Bionic tetra-coordinated copper monatomic nano-enzyme as well as preparation method and function of bionic tetra-coordinated copper monatomic nano-enzyme

Abstract

The invention provides a bionic tetra-coordinated copper monatomic nano-enzyme. The structural characterization of the bionic tetra-coordinated copper monatomic nano-enzyme is as follows: the copper monatomic nano-enzyme is a nanosphere particle with a Keggin type structure; in the copper monatomic nano-enzyme, the loading capacity (wt) of copper monatomic is 0.5%-2.5%, and four coordination atoms are arranged on the periphery of each copper monatomic. The invention further provides a preparation method of the copper monatomic nano-enzyme. Experiments prove that the copper monatomic nano-enzyme has a good anti-oxidation effect, can inhibit cis-platinum induced hair cell damage so as to achieve the effect of protecting hair cells, and is a potential medicine for preventing and treating sensorineural deafness.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and mainly relates to a method for preparing a novel copper single-atom enzyme targeted drug and its function in repairing hair cell damage. Background Art

[0002] Hearing loss is one of the most common disabilities in the world today. According to a 2021 report by the World Health Organization, hearing loss currently affects more than 1.5 billion people, of which 430 million experience moderate or more severe hearing loss. There are currently 220 million people in my country suffering from varying degrees of hearing loss, and aging has accelerated the increase in the number of people with hearing impairment. Hearing loss not only seriously affects the quality of life of patients, but also brings a heavy burden to society. The global economic burden caused by hearing loss is as high as 750 billion US dollars each year. Therefore, the efficient prevention and treatment of hearing loss is not only conducive to improving national public health security, but also improves people's life and health and sustainable economic development.

[0003] Nearly 70% of the current hearing-impaired population suffers from sensorineural hearing loss. Cochlear hair cells are important components of auditory perception and conduction. Noise, ototoxic drugs (anti-tumor chemotherapy drugs, aminoglycoside antibiotics, etc.), heavy metals, and aging, among other factors, cause irreversible damage and loss of hair cells and neurons, which are important causes of sensorineural hearing loss. A large number of studies have shown that various damaging factors induce stress responses in hair cells and neurons, which in turn produce a large amount of reactive oxygen species (ROS), which is the main mechanism leading to hair cell damage. Preventing or delaying the excessive accumulation of ROS and efficiently removing ROS in cells are critical to the protection and repair of hair cells.

[0004] For example, patients treated with the anti-tumor drug cisplatin are prone to tinnitus and hearing loss. Over the past two decades, clinical trials have explored various repurposed drugs and new drug candidates to mitigate cisplatin-induced hearing loss. So far, most treatment options have failed to demonstrate efficacy in preclinical animal studies and randomized controlled clinical trials. Therefore, there is an urgent need to develop structurally clear, safe, effective, and intelligently controllable antioxidant drugs to target and protect hair cells.

[0005] Currently, there are very limited treatments for hearing loss. The introduction of antioxidant drugs for treatment has become one of the important treatments for sensorineural hearing loss. Although there have been many research results in the field of the pathogenesis and drug treatment of sensorineural hearing loss, the hearing protection effect of the antioxidant drugs used in these results is limited. The main reasons are: (1) The special physiological and anatomical characteristics of the inner ear seriously hinder the supply and absorption of drugs to cochlear target cells. Systemic administration is restricted by the blood-labyrinth barrier (BLB), which greatly reduces the utilization rate of drugs and limits the duration of drug action; (2) The drugs have poor targeting, making it difficult to effectively track the delivery and metabolic pathways. They are easy to degrade, resulting in a short retention time and inability to efficiently remove ROS in the inner ear; (3) The clinical stability and safety of most drugs still need to be further verified. Therefore, the development of new drugs with high targeting, visualized drug delivery pathways, safety and long-term effects is of great significance for the protection of auditory hair cells and the treatment of hearing loss.

[0006] Nanozymes have catalytic properties similar to those of natural enzymes. Unlike natural organic enzymes, which have defects such as high sensitivity to the reaction environment, low stability, and long separation and preparation time, nanozymes have higher enzyme activity, stability, and adjustability and are widely used in the biomedical field.

