom cluster enzyme comprises the following steps:

[0028] (a) dissolving a metal salt corresponding to the metal in the heterostructure superatom cluster enzyme and a polymer dispersant in an organic solvent; the organic solvent is N-methylpyrrolidone, dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, dimethylacetamide, anhydrous toluene or water; wherein the metal salt is selected from one or more than two according to the required heterostructure, and when there are two, the ratio is 1:1 to 1:5, and when there are more than two, the ratio is adjusted according to the actual situation; if the heterostructure superatom cluster enzyme contains other elements besides metal and oxygen, a precursor compound of the corresponding element is also added at the same time, such as when containing SiN, a precursor of Si corresponding to tetraethyl orthosilicate, tetraethyl orthosilicate, etc. is added, and a precursor of N corresponding to N, such as ammonium salt, etc. is added;

[0029] (b) dissolving the reducing agent in N-methylpyrrolidone, dimethyl sulfoxide, dimethylformamide, tetrahydrofuran or dimethylacetamide to obtain a reducing agent solution; wherein each 0.1 g to 3.5 g of the reducing agent corresponds to 50 ml of solvent; and the amount of the reducing agent used is 1.5 to 2 times the equivalent of the metal salt.

[0030] (c) Figure 1 The reaction shown is prepared by using a Y-type microfluidic reactor, and the reaction solutions prepared in step (a) and step (b) are respectively extracted by a syringe and placed in a syringe pump system. The two reaction solutions are reacted after being merged through a Y-type connector, and a stable and dispersed superatomic cluster enzyme solution is obtained through a gas-protected quenching and collecting device. The capillary length from the outlet of the Y-type connector to the quenching and collecting device is 80 cm, and the inner diameter d is 0.5-1.2 mm; the flow rate of the syringe pump is: 9 ml / min-15 ml / min; the reaction temperature of the thermostat is set to: 80-100°C; the quenching and collecting device is placed in an ice-water mixture or liquid nitrogen at 0°C, and the gas introduced is nitrogen, argon, carbon dioxide or air, etc.; according to the formula Where V is the unit flow volume, t is the unit time, v is the average flow rate in the capillary, and A is the cross-sectional area of ​​the pipe; the preparation method controls the flow rate of the reaction solution in the tube through the injection pump system, adjusts the inner diameter of the capillary to control A, and fixes V to 100ml. In this way, the reaction time required for the reaction solution to reach the quenching collection device is strictly controlled, the reaction is terminated by suddenly dropping the temperature, and the size of the heterogeneous structure superatomic cluster enzyme is controlled below 2nm; then the collected solution is centrifuged, the supernatant is poured out, washed with ethanol and deionized water, and then dried using a freeze vacuum dryer to obtain a sample powder;

[0031] The polymer material mentioned above is selected from polyvinyl pyrrolidone and the like.

[0032] If the heterogeneous structure superatom cluster enzyme contains other elements besides metal and oxygen, and the above freezing can form the corresponding heterogeneous structure superatom cluster enzyme, then it is OK; if not, the sample powder obtained after cooling can be heated as needed (the heating temperature varies with the specific material as long as the corresponding compound can be formed) to form the corresponding element compound, and after the reaction is completed, it can be immediately frozen or directly carried out to the next step of surface modification reaction;

[0033] The surface modifier is selected from at least one of 3-aminopropyltrimethoxysilane (APTMS), vinyltrimethoxysilane, 3-mercaptopropyltriethoxysilane, 3-aminopropyltriethoxysilane, N-(β-aminoethyl)-γ-aminopropyltrimethoxysilane, N-(β-aminoethyl)-γ-aminopropyltriethoxysilane, di(β-sulfur-α-aminopropionic acid), and L-α-amino-β-mercaptopropionic acid; preferably at least one of 3-aminopropyltrimethoxysiloxane, 3-mercaptopropyl-triethoxysilane and di(β-sulfur-α-aminopropionic acid).

[0034] The cross-linking agent includes at least one of disuccinimidyl suberate (DSS), disuccinimidyl glutarate (DSG), polyethylene glycol-bis(succinimidyl succinate) (SS-PEG-SS), methoxypolyethylene glycol succinimidyl succinate (mPEG-SS), ethylene glycol bis(succinic acid N-hydroxysuccinimide ester) (EGS), N-hydroxysuccinimide (NHS), N-hydroxysulfosuccinimide (Sulfo-NHS), N-ethylmaleimide (NEM) and aziridine cross-linking agent XR-100; preferably at least one of DSS, SS-PEG-SS and NHS.

[0035] The condensing agent is selected from carbodiimide condensing agents, such as at least one of dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI);

[0036] The reaction terminator is selected from one or more of 2-mercaptoethanol, 20 kinds of amino acids (glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine), and tris(hydroxymethyl)aminomethane hydrochloride.

[0037] Another object of the present invention is to provide the use of the superatom cluster enzyme drug of the present invention in the preparation of anticancer drugs that target and autonomously target tumor cells.

[0038] The advantages of the present invention are: super-atomic clusters are prepared by regulating raw materials, reaction speed, reaction time (short reaction time and fast reaction speed, i.e., fast jet speed of the Y-type reactor, rapid cooling and other conditions can obtain heterogeneous structure super-atomic cluster enzymes), and then the prepared enzyme (the prepared heterogeneous structure super-atomic cluster enzyme is immediately modified and packaged with a modifier to maintain a particle size of less than 2nm) drug realizes the regulation of the composition, hierarchical structure, physicochemical properties, biocompatibility and the like of the material on the atomic scale, and its ultra-small size structure can improve its biocompatibility with cancer cells (such as avoiding the discovery and destruction of various immune bodies or macrophages in the body and having a self-stealth function) and its ability to enter cancer cells; according to the coupled special-needed drugs, autonomous targeting and specific targeting of cancer cells can be achieved, and in addition, the coupled traditional Chinese medicine organic molecules (such as ginsenoside, sea cucumber saponin, paclitaxel) have the function of improving the activity of normal cells and have a certain effect on improving the body's immunity, and some even have the opposite killing effect of ordinary chemotherapy drugs on normal cells of the body, greatly improving the health level of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Schematic diagram of the process of the microfluidic reactor.

