Photo-controlled nitric oxide releasing hollow single-atom nanoszyme, preparation method and application

Abstract

The application discloses a light-controlled nitric oxide releasing hollow single-atom nanoscale enzyme, a preparation method and application, and belongs to the technical field of biomedical materials. In the application, spherical silica nanoparticles are used as a template, and a structure of copper ion-polydopamine coated silica nanoparticles is formed on the surface of the silica nanoparticles through self-polymerization of dopamine and chelation of copper ions, and the spherical structure is maintained with the support of the silica nanoparticles. The copper ion-polydopamine coated silica nanoparticles are pyrolyzed to obtain a copper-based single-atom nanoscale enzyme with a carbon skeleton, and a hollow spherical single-atom nanoscale enzyme is obtained after etching. Nitric oxide donor molecules are loaded into the hollow of the hollow spherical single-atom nanoscale enzyme to obtain the light-controlled nitric oxide releasing hollow single-atom nanoscale enzyme. The application can realize the synergistic treatment effect of photothermal therapy, nitric oxide gas therapy and biological catalytic therapy, is used for efficiently removing bacteria and biofilms at an infected wound site, and effectively promotes the healing of the bacteria-infected wound.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biomedical materials, and particularly relates to a light-controlled hollow single-atom nanoscale enzyme for releasing nitric oxide, a preparation method and application. BACKGROUND

[0002] Bacterial infection diseases seriously threaten human life and health, and antibiotics are the most commonly used and effective drugs for treating bacterial infection diseases. However, due to problems such as antibiotic resistance, it is urgent to develop nanomaterials with excellent antibacterial activity and novel antibacterial mechanism to achieve more efficient antibacterial strategies, thereby maximizing the efficacy of bacterial infection diseases and reducing the side effects of treatment.

[0003] In recent years, gas therapy has attracted widespread attention from researchers in the fields of antibiosis and antitumor biomedical applications due to its green, safe and efficient advantages. Nitric oxide (NO) gas is closely related to the healing of bacterial infection wounds and plays a crucial role in regulating inflammatory responses, angiogenesis and cell metabolism. NO can make intracellular DNA and RNA lose biological activity by oxidation, affect the process of protein synthesis, and directly lead to the death of bacteria. However, as a highly active gas molecule, how to efficiently load and effectively control the release of NO and maximize its antibacterial performance has become the key to the development of current NO antibacterial materials. At present, a variety of nanomaterials have been designed and synthesized for the delivery and controlled release of NO. These nanosystems can achieve the controlled release of NO using exogenous or endogenous stimuli, such as X-rays, ultrasound, ultraviolet light, near-infrared light, pH, temperature, GSH and H2O2. Compared with endogenous stimuli, exogenous stimuli are more conducive to achieving the controlled release of gas. Among them, light-controlled release nanomaterials with near-infrared response performance are considered as ideal materials for the controlled release of NO. However, single NO therapy is insufficient to achieve the ideal antibacterial effect. SUMMARY

[0004] To solve the above problems, the application provides a light-controlled hollow single-atom nanoscale enzyme for releasing nitric oxide, a preparation method and application, to improve the antibacterial effect.

[0005] The application is achieved by the following technical solutions:

[0006] The application discloses a hollow single-atom nanoscale enzyme for releasing nitric oxide under light control, and relates to the technical field of nanoscale enzyme.

[0007] Preferably, the preparation method of the hollow single-atom nanoscale enzyme for releasing nitric oxide under light control comprises the following steps.

[0008] (1) mixing hydrochloric acid dopamine, spherical silica nanoparticles and copper chloride dihydrate to form a copper ion-polydopamine coated silica nanoparticle structure on the surface of the spherical silica nanoparticles through self-polymerization of dopamine and chelation of copper ions; centrifuging, washing and collecting the precipitate to obtain the copper ion-polydopamine coated silica nanoparticles; the mass ratio of the hydrochloric acid dopamine, the spherical silica nanoparticles and the copper chloride dihydrate is 10-20:3-10:1-5.

[0009] (2) freeze-drying the copper ion-polydopamine coated silica nanoparticles obtained in the step (1) and pyrolyzing the same in a pyrolysis environment to obtain a copper-based single-atom nanoscale enzyme with a carbon skeleton;

[0010] (3) etching the copper-based single-atom nanoscale enzyme obtained in the step (2) for 12-24 hours using an etching solution, and centrifuging and washing the same to obtain a hollow spherical single-atom nanoscale enzyme; the volume-mass ratio of the etching solution to the copper-based single-atom nanoscale enzyme is 1-5:10-20.