[0007] "Single-atom catalysis". In single-atom catalysts, metal atoms only interact electronically with the carrier. This strong interaction significantly modulates the electronic structure of the metal and the carrier, making it easy to control, easy to optimize and improve catalytic performance, simple and uniform structure, low cost and suitable for mass production.

[0008] Based on this, in response to the current lack of FDA-approved drugs for the prevention or treatment of sensorineural hearing loss, we selected metal ions with multivalent states and good catalytic activity, simulated the metal active center structure of natural antioxidant enzymes (superoxide dismutase, catalase, etc.), simulated the stable environment of metal atoms and nitrogen bases to form a four-coordinated copper single-atom nanozyme with antioxidant activity, safety and long-term effect. This copper single-atom nanozyme was used to protect hair cells and innovatively applied to the field of hearing protection. Summary of the invention

[0009] In view of the above technical problems, the present invention provides a bionic four-coordinated copper single-atom nanozyme and its preparation method and application. The specific technical solution is as follows:

[0010] The present invention provides a bionic four-coordinated copper single-atom nanozyme, whose structural characterization is as follows: the copper single-atom nanozyme is a nanosphere particle with a Keggin-type structure; in the copper single-atom nanozyme, the loading amount (wt) of the copper single atom is 0.5% to 2.5%, and each of the copper single atom has 4 coordinated atoms on the periphery.

[0011] Furthermore, the monomer structure of the copper single-atom nanozyme is based on the structure of Keggin-type silicotungstic acid as the supporting structure, and the copper single atom is loaded in the Keggin-type silicotungstic acid. The chemical structure of the monomer is Cu 0.25 Cs 2.5 HkDJ 12 O 40 , chemical name abbreviated as CuSTA.

[0012] The specific structure is characterized as follows: with 1 silicon atom as the central atom, 12 tungsten atoms are coordinated around the silicon atom, 5 to 6 oxygen atoms are coordinated around each tungsten atom and there are 40 coordinated oxygen atoms in total, the copper atom is loaded between the oxygen atoms and each copper atom is coordinated with 4 oxygen atoms around it.

[0013] The present invention also provides a method for preparing the above-mentioned biomimetic four-coordinated copper single-atom nanozyme, comprising the following steps:

[0014] S1: Keggin-type polyoxometalates (POMs) are used as copper single-atom support materials and are miscible with copper salts to form a four-coordinated stable environment around copper metal single atoms to form a copper single-atom nanozyme primary mixed solution;

[0015] S2: placing the copper single-atom nanozyme initial mixed solution in an ice bath and stirring, dripping an alkali metal salt solution while stirring, and continuing to incubate under ice bath and stirring conditions to convert the copper single-atom nanozyme initial mixed solution into a colloidal solution; centrifuging the colloidal solution to obtain solid I;

[0016] S3: After washing the solid I with deionized water, placing it in a deionized water solution environment, adding DSPE-PEG for sufficient reaction, adding A666 polypeptide, and the A666 polypeptide is combined with PEG through an amide bond; the solid after centrifugation of the above solution is washed with deionized water to obtain solid II, and the solid II is freeze-dried to finally obtain the copper single-atom nanozyme.

[0017] Preferably, the Keggin type polyoxometallate is selected from silicotungstic acid (H4SiW 12 O 40 , STA), phosphotungstic acid (H3PW 12 O 40 , PTA), silicomolybdic acid (H4SiMo12 O 40 , SMA), phosphomolybdic acid (H3PMo 12 O 40 , PMA)), etc., can be a mixture of one or more of them.

[0018] More preferably, the Keggin type polyoxometallate is silicotungstic acid (H4SiW 12 O 40 , STA).

[0019] Preferably, the copper salt is selected from: copper nitrate, copper sulfate, copper chloride, etc., and can be a mixture of one or more thereof.

[0020] Preferably, the alkali metal salt solution is a non-carbonate alkali metal salt selected from: cesium nitrate, cesium bromide, sulfuric acid, potassium nitrate, potassium bromide, potassium sulfate, sodium nitrate, sodium bromide, sodium sulfate, etc., and can be a mixture of one or more thereof.

[0021] More preferably, the alkali metal salt solution is a cesium salt, because the cesium salt can form a precipitate with POM and has a large specific surface area.