[0040] Figure 2 Au@WO prepared in Example 1 x TEM image (a) and size statistical distribution diagram (b);

[0041] Figure 3 Au@WO prepared in Example 1 x TEM images of conjugated ginsenosides;

[0042] Figure 4 The Au@WO prepared in Example 1 x , ginsenoside (Rg3) and Au@WO x Conjugated ginsenosides (Au@WO x -Rg3) cytotoxicity to human normal liver cells QSJ-7701 (a) and liver cancer cells LM3 (b) at concentrations of 0, 10, 20, 50, 100, 200 μg / ml;

[0043] Figure 5 Au@WO prepared in Example 2 x STEM image (a) and size statistical distribution diagram (b);

[0044] Figure 6 Au@MnO prepared in Example 3 x TEM image (a) and size statistical distribution diagram (b);

[0045] Figure 7A technical roadmap for constructing specific spatial stereostructures for modified superatomic cluster enzymes and organic ligands. The topological structure types constructed by coupling organic ligands with superatomic clusters as the center include tetrahedron, octahedron, icosahedron, planar quadrilateral and hexagonal spatial stereostructures. DETAILED DESCRIPTION

[0046] The present invention will be further described below in conjunction with examples, but the present invention is not limited to the following examples.

[0047] Example 1

[0048] The present invention uses Au@WO x Superatom cluster enzyme, taking ginsenoside as a coupled special drug as an example to illustrate the process of the invention.

[0049] Step 1: Preparation of superatom cluster enzyme Au@WO x (WO x represents the oxide of W)

[0050] First, 1g HAuCl4 powder was dissolved in 10ml N-methylpyrrolidone to prepare a 0.1g / ml HAuCl4 solution. After weighing 0.5ml HAuCl4 solution, 0.17g WCl6 and 0.35g polyvinylpyrrolidone (PVP, K30, molecular weight 30000), the three were dissolved in 50ml N-methylpyrrolidone as a metal salt solution. At the same time, 0.35g reducing agent NaBH4 was weighed and dissolved in the same volume of N-methylpyrrolidone as a reducing solution. The system reaction temperature was set to 90°C, the injection pump flow rate was set to 11ml / min, and the entire collection process was carried out under a nitrogen atmosphere.

[0051] Use a syringe to extract 50ml of metal salt solution and 50ml of reducing solution, fix them with a syringe pump and set the parameters of the reaction program. After the synthesis starts, the metal salt solution and reducing solution converge in the Y-shaped pipe, react and rapidly nucleate in the transparent microfluidic channel to form Au@WO x Particles (such as Figure 2 When collecting the product, the collecting device needs to be placed in a 0°C ice-water mixture for cooling to achieve the purpose of terminating the reaction.

[0052] The collected solution was then centrifuged at 13,000 rpm for 10 minutes, the supernatant was poured out and washed twice with ethanol and deionized water in a ratio of 8:2. After washing, it was dried using a freeze vacuum dryer to obtain sample powder for subsequent experiments.

[0053] Step 2: Superatom cluster enzyme conjugation with specific drugs

[0054] The Au@WO prepared in step 1 was treated with 3-aminopropyltrimethoxysilane. x(generally, the surface modification reaction is carried out immediately) and ginsenosides are used for surface modification respectively. x The powder is added to a certain amount of toluene (e.g., 20 ml), and then 3-aminopropyltrimethoxysilane accounting for 1 wt% of the solvent is added, and the mixture is sealed and placed at room temperature and stirred with a magnetic bar for 24 hours to complete the modification. After the reaction is completed, the mixture is centrifuged, washed twice with a mixed solution of water and ethanol, and dried for later use;

[0055] Will be with Au@WO x Ginsenoside powder with a powder mass ratio of 1:1 is dissolved in a certain amount (e.g., 20 ml) of toluene, and then 3-aminopropyltrimethoxysilane is added thereto at 1 wt% relative to the solvent, and the mixture is sealed and placed at room temperature and stirred with a magnetic bar for 24 hours to completely modify the mixture. After the reaction is completed, the mixture is centrifuged, washed twice with a mixed solution of water and ethanol, and dried for later use.

[0056] Modified Au@WO x Then, the Au@WO obtained in step 2 was activated by disuccinimidyl suberate: x The powder is dissolved in a certain amount (e.g., 20 ml) of dimethyl sulfoxide solution, and a certain amount (e.g., 1 gram) of disuccinimidyl suberate is added, and the mixture is sealed and placed under magnetic stirring at room temperature for 1 hour, and then centrifuged and washed twice with a mixed solution of water and ethanol. The centrifugal washing here is mainly to remove the remaining disuccinimidyl suberate in the solution.

[0057] The activated Au@WO x The powder and the modified ginsenoside powder obtained in step 2 were dissolved in a certain amount (e.g., 50 ml) of dimethyl sulfoxide solution, sealed and placed under magnetic stirring at room temperature for 1 hour to couple them together, and finally centrifuged and washed to obtain a novel anti-tumor drug (Au@WO x -Rg3)(such as Figure 3 shown).

[0058] Experimental study on the effect of the anti-tumor drug obtained:

[0059] The prepared Au@WO x , ginsenoside (Rg3) and Au@WO x Conjugated ginsenosides (Au@WO x -Rg3) powder is sterilized. The specific process is as follows: the powder is soaked in 75% alcohol solution (2-3ml) for 24 hours, centrifuged at 800rmp for 4 minutes, and then irradiated with ultraviolet light for one hour after removing the supernatant to achieve the purpose of sterilization.

[0060] A control group and five experimental groups with drug concentration gradients were set up, with drug concentrations of 0, 10, 20, 50, 100, and 200 μg / ml, respectively. Five replicate samples were set up in each group to eliminate experimental errors caused by single detection. In addition, a zero adjustment well should be set for each drug concentration as a blank control to avoid errors caused by the absorbance of the drug itself. Normal cells and liver cancer cells were inoculated into a 96-well cell plate and placed in a 37° carbon dioxide incubator for 24 hours before drug administration. 10 μl of MTT was added 24, 48, and 72 hours after administration, respectively, and placed in a 37° carbon dioxide constant temperature incubator for incubation for 4 hours. The liquid was removed with a 1 ml syringe, and 200 μl of DMSO was added. The reaction was continued for 1 hour, and a horizontal shaker was used for 15 minutes to allow DMSO to fully contact the liver cancer cells. The absorbance of each cell well was measured at 490 nm using an ELISA reader, and the cell survival rate at different drug concentrations was obtained by data processing using the formula: Cell survival rate = [(A2-A0) / (A1-A0)] × 100%, where A2 represents the absorbance of the experimental group, A1 is the absorbance of the control group, and A0 is the absorbance of the blank well.

[0061] Figure 4 The results are shown in Figure 24h after administration of different concentrations of Au@WO3, Rg3 and Au@WO x - Cytotoxicity of Rg3 to normal human liver cells QSG-7701 (a) and LM3 liver cancer cells (b). Figure 4 In (a), Au@WO3, Rg3 and Au@WO can be clearly observed. x The overall trend of the effect of -Rg3 on the cell activity of normal human liver cells decreased with the increase of drug concentration, but the cell survival rate was higher than 80%, proving that Au@WO3, Rg3 and Au@WO x -Rg3 has less toxicity to normal human liver cells. Figure 4 (b) The change in the concentration of Au@WO3 has little effect on the survival rate of liver cancer cells; and the survival rate of liver cancer cells decreases significantly with the increase in the concentration of Rg3 drug. x -Rg3 concentration also significantly reduced the survival rate of liver cancer cells. x -Rg3 has less toxicity to normal human liver cells, and after being coupled to Rg3, it has a higher lethality rate to liver cancer cells, thereby improving the drug's anti-liver cancer effect.