[0011] (4) freeze-drying the hollow spherical single-atom nanoscale enzyme obtained in the step (3), resuspending the same for the first time, adding a nitric oxide donor molecule into the hollow spherical single-atom nanoscale enzyme, and loading the nitric oxide donor molecule into the hollow of the hollow spherical single-atom nanoscale enzyme; centrifuging, washing and resuspending the same for the second time to obtain a hollow single-atom nanoscale enzyme for releasing nitric oxide under light control.

[0012] Preferably, the pyrolysis environment is as follows: first, raising the temperature to 340-360 DEG C under a nitrogen atmosphere and keeping the temperature for 2.5-3.5 hours; and then, raising the temperature to 790-810 DEG C and calcining and pyrolyzing for 1.5-2.5 hours.

[0013] Preferably, the etching solution is obtained by mixing hydrofluoric acid and ammonium fluoride in a volume-mass ratio of 1-5:2-10.

[0014] Preferably, the concentration of the spherical silica nanoparticles is 100 mg / mL to 200 mg / mL; the concentration of the copper-based single-atom nanozyme is 100 mg / mL to 200 mg / mL; and the concentration of the nitric oxide donor is 1 mg / mL to 5 mg / mL.

[0015] Preferably, the mixing reaction condition is a room-temperature alkaline condition, which is provided by a Tris buffer with a concentration of 9 mM to 11 mM and a pH of 8.7 to 8.9; and the room temperature is 25℃.

[0016] Preferably, the first reagent for resuspension is a mixed solution of pure water and anhydrous ethanol, the pure water and the anhydrous ethanol are in a volume ratio of 1 to 1.5:1, and the second reagent for resuspension is a PBS buffer with a pH of 7.2 to 7.6.

[0017] Preferably, the spherical silica nanoparticles are prepared by the following method:

[0018] The anhydrous ethanol, pure water, and ammonia water are mixed, tetraethyl orthosilicate is added, and the mixture is reacted at 45℃ to 60℃ for 2 h to 3 h, followed by centrifugal washing to collect the precipitate to obtain the spherical silica nanoparticles; the volume ratio of the anhydrous ethanol, pure water, and ammonia water is 90 to 100:10 to 20:2 to 12; and the volume ratio of the tetraethyl orthosilicate to the ammonia water is 1.0 to 2.0:1.0.

[0019] Preferably, the nitric oxide donor molecule includes at least one of nitroprusside, S-nitrosoglutathione, diol diazeniumdioxide, and N,N'-di-sec-butyl-N,N'-dinitroso-1,4-phenylenediamine.

[0020] The application of the light-controlled nitric oxide releasing hollow single-atom nanozyme in the preparation of biomedical materials.

[0021] The present application provides a hollow single-atom nanoscale enzyme for light-controlled nitric oxide release, which is prepared by using spherical silica nanoparticles as a template, forming a copper ion-polydopamine coated silica nanoparticle structure on the surface of the silica nanoparticles through self-polymerization of dopamine and chelation of copper ions, and maintaining the spherical structure with the support of the silica nanoparticles; pyrolyzing the copper ion-polydopamine coated silica nanoparticles to obtain a copper-based single-atom nanoscale enzyme with a carbon skeleton, and etching to obtain a hollow spherical single-atom nanoscale enzyme; loading a nitric oxide donor molecule into the hollow of the hollow spherical single-atom nanoscale enzyme to obtain a hollow single-atom nanoscale enzyme for light-controlled nitric oxide release. The hollow single-atom nanoscale enzyme for light-controlled nitric oxide release loaded with the nitric oxide donor molecule can realize the controlled release of nitric oxide under near-infrared II (NIR II) laser irradiation due to the excellent photothermal performance of the carbon-based carrier in the NIR II region, and can simultaneously generate nitric oxide and O2 ·- under NIR II laser irradiation, and then the two substances undergo a cascade reaction to generate peroxynitrite anion (ONOO - ), thereby enhancing the antibacterial treatment effect of the nanosystem.

[0022] In addition, the hollow spherical single-atom nanoscale enzyme for loading the nitric oxide donor molecule in the preparation process of the present application has excellent oxygenase-like (OXD) activity, and can generate O2 ·- through catalysis. Moreover, under NIR II laser irradiation, the performance of the hollow spherical single-atom nanoscale enzyme for catalyzing the generation of O2 ·- is greatly improved.