[0022] The present invention also provides the efficacy and application of the above-mentioned biomimetic four-coordinated copper single-atom nanozyme. The copper single-atom nanozyme has antioxidant properties. In a cisplatin environment, the copper single-atom nanozyme protects hair cells by resisting oxidative stress, has anti-ototoxicity, and can be used to prevent and treat sensorineural deafness.

[0023] The advantages of the present invention are:

[0024] (1) A copper single-atom nanozyme is provided, which is constructed by simulating the active center of natural antioxidant enzymes. The active center is the Cu-O4 site, which has similar properties and comparable activities to natural superoxide dismutase (SOD) and catalase (CAT). At the same time, the selected heteropolyacid carrier itself has a certain proton acidity, which can effectively promote the activity of CAT and synergistically exert an antioxidant therapeutic effect in hair cell damage. Compared with the current treatment strategy for hearing loss repair, the copper single-atom nanozyme has a better effect.

[0025] (2) Due to its surface electrical properties, copper single-atom nanozymes can be effectively connected to functionalized A666 targeting agents such as amino groups and dye molecules such as FITC, giving them better targeting and visualization effects. At the same time, the system is controlled to be a spherical material with a size less than 50 nm. The smaller size enables it to better cross the blood labyrinth barrier and effectively enter the hair cells of the inner ear. This high permeability enables it to play a role in the lesion site, and it has certain advantages in targeting and entering the hair cells, thereby improving the treatment efficiency.

[0026] (3) In addition, the selected carrier has a negatively charged surface, which can effectively combine with amino-functionalized targeting agents and fluorescent agents, giving them stronger targeting ability and a visualized delivery pathway.

[0027] (4) The constructed copper single-atom nanozyme can be degraded in the body. The nanozyme itself and its degradation products are non-toxic and harmless, with low toxicity and a certain degree of clinical safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1A This is the electrospray ionization time-of-flight mass spectrum of the mixture of silicotungstic acid and copper nitrate of Experimental Example 1;

[0029] Figure 1B This is the electrospray ionization time-of-flight mass spectrum of the mixture of phosphotungstic acid and copper nitrate of Experimental Example 1;

[0030] Figure 1C This is the electrospray ionization time-of-flight mass spectrum of the mixture of silicomolybdic acid and copper nitrate of Experimental Example 1;

[0031] Figure 1D This is the electrospray ionization time-of-flight mass spectrum of the mixture of phosphomolybdic acid and copper nitrate of Experimental Example 1;

[0032] Figure 1E The average particle size diagram of CuSTA and CuPTA obtained in Experimental Example 1;

[0033] Figure 1F ζ-potential diagram of CuSTA and CuPTA obtained in Experimental Example 1.

[0034] Figure 2A is a transmission electron microscope (TEM) image of CuSTA of Experimental Example 2;

[0035] Figure 2B is the EDX mapping image of the Cu element of Experimental Example 2;

[0036] Figure 2C is the EDX mapping image of W element in Experimental Example 2;

[0037] Figure 2D This is the XPS survey spectrum of CuSTA of Experimental Example 2;

[0038] Figure 2E This is the high-resolution Cu XPS 2p-level photoelectron spectrum of CuSTA of Experimental Example 2;

[0039] Figure 2F is the attenuated total reflection infrared spectrum (ATR-IR) of STA and CuSTA of Experimental Example 2;

[0040] Figure 2G is the model diagram of CuSTA of Experimental Example 2;

[0041] Figure 2H is the superoxide dismutase (SOD)-like activity of CuSTA at different copper loadings of Experimental Example 2;

[0042] Fig.2I The catalase-like (CAT) activity of CuSTA of Experimental Example 2 with a copper loading of 1.5 wt%.