[0062] Example 2

[0063] The present invention uses Au@WO x Superatom cluster enzyme, taking ginsenoside as a coupled special drug as an example to illustrate the process of the invention.

[0064] Step 1: Preparation of superatom cluster enzyme Au@WO x

[0065] First, 1g HAuCl4 powder was dissolved in 10ml N-methylpyrrolidone to prepare a 0.1g / ml HAuCl4 solution. After weighing 0.5ml HAuCl4 solution, 0.17g WCl6 and 0.35g polyvinylpyrrolidone (PVP, K30, molecular weight 30000), the three were dissolved in 50ml N-methylpyrrolidone as a metal salt solution. At the same time, 0.35g reducing agent NaBH4 was weighed and dissolved in the same volume of N-methylpyrrolidone as a reducing solution. The system reaction temperature was set to 85°C, the injection pump flow rate was set to 15ml / min, and the entire collection process was carried out under a nitrogen atmosphere.

[0066] Use a syringe to extract 50ml of metal salt solution and 50ml of reducing solution, fix them with a syringe pump and set the parameters of the reaction program. After the synthesis starts, the metal salt solution and reducing solution converge in the Y-shaped pipe, react and rapidly nucleate in the transparent microfluidic channel to form Au@WO x Particles (such as Figure 5 When collecting the product, the collecting device needs to be placed in a 0°C ice-water mixture for cooling, and then the collected solution is centrifuged at 13000 rpm for 10 minutes, the supernatant is poured out and washed twice with ethanol and deionized water in a ratio of 8:2, and then dried using a freeze vacuum dryer to obtain a sample powder for subsequent experiments.

[0067] Step 2: Superatom cluster enzyme conjugation with specific drugs

[0068] The Au@WO prepared in step 1 was treated with 3-aminopropyltrimethoxysilane. x and ginsenosides were surface modified. x The powder is added to a certain amount of toluene (e.g., 20 ml), and 3-aminopropyltrimethoxysilane is added at 1 wt% of the solvent. The mixture is sealed and placed at room temperature and stirred with a magnetic bar for 24 hours to completely modify the mixture. After the reaction is completed, the mixture is centrifuged, washed twice with a mixed solution of water and ethanol, and dried for later use;

[0069] Will be with Au@WO x Ginsenoside powder with a powder mass ratio of 1:1 is dissolved in a certain amount (e.g., 20 ml) of toluene, and then 3-aminopropyltrimethoxysilane is added thereto at 1 wt% relative to the solvent, and the mixture is sealed and placed at room temperature and stirred with a magnetic bar for 24 hours to completely modify the mixture. After the reaction is completed, the mixture is centrifuged, washed twice with a mixed solution of water and ethanol, and dried for later use.

[0070] Modified Au@WO xThen, the Au@WO obtained in step 2 was activated by disuccinimidyl suberate: x The powder is dissolved in a certain amount (e.g., 20 ml) of dimethyl sulfoxide solution, and a certain amount (e.g., 1 gram) of disuccinimidyl suberate is added, and the mixture is sealed and placed under magnetic stirring at room temperature for 1 hour, and then centrifuged and washed twice with a mixed solution of water and ethanol. The centrifugal washing here is mainly to remove the remaining disuccinimidyl suberate in the solution.

[0071] The activated Au@WO x The powder and the modified ginsenoside powder obtained in step 2 were dissolved in a certain amount (e.g., 50 ml) of dimethyl sulfoxide solution, sealed and placed under magnetic stirring at room temperature for 1 hour to couple them together, and finally centrifuged and washed to obtain a novel anti-tumor drug (Au@WO x -Rg3).

[0072] Example 3

[0073] The present invention uses Au@MnO x Superatom cluster enzyme, taking ginsenoside as a coupled special drug as an example to illustrate the process of the invention.

[0074] Step 1: Preparation of superatom cluster enzyme Au@MnO x

[0075] First, 1g HAuCl4 powder was dissolved in 10ml N-methylpyrrolidone to prepare a 0.1g / ml HAuCl4 solution. After weighing 1.2ml HAuCl4 solution, 0.11g MnCl2 and 0.35g polyvinylpyrrolidone (PVP, K30, molecular weight 30000), the three were dissolved in 50ml N-methylpyrrolidone as a metal salt solution. At the same time, 0.35g reducing agent NaBH4 was weighed and dissolved in the same volume of N-methylpyrrolidone as a reducing solution. The system reaction temperature was set to 95°C, the injection pump flow rate was set to 13ml / min, and the entire collection process was carried out under a nitrogen atmosphere.

[0076] Use a syringe to extract 50ml of metal salt solution and 50ml of reducing solution, fix them with a syringe pump and set the parameters of the reaction program. After the synthesis starts, the metal salt solution and reducing solution converge in the Y-shaped pipe, react and rapidly nucleate in the transparent microfluidic channel to form Au@MnO x Particles (such as Figure 6 When collecting the product, the collecting device needs to be placed in a 0°C ice-water mixture for cooling, and then the collected solution is centrifuged at 12000 rpm for 10 minutes, the supernatant is poured out and washed twice with ethanol and deionized water in a ratio of 8:2, and then dried using a freeze vacuum dryer to obtain a sample powder for subsequent experiments.

[0077] Step 2: Superatom cluster enzyme conjugation with specific drugs

[0078] The Au@MnO prepared in step 1 was treated with 3-aminopropyltrimethoxysilane. x and ginsenosides were surface modified. x The powder is added to a certain amount of toluene (e.g., 20 ml), and 3-aminopropyltrimethoxysilane is added at 1 wt% of the solvent. The mixture is sealed and placed at room temperature and stirred with a magnetic bar for 24 hours to completely modify the mixture. After the reaction is completed, the mixture is centrifuged, washed twice with a mixed solution of water and ethanol, and dried for later use;

[0079] With Au@MnO x Ginsenoside powder with a powder mass ratio of 1:1 is dissolved in a certain amount (e.g., 20 ml) of toluene, and then 3-aminopropyltrimethoxysilane is added thereto at 1 wt% relative to the solvent, and the mixture is sealed and placed at room temperature and stirred with a magnetic bar for 24 hours to completely modify the mixture. After the reaction is completed, the mixture is centrifuged, washed twice with a mixed solution of water and ethanol, and dried for later use.