[0023] Therefore, the hollow single-atom nanoscale enzyme for light-controlled nitric oxide release of the present application can realize the synergistic treatment effect of photothermal therapy, nitric oxide gas therapy and biological catalytic therapy, and can be used for efficiently removing bacteria and biofilms in infected wound sites and effectively promoting the healing of bacterial infected wounds. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the following embodiment or prior art description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0025] Figure 1 The transmission electron microscope image of the hollow spherical single-atom nanoscale enzyme (Cu HSAz) obtained in Example 1.

[0026] Figure 2 The aberration-corrected high-angle annular dark-field scanning transmission electron microscope image of the Cu HSAz obtained in Example 1.

[0027] Figure 2 In the figure, A is the aberration-corrected high-angle annular dark-field scanning transmission electron microscopy image of Cu HSAz at 100 nm; B is the aberration-corrected high-angle annular dark-field scanning transmission electron microscopy image of Cu HSAz at 5 nm.

[0028] Figure 3 The Cu HSAz particle size distribution chart obtained in Example 1.

[0029] Figure 4 The X-ray powder diffraction chart and the X-ray photoelectron spectroscopy chart of Cu HSAz obtained in Example 1.

[0030] Figure 4 In the figure, A is the X-ray diffraction chart of Cu HSAz; B is the XPS Cu2p3 / 2 spectrum chart of Cu HSAz; C is the N1s spectrum chart of Cu HSAz; D is the C1s spectrum chart of Cu HSAz;

[0031] Figure 5 The ultraviolet-near-infrared-visible absorption spectrum chart of Cu HSAz@BNN6 obtained in Example 1, wherein the abscissa is the absorption wavelength (Wavelength), and the ordinate is the absorbance (Absorbance).

[0032] Figure 6 The temperature-time curve chart of Cu HSAz@BNN6 with different concentrations after irradiation by different laser powers.

[0033] Figure 6 In the figure, A is the infrared thermal imaging chart of Cu HSAz@BNN6; B is the time-temperature curve chart of Cu HSAz@BNN6 with different powers; C is the time-temperature curve chart of Cu HSAz@BNN6 with different concentrations; D is the photothermal stability curve chart of Cu HSAz@BNN6.

[0034] In the figure, A is the infrared thermal imaging chart of Cu HSAz@BNN6; B is the time-temperature curve chart of Cu HSAz@BNN6 with different powers; C is the time-temperature curve chart of Cu HSAz@BNN6 with different concentrations; D is the photothermal stability curve chart of Cu HSAz@BNN6. Figure 6 In the figure, B, C, and D, the abscissa is time (Time), and the ordinate is temperature (Temperature).

[0035] Figure 7 The NO release result chart of 200 μg / mL Cu HSAz@BNN6 after irradiation by 0.5 W / cm 2 power.

[0036] Figure 7 In the figure, A is the nitric oxide release concentration curve chart with or without laser irradiation; B is the laser-controlled NO release “on-off” curve chart.

[0037] Wherein the abscissa is time (Time), the ordinate is the concentration of nitric oxide release (NO release concentration).

[0038] Figure 8 The results of Cu HSAz@BNN6 superoxide anion (O2 ·- release under the condition of laser irradiation or not, and the results of different materials catalyzing the generation of peroxynitrite RNS.

[0039] Figure 8 A is the time concentration curve of Cu HSAz@BNN6 releasing O2 ·- , B is the ESR spectrum of O2 ·- , and C is the fluorescence intensity diagram of DHR123 probe studying the generation of RNS catalyzed by different materials.

[0040] Wherein Figure 8 A in the abscissa is time (Time), the ordinate is the concentration of superoxide anion (O2 ·- concentration), Figure 8 B in the abscissa is magnetic field (Magnetic Field), the ordinate is intensity (Intensity), Figure 8 C in the abscissa is fluorescence wavelength (Wavelength), the ordinate is fluorescence intensity (Fluorescent intensity).

[0041] Figure 9 The antibacterial effect diagram of different material treatment on S. aureus, E. coli and MRSA.

[0042] Figure 9 A in the diagram is the antibacterial effect of different material treatment on S. aureus, E. coli and MRSA; B is the survival rate diagram of MRSA bacteria; C is the survival rate diagram of E. coli bacteria; D is the survival rate diagram of S. aureus bacteria.