[0043] Figure 3A The DCFH-DA probe staining and flow cytometry profile of the ROS kit of Experimental Example 3;

[0044] Figure 3B This is a data graph showing the results of identifying the ROS level in HEI-OC1 cells in Experimental Example 3;

[0045] Figure 3C This is a data graph showing the CAT enzyme activity detection results of CuSTA in HEI-OC1 cell model in Experimental Example 3;

[0046] Figure 3D This is a data graph showing the SOD enzyme activity detection results of CuSTA in HEI-OC1 cell model of Experimental Example 3;

[0047] Figure 3E This is a staining image of the Mito-sox Red reagent used in Experimental Example 3 to detect the level of ROS induced by mitochondria in mouse basal membrane hair cells;

[0048] Figure 3F This is a data graph of quantifying ROS levels using fluorescence detection in Experimental Example 3;

[0049] Figure 3G This is a data graph showing the results of the CuSTA SOD enzyme activity test in the mouse basement membrane model of Experimental Example 3;

[0050] Figure 3H This is a data graph showing the results of CAT enzyme activity detection by CuSTA in the mouse basement membrane model of Experimental Example 3.

[0051] Figure 4A The cell viability assay of HEI-OC1 cells incubated with 30 μM cisplatin and different concentrations of CuSTA in Experimental Example 4;

[0052] Figure 4B Quantitative analysis of relative fluorescence intensity of live / dead staining of HEI-OC1 cells in Experimental Example 4;

[0053] Figure 4C Live / dead cell staining images of HEI-OC1 cells treated with different control conditions in Experimental Example 4;

[0054] Figure 4DThe morphology and number of HEI-OC1 cells under different control conditions of Experimental Example 4;

[0055] Figure 4E The immunofluorescence staining images of the apical gyrus (APEX), middle gyrus (MIDDLE) and basal gyrus (BASE) of the mouse basilar membrane cochlear hair cells of Experimental Example 4 are shown;

[0056] Figure 4F This is the data analysis of surviving hair cells under different control conditions of Experimental Example 4.

[0057] Figure 5A This is a schematic diagram of the experiment of CuSTA treatment of mice in vivo in Experimental Example 5;

[0058] Figure 5B The changes in hearing threshold of C57BL / 6J mice after different treatments in Experimental Example 5;

[0059] Figure 5C This is the immunofluorescence staining of the apical gyrus (APEX), middle gyrus (MIDDLE) and basal gyrus (BASE) of the mouse cochlear hair cells of Experimental Example 5;

[0060] Figure 5D The statistical analysis results of the surviving hair cells in Experimental Example 5 are shown in FIG. DETAILED DESCRIPTION

[0061] The following test examples and embodiments of the present invention are given to specifically illustrate the schemes that can be implemented in the present invention, but the protection scope of the present invention is not limited to the description of the specific embodiments. Technical structures with the same or similar functions as the present invention obtained by other equivalent technical means or equivalent replacement should also fall within the protection scope of the present invention.

[0062] In the following test examples, the text explanations in the figures are as follows: CTR is the negative control group; CIS is the injury group (only cisplatin is added); CuSTA is the drug control group (only CuSTA is added); CuSTA+CIS is the drug treatment group (CuSTA and cisplatin co-treatment group).

[0063] Test Example 1:

[0064] This test example is to screen out POMs that can effectively stabilize copper cations.

[0065] Experimental method: First, a series of stable Keggin-type polyoxometalates (POMs), silicotungstic acid (H4SiW 12 O 40 , STA), phosphotungstic acid (H3PW 12 O 40 , PTA), silicomolybdic acid (H4SiMo 12 O 40, SMA), phosphomolybdic acid (H3PMo 12 O 40 , PMA)) was mixed with copper nitrate (Cu(NO3)2) and after mixing for 10 minutes, the electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) spectrum was collected. The results are shown in Figure 1A-Figure 1D shown.

[0066] Then, the mixing time was extended to 5 hours to form a large amount of [Cu+POM] complexes, and cesium salt was added to form nanospheres composed of these complexes, and this process was kept in an ice bath. Dynamic light scattering technology was used to examine the size in the solution phase. The results are shown in Figure 1E-1F shown.

[0067] Results analysis: (1) Figure 1A-Figure 1D As shown in Figure 2, when interacting with two tungsten-containing POMs, the [Cu+POM] complex is more significantly formed, and the molar ratio of copper to POM ideally reaches 1:1. It is worth noting that when reacting with copper nitrate solution, PTA decomposes into PW 11 O 385 -. (2) Figure 1E As shown in Figure 2, the average hydrodynamic diameter of CuSTA is only 89 nanometers, which is much smaller than that of CuPTA (~150 nanometers). Figure 1F As shown in Figure 2, the zeta potential of CuSTA is less negative than that of CuPTA, which indicates that Cu2+ forms a stable coordination with STA. Therefore, based on the above screening, CuSTA was selected as the POM for the copper center.