[0080] Modified Au@MnO x Then, the Au@MnO obtained in step 2 was activated by disuccinimidyl suberate: x The powder is dissolved in a certain amount (e.g., 20 ml) of dimethyl sulfoxide solution, and a certain amount (e.g., 1 gram) of disuccinimidyl suberate is added, and the mixture is sealed and placed under magnetic stirring at room temperature for 1 hour, and then centrifuged and washed twice with a mixed solution of water and ethanol. The centrifugal washing here is mainly to remove the remaining disuccinimidyl suberate in the solution.

[0081] The activated Au@MnO x The powder and the modified ginsenoside powder obtained in step 2 were dissolved in a certain amount (e.g., 50 ml) of dimethyl sulfoxide solution, sealed and placed under magnetic stirring at room temperature for 1 hour to couple them together, and finally centrifuged and washed to obtain a novel anti-tumor drug (Au@MnO x -Rg3).

[0082] Example 4

[0083] The present invention uses Au@MnO x Superatom cluster enzyme, taking ginsenoside as a coupled special drug as an example to illustrate the process of the invention.

[0084] Step 1: Preparation of superatom cluster enzyme Au@MnO x

[0085] First, 1g HAuCl4 powder was dissolved in 10ml N-methylpyrrolidone to prepare a 0.1g / ml HAuCl4 solution. After weighing 0.5ml HAuCl4 solution, 0.11g MnCl2 and 0.35g polyvinylpyrrolidone (PVP, K30, molecular weight 30000), the three were dissolved in 50ml N-methylpyrrolidone as a metal salt solution. At the same time, 0.3g reducing agent NaBH4 was weighed and dissolved in the same volume of N-methylpyrrolidone as a reducing solution. The system reaction temperature was set to 100°C, the injection pump flow rate was set to 12ml / min, and the entire collection process was carried out under a nitrogen atmosphere.

[0086] Use a syringe to extract 50ml of metal salt solution and 50ml of reducing solution, fix them with a syringe pump and set the parameters of the reaction program. After the synthesis starts, the metal salt solution and reducing solution converge in the Y-shaped pipe, react and rapidly nucleate in the transparent microfluidic channel to form Au@MnO x Particles. When collecting the product, the collection device needs to be placed in a 0°C ice-water mixture for cooling, and then the collected solution is centrifuged at 13,000 rpm for 10 minutes, the supernatant is poured out and washed twice with ethanol and deionized water in a ratio of 8:2, and then dried using a freeze vacuum dryer to obtain sample powder for subsequent experiments.

[0087] Step 2: Superatom cluster enzyme conjugation with specific drugs

[0088] The Au@MnO prepared in step 1 was treated with 3-aminopropyltrimethoxysilane. x and ginsenosides were surface modified. x The powder is added to a certain amount of toluene (e.g., 20 ml), and 3-aminopropyltrimethoxysilane is added at 1 wt% of the solvent. The mixture is sealed and placed at room temperature and stirred with a magnetic bar for 24 hours to completely modify the mixture. After the reaction is completed, the mixture is centrifuged, washed twice with a mixed solution of water and ethanol, and dried for later use;

[0089] With Au@MnO x Ginsenoside powder with a powder mass ratio of 1:1 is dissolved in a certain amount (e.g., 20 ml) of toluene, and then 3-aminopropyltrimethoxysilane is added thereto at 1 wt% relative to the solvent, and the mixture is sealed and placed at room temperature and stirred with a magnetic bar for 24 hours to completely modify the mixture. After the reaction is completed, the mixture is centrifuged, washed twice with a mixed solution of water and ethanol, and dried for later use.

[0090] Modified Au@MnO x Then, the Au@MnO obtained in step 2 was activated by disuccinimidyl suberate: xThe powder is dissolved in a certain amount (e.g., 20 ml) of dimethyl sulfoxide solution, and a certain amount (e.g., 1 gram) of disuccinimidyl suberate is added, and the mixture is sealed and placed under magnetic stirring at room temperature for 1 hour, and then centrifuged and washed twice with a mixed solution of water and ethanol. The centrifugal washing here is mainly to remove the remaining disuccinimidyl suberate in the solution.

[0091] The activated Au@MnO x The powder and the modified ginsenoside powder obtained in step 2 were dissolved in a certain amount (e.g., 50 ml) of dimethyl sulfoxide solution, sealed and placed under magnetic stirring at room temperature for 1 hour to couple them together, and finally centrifuged and washed to obtain a novel anti-tumor drug (Au@MnO x -Rg3).

[0092] Example 5

[0093] The present invention uses CoFe@WO x Superatom cluster enzyme, taking ginsenoside as a coupled special drug as an example to illustrate the process of the invention.

[0094] Step 1: Preparation of superatom cluster enzyme CoFe@WO x

[0095] Weigh 0.080g FeCl2·4H2O, 0.095g CoCl2·6H2O, 0.079g WCl6 and 0.42g polyvinyl pyrrolidone (PVP, K30, molecular weight 30000) and dissolve them in 50ml N-methylpyrrolidone as metal salt solution. At the same time, weigh 0.4g reducing agent NaBH4 and dissolve it in the same volume of N-methylpyrrolidone as reducing solution. The system reaction temperature is set to 90℃, the injection pump flow rate is set to 10ml / min, and the entire collection process is carried out under nitrogen atmosphere.

[0096] Use a syringe to extract 50ml of metal salt solution and 50ml of reducing solution, fix them with a syringe pump and set the parameters of the reaction program. After the synthesis starts, the metal salt solution and reducing solution converge in the Y-shaped pipe, react and rapidly nucleate in the transparent microfluidic channel to form CoFe@WO x Particles. When collecting the product, the collection device needs to be placed in a 0°C ice-water mixture for cooling, and then the collected solution is centrifuged at 13,000 rpm for 10 minutes, the supernatant is poured out and washed twice with ethanol and deionized water in a ratio of 8:2, and then dried using a freeze vacuum dryer to obtain sample powder for subsequent experiments.

[0097] Step 2: Superatom cluster enzyme conjugation with specific drugs

[0098] The CoFe@WO prepared in step 1 was treated with 3-aminopropyltrimethoxysilane. x and ginsenosides were used for surface modification. x The powder is added to a certain amount of toluene (e.g., 20 ml), and 3-aminopropyltrimethoxysilane is added at 1 wt% of the solvent. The mixture is sealed and placed at room temperature and stirred with a magnetic bar for 24 hours to completely modify the mixture. After the reaction is completed, the mixture is centrifuged, washed twice with a mixed solution of water and ethanol, and dried for later use;

[0099] Will be combined with CoFe@WO x Ginsenoside powder with a powder mass ratio of 1:1 is dissolved in a certain amount (e.g., 20 ml) of toluene, and then 3-aminopropyltrimethoxysilane is added thereto at 1 wt% relative to the solvent, and the mixture is sealed and placed at room temperature and stirred with a magnetic bar for 24 hours to completely modify the mixture. After the reaction is completed, the mixture is centrifuged, washed twice with a mixed solution of water and ethanol, and dried for later use.