[0043] Figure 10 The crystal violet staining diagram of MRSA bacterial biofilm treated by different materials and different concentrations of Cu HSAz@BNN6.

[0044] Figure 10In the figure, A is the crystal violet staining diagram of MRSA bacterial biofilm treated by different materials; B is the crystal violet staining diagram of MRSA bacterial biofilm treated by different concentrations of CuHSAz@BNN6; C is the crystal violet absorbance analysis diagram of MRSA bacterial biofilm treated by different materials; D is the crystal violet absorbance analysis diagram of MRSA bacterial biofilm treated by different concentrations of CuHSAz@BNN6.

[0045] wherein Figure 10 In the figure, the abscissa of A, B, C and D is different materials and different concentrations of CuHSAz@BNN6, and the ordinate is the crystal violet absorbance.

[0046] Figure 11 In the figure, A is the wound diagram of rats treated by different materials on different days; B is the wound area diagram of rats; wherein

[0047] Figure 11 In the figure, A is the wound diagram of rats treated by different materials on different days; B is the wound area diagram of rats; wherein Figure 11 In the figure, the abscissa of A and B is the treatment day (Day), Figure 11 The ordinate of B is the relative wound area (Relative wound area). DETAILED DESCRIPTION

[0048] In order to facilitate the understanding of the present application, the present application will be described more fully below, and the preferred embodiments of the present application will be given. However, the present application can be realized in many different forms, and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present application more thorough and comprehensive.

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. The terminology used in the specification of the present application herein is only for the purpose of describing specific embodiments and is not intended to limit the present application.

[0050] The inventive concept of the present application is as follows:

[0051] At present, a variety of nanomaterials have been designed and synthesized for the delivery and controlled release of NO. These nanosystems can achieve controlled release of NO using exogenous or endogenous stimulation conditions, such as X-rays, ultrasound, ultraviolet light, near-infrared light, pH, temperature, GSH and H2O2, etc. Compared with endogenous stimulation, exogenous stimulation is more conducive to achieving controlled release of gas. Among them, light-controlled release nanomaterials with near-infrared response performance are considered as ideal materials for controlled release of NO. However, single NO therapy is not enough to achieve the ideal antibacterial effect.

[0052] Based on this, the application provides a light-controlled nitric oxide releasing hollow single-atom nanoscale enzyme, which is prepared by using spherical silica nanoparticles as a template, forming a copper ion-polydopamine wrapped silica nanoparticle structure on the surface of the silica nanoparticles through self-polymerization of dopamine and chelation of copper ions, and maintaining a spherical structure with the support of the silica nanoparticles; pyrolyzing the copper ion-polydopamine wrapped silica nanoparticle to obtain a copper-based single-atom nanoscale enzyme with a carbon skeleton, and etching to obtain a hollow spherical single-atom nanoscale enzyme; loading a nitric oxide donor molecule into the hollow of the hollow spherical single-atom nanoscale enzyme to obtain the light-controlled nitric oxide releasing hollow single-atom nanoscale enzyme.

[0053] The application prepares a copper single-atom nanoscale enzyme with a hollow structure, and for the first time, a light-controlled nitric oxide releasing hollow single-atom nanoscale enzyme with nitric oxide controlled release performance is obtained by loading a nitric oxide donor molecule, and the gas therapy and biological catalytic therapy are combined for efficient treatment of bacterial infection diseases. On the one hand, due to the hollow structure of the copper-based single-atom nanoscale enzyme, the hollow structure can be used for efficient loading of the nitric oxide donor molecule. On the other hand, the hollow spherical single-atom nanoscale enzyme has photo-thermal catalytic performance, and under the irradiation of a 1064 nm laser, the photo-thermal performance and catalytic performance of generating O2 ·- are generated. Further, the nitric oxide and O2 ·- occur in a cascade reaction to generate peroxynitrite anion (ONOO - ), which further enhances the antibacterial treatment effect of the nanosystem. The light-controlled nitric oxide releasing hollow single-atom nanoscale enzyme of the application can realize the synergistic treatment effect of photo-thermal therapy, nitric oxide gas therapy and biological catalytic therapy, and can be used for efficient removal of bacteria and biofilms in an infected wound site and effective promotion of the healing of a bacterial infection wound.

[0054] The application will be further described in detail below in combination with examples and drawings, but the embodiments of the application are not limited thereto.

[0055] The reagents used in the application are as follows:

[0056] The purity of the ammonia water is analytical reagent (AR), and the concentration is 25% to 28%.