[0068] Example:

[0069] This example is the preparation of copper single-atom nanozyme, and the specific steps are as follows:

[0070] S1: 32 mg of copper nitrate powder (Cu(NO3)2) and 2.16 g of tungstosilicic acid powder (H4SiW12O40, STA) were mixed and dissolved in 30 mL of deionized water, and stirred for 30 min under ice bath conditions to form a preliminary copper single-atom nanozyme pre-mixed solution;

[0071] S2: placing the copper single-atom nanozyme premixed solution in an ice bath and stirring; dissolving 0.32 g of cesium chloride in 30 mL of deionized water, and then injecting it into the copper single-atom nanozyme premixed solution at a rate of 1 mL / min, and continuing to incubate for 4 hours under ice bath and stirring conditions to turn the copper single-atom nanozyme premixed solution into a colloidal solution; centrifuging the colloidal solution at 7000 rpm for 4 minutes to obtain a bluish-white solid Ⅰ;

[0072] S3: After washing the solid I with deionized water for 3 times, a blue solid II is obtained; 5 mg of DSPE-PEG is added to the obtained blue solid II in a deionized water solution environment, and after sufficient reaction for 2 hours, an A666 polypeptide is added; the A666 polypeptide is combined with PEG through an amide bond, and then connected to CuSTA; the obtained CuSTA modified with A666 targeting peptide is dried by a freeze dryer to finally form the copper single-atom nanozyme (CuSTA).

[0073] Test Example 2:

[0074] This test example is to characterize the copper single atom nanozyme (CuSTA) obtained in the example, and the specific method and results are as follows:

[0075] Firstly, the morphology and particle size of CuSTA were studied and analyzed using transmission electron microscopy (TEM) and energy dispersive X-ray fluorescence spectrometer (EDX). Figure 2A As shown, the size of the final CuSTA was about 90 nm.

[0076] Energy dispersive x-ray spectroscopy (EDX) elemental mapping was performed in the same area. The results are shown in Figure 2B-2C As shown, it indicates that Cu and STA are well mixed and form a good distribution.

[0077] X-ray photoelectron spectroscopy (XPS) was used to identify the electronic structure and chemical composition of CuSTA. Figure 2D As shown in Figure 2, the coexistence of Cu, STA and Cs in the nanospheres is demonstrated. Figure 2E It is shown that Cu in CuSTA exists in the +2 valence state, which is confirmed by the satellite peaks and the binding energy of 933.7 eV.

[0078] The formation of Cu-O bonds was further confirmed by attenuated total reflectance infrared spectroscopy (ATR-IR), as shown in Figure 2F As shown, located at 870cm -1 .

[0079] like Figure 2G This is the model diagram of CuSTA.

[0080] CuSTA was detected by catalase assay kit (KGA7312-100, KeyGEN) and superoxide dismutase assay kit (S0101S, Beyotime), respectively. Figure 2H-Figure 2I As shown, CuSTA exhibited superior CAT- and SOD-like enzyme activities in vitro.

[0081] Test Example 3:

[0082] This test example is an analysis of the antioxidant properties of the copper single-atom nanozyme (CuSTA) obtained in the example:

[0083] In order to verify the antioxidant properties of the optimized CuSTA, this case used HEI-OC1 (House Ear Institute Organ of Corti l cell, HEI-OC1) cells and C57BL / 6J mouse basement membrane to construct an in vitro model of cisplatin damage, and identified the antioxidant activity of CuSTA in the field of auditory cell protection.

[0084] 30 μM cisplatin was selected to construct the hair cell injury model. Before cisplatin injury, the cells were pretreated with 2 μg / mL CuSTA, and then the hair cells were co-treated with 30 μM cisplatin and 2 μg / mL CuSTA for 24 h. The antioxidant properties of CuSTA were then tested using ROS detection reagent, CAT detection kit, and SOD detection kit, respectively. The results are shown in Figure 3A-3B As shown in Figure 2, DCFH-DA probe staining and flow cytometry results showed that in the HEI-OC1 cell model, CuSTA could reduce the accumulation of ROS induced by cisplatin, thereby protecting hair cells. Figure 3C-Figure 3D As shown, we also used CAT detection kit and SOD detection kit to prove that CuSTA has antioxidant enzyme activity in in vitro cell models.