[0100] Modified CoFe@WO x Then, the CoFe@WO obtained in step 2 was activated by disuccinimidyl suberate: x The powder is dissolved in a certain amount (e.g., 20 ml) of dimethyl sulfoxide solution, and a certain amount (e.g., 1 gram) of disuccinimidyl suberate is added, and the mixture is sealed and placed under magnetic stirring at room temperature for 1 hour, and then centrifuged and washed twice with a mixed solution of water and ethanol. The centrifugal washing here is mainly to remove the remaining disuccinimidyl suberate in the solution.

[0101] The activated CoFe@WO x The powder and the modified ginsenoside powder obtained in step 2 were dissolved in a certain amount (e.g., 50 ml) of dimethyl sulfoxide solution, sealed and placed under magnetic stirring at room temperature for 1 hour to couple them together, and finally centrifuged and washed to obtain a novel anti-tumor drug (CoFe@WO x -Rg3).

[0102] Example 6

[0103] The present invention takes AuPt superatom cluster enzyme and ginsenoside as a coupled special drug as an example to illustrate the process of the invention.

[0104] Step 1: Preparation of superatom cluster enzyme AuPt

[0105] First, 1g HAuCl4 powder was dissolved in 10ml N-methylpyrrolidone to prepare a 0.1g / ml HAuCl4 solution. After weighing 0.4ml HAuCl4 solution, 0.079g H2PtCl6 and 0.4g polyvinylpyrrolidone (PVP, K30, molecular weight 30000), the three were dissolved in 50ml N-methylpyrrolidone as a metal salt solution. At the same time, 0.35g reducing agent NaBH4 was weighed and dissolved in the same volume of N-methylpyrrolidone as a reducing solution. The system reaction temperature was set to 100°C, the injection pump flow rate was set to 12ml / min, and the entire collection process was carried out under a nitrogen atmosphere.

[0106] Use a syringe to extract 50ml of metal salt solution and 50ml of reducing solution, fix it with a syringe pump and set the parameters of the reaction program. After the synthesis starts, the metal salt solution and the reducing solution converge in the Y-shaped pipe, react and quickly nucleate in the transparent microfluidic channel to form AuPt particles. When collecting the product, the collection device needs to be placed in a 0℃ ice-water mixture for cooling, and then the collected solution is centrifuged at 13000rpm for 10 minutes, the supernatant is poured out and washed twice with ethanol and deionized water in a ratio of 8:2. After washing, it is dried using a freeze vacuum dryer to obtain sample powder for subsequent experiments.

[0107] Step 2: Superatom cluster enzyme conjugation with specific drugs

[0108] 3-aminopropyltrimethoxysilane is used to modify the surface of AuPt and ginsenoside in step 1. A certain amount of AuPt powder is added to a certain amount of toluene (e.g., 20 ml), and 3-aminopropyltrimethoxysilane is added to account for 1 wt% of the solvent. The mixture is sealed and placed at room temperature and stirred with a magnetic bar for 24 hours to completely modify the AuPt. After the reaction is completed, the mixture is centrifuged, washed twice with a mixed solution of water and ethanol, and dried for later use.

[0109] Dissolve ginsenoside powder in a 1:1 mass ratio with AuPt powder in a certain amount (e.g., 20 ml) of toluene, add 3-aminopropyltrimethoxysilane at 1 wt% relative to the solvent, seal and place at room temperature and stir with a magnetic bar for 24 hours to complete the modification. After the reaction is completed, centrifuge, wash twice with a mixed solution of water and ethanol, and dry for later use.

[0110] The modified AuPt is then activated by disuccinimidyl suberate: the AuPt powder obtained in step 2 is dissolved in a certain amount (e.g., 20 ml) of dimethyl sulfoxide solution, and a certain amount (e.g., 1 gram) of disuccinimidyl suberate is added, and the mixture is sealed and placed under magnetic stirring at room temperature for 1 hour, and then centrifuged and washed twice with a mixed solution of water and ethanol. The centrifugal washing here is mainly to remove the remaining disuccinimidyl suberate in the solution.

[0111] The activated AuPt powder and the modified ginsenoside powder obtained in step 2 are dissolved in a certain amount (eg, 50 ml) of dimethyl sulfoxide solution, sealed and placed under magnetic stirring at room temperature for 1 hour to couple them together, and finally centrifuged and washed to obtain a new anti-tumor drug (AuPt-Rg3).

[0112] Example 7

[0113] The ginsenosides in Example 1 were replaced with sea cucumber saponins, and the superatom cluster enzyme and sea cucumber saponins (SCG) were coupled by the same process to obtain a new anti-tumor drug (Au@WO x -SCG).

[0114] Example 8

[0115] The ginsenosides in Example 3 were replaced with sea cucumber saponins, and the superatom cluster enzyme and sea cucumber saponins (SCG) were coupled by the same process to obtain a new anti-tumor drug (Au@MnO x -SCG).

[0116] Example 9

[0117] The ginsenosides in Example 1 were replaced with quillaja saponins, and the superatom cluster enzyme and quillaja saponins (QSS) were coupled by the same process to obtain a novel anti-tumor drug (Au@WO x -QSS).

[0118] Example 10

[0119] The ginsenosides in Example 3 were replaced with quillaja saponins, and the superatom cluster enzyme and quillaja saponins (QSS) were coupled by the same process to obtain a novel anti-tumor drug (Au@MnO x -QSS).

[0120] Embodiment 11

[0121] The ginsenosides in Example 1 were replaced with paclitaxel, which also contains multiple hydroxyl groups. The superatom cluster enzyme and paclitaxel (PTX) were coupled by the same process to obtain a new anti-tumor drug (Au@WO x -PTX). Paclitaxel can inhibit the proliferation of cancer cells by inducing cell cycle arrest and inducing mitotic catastrophe, and has a good anti-tumor effect, especially for ovarian cancer, uterine cancer and breast cancer, which have a high incidence of cancer.

[0122] Example 12

[0123] The ginsenosides in Example 3 were replaced with paclitaxel, which also contains multiple hydroxyl groups. The superatom cluster enzyme and paclitaxel (PTX) were coupled by the same process to obtain a new anti-tumor drug (Au@MnO x -PTX).

[0124] Embodiment 13

[0125] The present invention uses Au@WO x Superatom cluster enzyme, taking ATZ as the coupled specific target molecule as an example to illustrate the process of the invention.