[0057] The purity of the tetraethyl silicate is chromatographically pure (GC), and the concentration is 99% to 100%.

[0058] The purity of the dopamine hydrochloride is 98% to 100%.

[0059] The concentration of the spherical silica nanoparticles is 100 mg / mL to 200 mg / mL.

[0060] The purity of the copper chloride dihydrate is analytical reagent (AR), and the concentration is 99% to 100%.

[0061] The purity of the hydrofluoric acid is 40% to 44%.

[0062] The purity of the ammonium fluoride is 96% to 100%.

[0063] The concentration of the copper-based single-atom nanoscale enzyme is 100 mg / mL to 200 mg / mL.

[0064] The concentration of the nitric oxide donor is 1 mg / mL to 5 mg / mL.

[0065] Embodiment 1, a preparation method of a light-controlled nitric oxide releasing hollow single-atom nanoscale enzyme

[0066] (1) 94.2 mL of anhydrous ethanol, 16.2 mL of pure water and 4.54 mL of ammonia water were sequentially measured and mixed under stirring for 10 min, 5.4 mL of tetraethyl orthosilicate (TEOS) was added, and water bath reaction was carried out at 45℃ for 3 h; after reaction, a cream-colored precipitate was obtained by centrifugation, and the precipitate was washed twice with a mixed solution of water and ethanol at a volume ratio of 1:1 at a speed of 10000 rmp for 10 min, and the precipitate was collected to obtain spherical silica nanoparticles.

[0067] (2) Dopamine hydrochloride, the spherical silica nanoparticles obtained in (2) and copper chloride dihydrate were mixed in a mass ratio of 10:3:1 under alkaline conditions at room temperature, i.e. in a Tris buffer solution with a concentration of 9 mM and pH=8.7 for 24 h, so that the surface of the spherical silica nanoparticles was wrapped with copper ion-polydopamine through self-polymerization of dopamine and chelation of copper ions, and then the precipitate was obtained by centrifugation and washed twice with water at a speed of 12000 rmp for 10 min, and the precipitate was collected to obtain copper ion-polydopamine wrapped silica nanoparticles, i.e. SiO2@PDA-Cu.

[0068] (3) The copper ion-polydopamine wrapped silica nanoparticles obtained in (2) were freeze-dried and then subjected to gradient temperature pyrolysis under nitrogen atmosphere, first heated to 340℃ and kept for 2.5 h, and then heated to 790℃ and calcined for 1.5 h to obtain copper-based single-atom nanoscale enzyme with carbon as the skeleton, i.e. SiO2@Cu-SAz.

[0069] (4) Hydrofluoric acid and fluorinated etching solution were prepared in a volume to mass ratio of 1 mL:2 mg, and the etching solution was used to etch the copper-based single-atom nanoscale enzyme obtained in (3) in a ratio of 1 mL:10 mg for 12 h, and then centrifuged and washed to obtain hollow spherical single-atom nanoscale enzyme, i.e. Cu HSAz.

[0070] (5) The hollow spherical single-atom nanoscale enzyme obtained in (4) is freeze-dried, then resuspended in an ethanol-water solvent for the first time, with a volume ratio of water to ethanol of 1:1, and 1 mg / mL N,N-di-sec-butyl-N,N-dinitrosopiperazin-1,4-amine (BNN6) is added under stirring to load the hollow spherical single-atom nanoscale enzyme with a nitric oxide donor molecule in the hollow, and after stirring at room temperature for 5 h, the reaction solution is centrifuged at 10,000 rpm for 15 min to obtain a precipitate, which is washed twice with water and then resuspended in 1 mL of PBS buffer with a pH of 7.2 for the second time to obtain a light-controlled nitric oxide releasing hollow single-atom nanoscale enzyme, namely Cu HSAz@BNN6.

[0071] Example 2, a preparation method of a light-controlled nitric oxide releasing hollow single-atom nanoscale enzyme

[0072] (1) 90 mL of anhydrous ethanol, 10 mL of pure water and 2 mL of ammonia water are sequentially measured, stirred and mixed for 10 min, 5.4 mL of tetraethyl silicate (TEOS) is added, and water bath reaction is carried out at 48°C for 2.5 h; after reaction, a white precipitate is obtained by centrifugation, and the precipitate is washed twice with a mixed solution of water and ethanol with a volume ratio of 1:1 at a speed of 10,000 rmp for 10 min, and the collected spherical silica nanoparticle precipitate is obtained.