[0085] In the mouse basement membrane, we selected basement membrane cells for in vitro culture and used cisplatin to induce an in vitro hair cell injury model. According to the results of Mitosox-Red staining, Figure 3E-Figure 3F As shown in Figure 2, after treatment with CuSTA, compared with the cisplatin-only treatment group, the CuSTA was able to reduce the accumulation of mitochondrial-induced ROS. Figure 3G-3H As shown, CuSTA also exhibited good CAT and SOD enzyme activities in mouse basal membrane cells.

[0086] In summary, the CuSTA used in this case showed good antioxidant properties in both the HEI-OC1 cell and C57BL / 6J mouse basement membrane injury models, proving that the material can protect hair cells from damage by resisting oxidative stress.

[0087] Test example 4:

[0088] In this test example, the copper single-atom nanozyme (CuSTA) obtained in the example was tested in an in vitro model induced by cisplatin to test whether CuSTA has the effect of protecting hair cells.

[0089] In this case, HEI-OC1 cells and mouse basement membrane cells were selected to construct an in vitro model of cisplatin injury, and 30μM cisplatin was selected to construct a hair cell injury model. Before cisplatin injury, we pretreated the cells with different concentrations of CuSTA for 2h. The different concentrations of CuSTA were 0.5μg / mL, 1μg / mL, 2μg / mL, 5μg / mL, 10μg / mL, 25μg / mL, 50μg / mL, and 100μg / mL; then, 30μM cisplatin and corresponding different concentrations of CuSTA were used to co-treat HEI-OC1 cells and mouse basement membrane cells for 24h. The results are shown in Figure 4A As shown, at a concentration of 2 μg / mL, CuSTA exhibited the most effective protective effect, increasing the survival rate of HEI-OC1 cells treated with 30 μM cisplatin to approximately 80%.

[0090] In addition, Calcein-AM (green) and PI (red) double staining and microscopic observation showed that Figure 4B-4D As shown, at a concentration of 2 μg / mL, the cell viability of the CuSTA-treated group was the best, and its cell morphology and number were significantly better than those of the cisplatin-only-treated group.

[0091] In vitro cochlear culture was used to observe the protective effect of CuSTA on cisplatin-damaged cochlear hair cells. The in vitro cochlear basement membrane was cultured overnight in DMEM / F12 culture medium supplemented with 2% B27, 1% N-2 and 50ug / m LAMP. The incubator conditions were 37°C, 5% CO2; on the second day, CuSTA was added to the drug protection group for pretreatment for 2 hours, and then 30μM cisplatin was added to the drug protection group and cisplatin damage group for treatment for 24 hours; the CTR group (control group) did not receive additional treatment. After 24 hours of injury, the culture medium was removed, and normal tissue culture medium was added to continue culturing for 48 hours, and then subsequent biological identification experiments were performed on the basement membrane. The results are shown in Figure 4E-4F As shown, compared with the control group, the loss of hair cells in various regions of the cochlea was significantly reduced after 24 hours of treatment with 30 μM cisplatin. CuSTA can significantly alleviate cisplatin-induced hair cell loss, and the protective effect is most significant at a concentration of 5 μg / mL.

[0092] The above experiments demonstrated that CuSTA has a good protective effect on hair cells in the cisplatin injury model in vitro.

[0093] Test Example 5:

[0094] In this test example, the copper single-atom nanozyme (CuSTA) obtained in the example was tested in a cisplatin-induced in vivo injury model to see whether it has the effect of protecting hair cells.