[0126] Au@WO was prepared by the method in Example 1. x Superatom cluster enzyme. x The superatom cluster enzyme was redispersed in anhydrous toluene solution, and 3-aminopropyltrimethoxysilane was added at 1 wt% relative to the solvent. The mixed solution was placed on a magnetic stirrer and magnetically stirred at room temperature for 24 hours. After stirring, it was transferred to a centrifuge tube for centrifugation and washing to finally obtain aminosilane-modified Au@WO x Superatom cluster enzyme. Dissolved in PBS to form 1mg / mL modified Au@WO x Solution.

[0127] Prepare ATZ solution with MES buffer to make it about 50mmol / mL. Take 20uMES buffer solution of 0.2mg / mL LEDC to 200μL ATZ solution. Add 20uMES buffer solution of 0.8mg / mL Sulfo-NHS to 1mL and mix at 4℃ for 20min to form solution A. Mix solution A and 10mL modified Au@WO x The solution mixture was used as solution B and incubated at 4°C overnight. After the incubation, 1 mL of lysine (concentration of 0.11 mg / mL) was added to solution B to terminate the reaction, and the final product Au@WO was obtained. x -ATZ.

[0128] Embodiment 14

[0129] The superatom cluster enzyme of Example 13 was replaced by Au@MnO x Superatom cluster enzymes to obtain new anti-tumor drugs (Au@MnO x -ATZ). Atezolizumab (ATZ) is a monoclonal antibody designed to bind to the PD-L1 protein. Atezolizumab binds to PD-L1 expressed on tumor cells and tumor-infiltrating immune cells, thereby preventing its interaction with PD-1 and B7.1 receptors. It currently has a good therapeutic effect in the treatment of liver cancer.

[0130] Embodiment 15

[0131] The present invention uses Au@WO x Superatom cluster enzyme, taking IGF1 as a coupled specific target molecule as an example to illustrate the process of the invention.

[0132] Au@WO was prepared by the method in Example 1. x Superatom cluster enzymes.

[0133] Au@WO x The superatom cluster enzyme was dispersed in anhydrous toluene solution, and 3-aminopropyltrimethoxysilane was added at 1.5 wt% relative to the solvent. The mixed solution was placed on a magnetic stirrer and magnetically stirred at room temperature for 24 hours. After stirring, it was transferred to a centrifuge tube and centrifuged at 10000r for 8 minutes, and washed by centrifugation 3 times to finally obtain the aminosilane-modified superatom cluster enzyme. It was resuspended in PBS to form 0.1 mg / mL modified Au@WO x The solution is ready for use.

[0134] Prepare IGF1 solution of about 50mmol / mL using MES buffer. Take 20uMES buffer solution of 0.2mg / mL LEDC to 200μL IGF1 solution. Add 20uMES buffer solution of 0.8mg / mL Sulfo-NHS to 1mL and mix at 4℃ for 20min to form solution A. Mix solution A and 10mL modified Au@WO x The solution mixture was used as solution B and incubated at 4°C overnight. After the incubation, 1 mL of lysine (concentration of 0.11 mg / mL) was added to solution B to terminate the reaction, and the final product Au@WO was obtained. x- IGF1.

[0135] Example 16

[0136] The superatom cluster enzyme of Example 15 was replaced by Au@MnO x Superatom cluster enzymes to obtain new anti-tumor drugs (Au@MnO x -IGF1). Insulin-like growth factor 1 (IGF1) is an endocrine hormone produced in the liver. In the presence of growth hormone, the production of IGF1 increases. There are many different types of cells in the body that are equipped with receptors that accept IGF1. This makes IGF1 a good protagonist in more autocrine cell signaling processes that target tissues to promote cell-to-cell communication (growth) or promote cell division. IGFL1 may be related to the regulatory process of multiple tumors. At present, IGFL1 is used in the treatment of tumor diseases mainly including Basal-like breast cancer (triple negative breast cancer), ovarian cancer, liver cancer, etc.

[0137] Embodiment 17

[0138] The present invention uses Au@WOx The process of the invention is illustrated by taking superatom cluster enzyme, ginsenoside as a special drug coupled, and ATZ as a specific target molecule coupled as an example.

[0139] Au@WO was prepared by the method in Example 1. x Superatom cluster enzyme. x The superatom cluster enzyme was dispersed in anhydrous toluene solution, and 1.5wt% of the surface modifier 3-aminopropyltrimethoxysilane relative to the solvent was added as mixed solution 1. At the same time, an equal mass of Rg3 was dispersed in anhydrous toluene solution, and 1.5wt% of the surface modifier 3-aminopropyltrimethoxysilane relative to the solvent was added as mixed solution 2. The mixed solution was placed on a magnetic stirrer and magnetically stirred at room temperature for 24 hours. After stirring, it was centrifuged and washed twice to finally obtain aminosilane-modified Au@WO x Superatom cluster enzyme and aminosilane-modified Rg3.

[0140] Modified Au@WO x The superatom cluster enzyme uses the crosslinker DSS activation reaction to activate the surface amino groups. The aminosilane-modified Au@WO is dissolved in DMSO (organic solvent) x Superatom cluster enzyme, and weigh an equal amount of DSS to add it. The mixture solution was magnetically stirred or incubated for 2 hours at room temperature, centrifuged and washed three times, and the precipitate was retained to obtain activated Au@WO x Superatom cluster enzyme, activated Au@WO x The powder and the modified ginsenoside powder obtained in step 2 were dissolved in a certain amount (e.g., 50 ml) of dimethyl sulfoxide solution, sealed and placed under magnetic stirring at room temperature for 1 hour to couple them together. After the stirring was completed, the mixture was transferred to a centrifuge tube and centrifuged at 10,000 r for 8 minutes, and washed by centrifugation 3 times to finally obtain Au@WO x -Rg3 superatom cluster enzyme drug. Resuspend in PBS to form 0.1mg / mL modified Au@WO x- The Rg3 solution was set aside. A solution with about 50 mmol / mL ATZ was prepared with MES buffer. 20 μL of 0.2 mg / mL EDC solution prepared with MES buffer was added to 200 μL ATZ solution. On this basis, 20 μL of 0.8 mg / mL Sulfo-NHS solution prepared with MES buffer was added and quantified to 1 mL. The mixture was mixed at 4°C for 20 min to form solution A. Solution A and 10 mL of modified Au@WO were mixed. x -Rg3 solution was mixed as solution B and incubated at 4°C overnight. After the incubation, 1 mL of 0.15 mg / mL lysine solution was added to solution B to terminate the reaction, and the final product Au@WO was obtained. x -Rg3-ATZ.

[0141] Embodiment 18

[0142] The present invention uses Au@WO x Superatom cluster enzyme, with ginsenoside as the special drug coupled, and IGF1 as the specific target molecule coupled, the preparation process is the same as Example 17, and the final product Au@WO is obtained x -Rg3-IGF1.

[0143] Embodiment 19

[0144] The present invention uses Au@MnO x Superatom cluster enzyme, with ginsenoside as the special drug coupled, and ATZ as the specific target molecule coupled, the preparation process is the same as Example 17, and the final product Au@MnO x -Rg3-ATZ.

[0145] Embodiment 20

[0146] The present invention uses Au@MnO x Superatom cluster enzyme, with paclitaxel as the special drug coupled, and IGF1 as the specific target molecule coupled, the preparation process is the same as Example 17, and the final product Au@MnO x -PTX-IGF1.

[0147] Embodiment 21

[0148] The present invention uses Au@WO x Superatom cluster enzyme, with paclitaxel as the special drug coupled, and IGF1 as the specific target molecule coupled, the preparation process is the same as Example 17, and the final product Au@WO is obtained x -PTX-IGF1.

[0149] Embodiment 22

[0150] The present invention uses Au@WO x Superatom cluster enzyme, with paclitaxel as the special drug coupled, and PDL1 as the specific target molecule coupled, the preparation process is the same as Example 17, and the final product Au@WO is obtained x -PTX-PDL1.

[0151] Embodiment 23

[0152] The present invention uses Au@MnO x Superatom cluster enzyme, with paclitaxel as the special drug coupled, and PDL1 as the specific target molecule coupled, the preparation process is the same as Example 17, and the final product Au@MnO x -PTX-PDL1.

Claims

1. A heterogeneous structure super-atom cluster enzyme and a heterogeneous structure super-atom cluster enzyme drug, characterized in that: Heterostructure superatom cluster enzyme drugs are prepared by modifying the surface of heterostructure superatom cluster enzymes with ligand groups to couple special drugs and / or specific targets; The heterogeneous structure superatom cluster enzyme is a heterogeneous structure superatom cluster enzyme composed of metal or metal atom cluster, ceramic atom cluster enzyme and / or metal compound atom cluster enzyme as units; Heterostructure superatom cluster enzyme drugs are constructed by coupling heterostructure superatom cluster enzymes with drugs or / and specific target molecules through surface-modified ligand groups.

2. A heterogeneous structure super-atom cluster enzyme and a heterogeneous structure super-atom cluster enzyme drug according to claim 1, characterized in that: The metal is selected from one or more elements of Au, Ag, Pt, Cu, Ru, Fe, Co, Ni, Gd, Bi, Sn, Mn, Ac, Sc, Y, or La series elements; the ceramic is selected from one or more ceramics of inorganic non-metallic materials such as Al2O3, ZrO2, HfO2, SiN, SiC, and ceramics of metal oxide materials; the metal compound is selected from metal sulfides / nitrides / borides / phosphides, etc., excluding metal oxides; the heterostructure superatom cluster enzyme at least includes metal atom clusters, such as heterojunctions of multi-metal atom cluster aggregates, core-shell structure heterojunctions with metal atom clusters as the core and ceramics or metal compound clusters as the shell, or gradient structure heterojunctions gradually transitioning from metal clusters to metal compound clusters.

3. The drug according to claim 1, characterized in that Superatomic cluster enzyme drugs have a specific spatial stereostructure: with the surface atoms of the heterogeneous superatomic cluster enzyme as the center or vertex, a topological structure similar to the spatial stereostructure of organic metal compounds is formed, such as tetrahedron, octahedron, icosahedron, pyramid rhombus, half pyramid, planar quadrilateral, pentagon and hexagon.

4. A heterogeneous structure super-atom cluster enzyme and a heterogeneous structure super-atom cluster enzyme drug according to claim 1, characterized in that: The size of the heterostructure superatom cluster is (i.e. 0.1 nm to 2 nm), more preferably (i.e. 0.5nm-1.5nm), the number of atoms is 2 to 300, and the optimal number is 2-150, or it has 0-3 shells.

5. The drug according to claim 1, characterized in that The surface modification ligand group of the superatom cluster enzyme is one or more of functional groups or zwitterionic compounds, wherein the functional groups are selected from hydroxyl, carboxyl, thiol, amine and amide groups; the zwitterionic compounds are selected from alkaloids (such as betaine), cystine, selenoic acid, selenocysteine ​​and 20 kinds of amino acids essential to the human body, preferably amino, hydroxyl, carboxyl, cysteine ​​and selenoic acid; The coupled special drug is selected from one or more of squalene, ginsenoside, paclitaxel, sea cucumber saponin, saponin, doxorubicin, ginkgo flavonoids, curcumin, polypeptide drugs, antibodies (such as mesothelin antibodies, V-ATPase antibodies, atezolizumab, insulin-like growth factor 1: IGF1), mRNA (such as mRNA-4157), iRNA (such as siRNA, miRNA), DNA (such as mtDNA), preferably ginsenoside, sea cucumber saponin, saponin, ginkgo flavonoids, PDL1, CD8, mRNA; The coupled specific target molecules are selected from: specific target molecules of specific cancers (such as lung cancer, liver cancer, pancreatic cancer, gastric cancer, colorectal cancer, breast cancer, etc.) (such as AKT target, EGFR target, NTRK target, GPC3 target, PD-1 / PD-L1 target, Claudin18.2 target, IGF1R target, MET target, UCK2 target, CD4 / 6 / 39 / 47 / 70 target, FGFR target and DDR1 target), and one or more of the related targeting substances targeting tumor microenvironment factors (such as pH value and temperature) (such as interpenetrating network biomacromolecule alloy substances composed of sodium alginate and modified chitosan, etc.).

6. The drug according to claim 1, characterized in that The heterogeneous structure superatomic cluster enzyme drug is a superatomic cluster enzyme drug constructed by coupling a heterogeneous structure superatomic cluster enzyme with a special drug through a surface-modified ligand group; or a superatomic cluster enzyme drug coupled with a heterogeneous structure superatomic cluster enzyme with a specific target through a surface-modified ligand group, or a superatomic cluster enzyme drug coupled with a special drug and a specific target through a surface-modified ligand group, namely, a superatomic cluster enzyme drug ②.