[0095] To study the effect of CuSTA in vivo, we used the cisplatin injury model of C57BL / 6J mice for verification. The specific experimental flow chart is shown in the figure. Figure 5A As shown. Before each treatment, all mice were tested for auditory brainstem response (ABR) to ensure the maintenance of normal hearing ability. Then the mice were divided into a control group, a cisplatin-only treatment group, a CuSTA control group, and a cisplatin and CuSTA co-treatment group for experimental treatment. Figure 5B As shown, after exposure to cisplatin, the auditory brainstem response (ABR) thresholds of the cisplatin-alone treated group showed significant hearing loss, while CuSTA treatment restored the cisplatin-induced hearing loss in mice. Notably, no significant difference was detected in the group treated with CuSTA alone, suggesting that CuSTA does not affect hair cell death in vivo. Figure 5C-5D As shown, myosin VIIa staining of hair cells showed that cisplatin caused significant damage to OHCs, among which the basal gyrus was most severely damaged. The above experiments demonstrated that CuSTA has an anti-ototoxic effect on cisplatin-induced hearing loss.

Claims

1. A biomimetic four-coordinated copper single-atom nanozyme, characterized in that: The copper single-atom nanozyme is a nanosphere particle with a Keggin-type structure; in the copper single-atom nanozyme, the loading amount (wt) of the copper single atom is 0.5% to 2.5%, and each of the copper single atoms has 4 coordinated atoms on the periphery.

2. A biomimetic four-coordinated copper single-atom nanozyme as claimed in claim 1, characterized in that: The monomer structure of the copper single-atom nanozyme is based on the structure of Keggin-type silicotungstic acid as a supporting structure, and copper single atoms are loaded in the Keggin-type silicotungstic acid; The chemical structure of its monomer is Cu 0.25 Cs 2.5 HkDJ 12 O 40 , its chemical name is abbreviated as CuSTA. .

3. A biomimetic four-coordinated copper single-atom nanozyme as claimed in claim 1, characterized in that: The particle size of the copper single-atom nanozyme is ≤50nm.

4. A biomimetic four-coordinated copper single-atom nanozyme as claimed in claim 2, characterized in that: The structure of the copper single-atom nanozyme is that a silicon atom is used as the central atom, 12 tungsten atoms are coordinated around the silicon atom, 5 to 6 oxygen atoms are coordinated around each of the tungsten atoms, and there are 40 coordinated oxygen atoms in total. The copper single atom is loaded between the oxygen atoms, and each of the copper single atom is coordinated with 4 oxygen atoms.

5. A method for preparing a biomimetic four-coordinated copper single-atom nanozyme as claimed in claim 1, characterized in that: The following steps are involved: S1: Keggin-type polyoxometalate is used as the copper single atom support material, which is miscible with copper salt to form a four-coordinated stable environment around the copper metal single atom to form a copper single atom nanozyme initial mixed solution; S2: placing the copper single-atom nanozyme initial mixed solution in an ice bath and stirring, dripping an alkali metal salt solution while stirring, and continuing to incubate under ice bath and stirring conditions to convert the copper single-atom nanozyme initial mixed solution into a colloidal solution; centrifuging the colloidal solution to obtain solid I; S3: After washing the solid I with deionized water, placing it in a deionized water solution environment, adding DSPE-PEG for sufficient reaction, adding A666 polypeptide, and the A666 polypeptide is combined with PEG through an amide bond; the solid after centrifugation of the above solution is washed with deionized water to obtain solid II, and the solid II is freeze-dried to finally obtain the copper single-atom nanozyme.

6. The method for preparing a biomimetic four-coordinated copper single-atom nanozyme according to claim 5, characterized in that: The Keggin type polyoxometallate is selected from silicotungstic acid (H4SiW 12 O 40 , STA), phosphotungstic acid (H3PW 12 O 40 , PTA), silicomolybdic acid (H4SiMo 12 O 40 , SMA), phosphomolybdic acid (H3PMo 12 O 40 , PMA)).

7. The method for preparing a biomimetic four-coordinated copper single-atom nanozyme according to claim 5, characterized in that: The alkali metal salt solution is a non-carbonate cesium salt.

8. The function of the bionic tetracoordinated copper single-atom nanozyme according to any one of claims 1 to 4.

9. The function of a biomimetic four-coordinated copper single-atom nanozyme as claimed in claim 8, characterized in that: The copper single-atom nanozyme has antioxidant properties.

10. The function of a biomimetic four-coordinated copper single-atom nanozyme as claimed in claim 8, characterized in that: In the environment of cisplatin, the copper single-atom nanozyme protects hair cells by resisting oxidative stress and is used to treat sensorineural deafness.

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