7. The method for preparing the heterogeneous structure super-atom cluster enzyme and the heterogeneous structure super-atom cluster enzyme drug according to any one of claims 1 to 6, characterized in that: The specific steps include: Step 1: Preparation of heterogeneous structure superatom cluster enzyme; Step 2: Heterostructure superatom cluster enzyme coupled with special drugs; (a) adding the heterogeneous structure superatom cluster enzyme prepared in step 1 immediately to an organic solution containing a surface modifier, stirring at room temperature to completely modify it, centrifuging after the reaction, and washing by centrifugation with a mixed solution of water and ethanol to obtain a surface-modified heterogeneous structure superatom cluster enzyme; at the same time, adding a special drug to an organic solution containing a surface modifier, stirring at room temperature to completely modify it, centrifuging after the reaction, and washing by centrifugation with a mixed solution of water and ethanol to obtain a surface-modified special drug; (b) dispersing the surface-modified heterogeneous structure superatom cluster enzyme and the cross-linking agent obtained in step (a) into an organic solvent, stirring and incubating, and then centrifuging and washing with a mixed solution of water and ethanol to obtain an activated heterogeneous structure superatom cluster enzyme for use; (c) dispersing or dissolving the activated heterogeneous structure superatom cluster enzyme obtained in step (b) and the special drug after surface modification in step (a) in an organic solvent, stirring at room temperature to couple them together, and then centrifugally washing with a mixed solution of water and ethanol and vacuum drying to obtain a superatom cluster enzyme drug of heterogeneous structure superatom cluster enzyme coupled with the special drug①; or / and step 3: conjugation to specific target (a) adding the heterogeneous structure superatom cluster enzyme prepared in step (1) or the superatom cluster enzyme drug ① prepared in step (2) to an organic solution containing a surface modifier, stirring at room temperature to completely modify it, centrifuging after the reaction, washing by centrifugation with a mixed solution of water and ethanol, and vacuum drying to obtain a surface-modified heterogeneous structure superatom cluster enzyme or a surface-modified superatom cluster enzyme drug ①; (b) dissolving the surface-modified heterogeneous superatom cluster enzyme or the surface-modified superatom cluster enzyme drug ① obtained in step (a) in PBS to prepare a solution A; (c) preparing a specific target solution and a condensing agent solution with MES buffer and mixing the two; preparing a crosslinking agent solution with MES buffer and then adding the crosslinking agent solution to the above solution, and the final mixed solution is solution B; (d) Mix solution A from step (b) and solution B from step (c) as solution C, and incubate at low temperature overnight; then dissolve the terminator in PBS to prepare a low concentration reaction terminator solution; add the reaction terminator solution to solution C to terminate the reaction to obtain the final product, and after centrifugal washing and vacuum drying, obtain a heterogeneous structure superatomic cluster enzyme-coupled superatomic cluster enzyme drug with a specific target or a heterogeneous structure superatomic cluster enzyme-coupled special drug and a superatomic cluster enzyme drug with a specific target, namely, superatomic cluster enzyme drug②.

8. The method according to claim 7, characterized in that Step 1: Preparation of heterostructure superatom cluster enzymes includes the following steps: (a) dissolving a metal salt corresponding to the metal in the heterostructure superatom cluster enzyme and a polymer dispersant in an organic solvent; the organic solvent is N-methylpyrrolidone, dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, dimethylacetamide, anhydrous toluene or water; wherein the metal salt is selected from one or more than two according to the required heterostructure, and when there are two, the ratio is 1:1 to 1:5, and when there are more than two, the ratio is adjusted according to the actual situation; if the heterostructure superatom cluster enzyme contains other elements besides metal and oxygen, a precursor compound of the corresponding element is also added at the same time, such as when containing SiN, a precursor of Si corresponding to tetraethyl orthosilicate, tetraethyl orthosilicate, etc. is added, and a precursor of N corresponding to N, such as ammonium salt, etc. is added; (b) dissolving a reducing agent in N-methylpyrrolidone, dimethyl sulfoxide, dimethylformamide, tetrahydrofuran or dimethylacetamide to obtain a reducing agent solution; wherein 0.1 g to 3.5 g of the reducing agent corresponds to 50 ml of the solvent; and the amount of the reducing agent used is 1.5 to 2 times the equivalent of the metal salt; (c) The reaction is prepared using a Y-type microfluidic reactor. The reaction solutions prepared in step (a) and step (b) are drawn out by a syringe and placed in a syringe pump system respectively. The two reaction solutions are combined through a Y-type connector to react, and a stably dispersed superatomic cluster enzyme solution is obtained through a gas-protected quenching collection device. The capillary length from the outlet of the Y-type connector to the quenching collection device is 80 cm, and the inner diameter d is 0.5-1.2 mm. The flow rate of the syringe pump is 9 ml / min-15 ml / min. The reaction temperature of the thermostat is set to 80-100°C. The quenching collection device is placed in an ice-water mixture or liquid nitrogen at 0°C, and the gas introduced is nitrogen, argon, carbon dioxide or air. The reaction time required for the reaction solution to reach the quenching collection device is strictly controlled, and the reaction is terminated by suddenly lowering the temperature to control the size of the heterogeneous structure superatomic cluster enzyme to be below 2 nm. The collected solution is then centrifuged, the supernatant is poured out, washed with ethanol and deionized water, and then dried using a freeze vacuum dryer to obtain a sample powder. The polymer material described above is selected from polyvinyl pyrrolidone and the like; If the heterogeneous structure superatomic cluster enzyme contains other elements besides metal and oxygen, and the corresponding heterogeneous structure superatomic cluster enzyme can be formed after the above freezing, then it is fine. If not, the sample powder obtained after cooling can be heated as needed (the heating temperature varies with the specific material as long as the corresponding compound can be formed) to form a compound of the corresponding element. After the reaction is completed, it can be frozen immediately or directly proceed to the next surface modification reaction.

9. The method according to claim 7, characterized in that The surface modifier is selected from at least one of 3-aminopropyltrimethoxysilane (APTMS), vinyltrimethoxysilane, 3-mercaptopropyltriethoxysilane, 3-aminopropyltriethoxysilane, N-(β-aminoethyl)-γ-aminopropyltrimethoxysilane, N-(β-aminoethyl)-γ-aminopropyltriethoxysilane, di(β-sulfur-α-aminopropionic acid), and L-α-amino-β-mercaptopropionic acid; preferably at least one of 3-aminopropyltrimethoxysiloxane, 3-mercaptopropyl-triethoxysilane and di(β-sulfur-α-aminopropionic acid); The cross-linking agent includes at least one of disuccinimidyl suberate (DSS), disuccinimidyl glutarate (DSG), polyethylene glycol-bis(succinimidyl succinate) (SS-PEG-SS), methoxypolyethylene glycol succinimidyl succinate (mPEG-SS), ethylene glycol bis(succinic acid N-hydroxysuccinimide ester) (EGS), N-hydroxysuccinimide (NHS), N-hydroxysulfosuccinimide (Sulfo-NHS), N-ethylmaleimide (NEM) and aziridine cross-linking agent XR-100; preferably at least one of DSS, SS-PEG-SS and NHS; The condensing agent is selected from carbodiimide condensing agents, such as at least one of dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI); The reaction terminator is selected from one or more of 2-mercaptoethanol, 20 kinds of amino acids (glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine), and tris(hydroxymethyl)aminomethane hydrochloride.

10. Use of the superatom cluster enzyme drug according to any one of claims 1 to 6 in the preparation of anticancer drugs that target and autonomously target tumor cells.

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