[0073] (2) Dopamine hydrochloride, the spherical silica nanoparticle obtained in (2) and copper chloride dihydrate are mixed in a mass ratio of 10:3:1 under alkaline conditions at room temperature, i.e. in a Tris buffer solution with a concentration of 10 mM and a pH of 8.8, for 24 h, so that the surface of the spherical silica nanoparticle is wrapped with a copper ion-polydopamine structure through self-polymerization of dopamine and chelation of copper ions, and then the precipitate is obtained by centrifugation, washed twice with water at a speed of 12,000 rmp for 10 min, and the collected copper ion-polydopamine wrapped silica nanoparticle, namely SiO2@PDA-Cu, is obtained.

[0074] (3) The copper ion-polydopamine wrapped silica nanoparticle obtained in (2) is freeze-dried, and then gradient temperature pyrolysis is carried out under a nitrogen atmosphere, first heated to 350°C for 3 h, and then heated to 800°C for 2 h to obtain a copper-based single-atom nanoscale enzyme with carbon as the skeleton, namely SiO2@Cu-SAz.

[0075] (4) Hydrofluoric acid and fluorination are configured in a volume to mass ratio of 3 mL:6 mg to obtain an etching solution, and the etching solution is used to etch the copper-based single-atom nanoscale enzyme obtained in (3) in a ratio of 1 mL:10 mg for 12 h, and then centrifuged and washed to obtain a hollow spherical single-atom nanoscale enzyme, namely Cu HSAz.

[0076] (5) The hollow spherical single-atom nanoscale enzyme obtained in (4) is freeze-dried, then resuspended in an ethanol-water solvent for the first time, with a volume ratio of water to ethanol of 1:1, and 1 mg / mL N,N-di-sec-butyl-N,N-dinitrosopiperazin-1,4-amine (BNN6) is added under stirring to load nitric oxide donor molecules in the hollow of the hollow spherical single-atom nanoscale enzyme, and after stirring at room temperature for 5 h, the reaction solution is centrifuged at 10,000 rpm for 15 min to obtain a precipitate, which is washed twice with water and then resuspended in 1 mL of PBS buffer with a pH of 7.4 for the second time to obtain a light-controlled nitric oxide releasing hollow single-atom nanoscale enzyme, namely Cu HSAz@BNN6.

[0077] Example 3, a preparation method of a light-controlled nitric oxide releasing hollow single-atom nanoscale enzyme

[0078] (1) 100 mL of anhydrous ethanol, 20 mL of pure water and 12 mL of ammonia water are sequentially measured, stirred and mixed for 10 min, 5.4 mL of tetraethyl silicate (TEOS) is added, and water bath reaction is carried out at 60°C for 3 h; after the reaction, a white precipitate is obtained by centrifugation, and the precipitate is washed twice with a mixed solution of water and ethanol with a volume ratio of 1:1 at a speed of 10,000 rpm for 10 min, and the precipitate is collected to obtain spherical silica nanoparticles.

[0079] (2) Dopamine hydrochloride, the spherical silica nanoparticles obtained in (2) and copper chloride dihydrate are mixed in a mass ratio of 20:10:5 under alkaline conditions at room temperature, i.e. in a Tris buffer solution with a concentration of 11 mM and a pH of 8.9, and reacted for 24 h, so that the surface of the spherical silica nanoparticles is wrapped with copper ion-polydopamine through self-polymerization of dopamine and chelation of copper ions to form a structure of copper ion-polydopamine wrapped silica nanoparticles, and then the precipitate is obtained by centrifugation, washed twice with water at a speed of 12,000 rpm for 10 min, and the precipitate is collected to obtain copper ion-polydopamine wrapped silica nanoparticles, namely SiO2@PDA-Cu.

[0080] (3) The copper ion-polydopamine wrapped silica nanoparticles obtained in (2) are freeze-dried, and then gradient temperature pyrolysis is carried out under a nitrogen atmosphere, first heated to 360°C for 3.5 h, and then heated to 810°C for 2.5 h for calcination and pyrolysis, to obtain a copper-based single-atom nanoscale enzyme with carbon as the skeleton, namely SiO2@Cu-SAz.

[0081] (4) Hydrofluoric acid and fluorination are configured in a proportion of 5 mL:10 mg, and the etching solution is used to etch the copper-based single-atom nanoscale enzyme obtained in (3) in a proportion of 5 mL:2 mg for 24 h, and then centrifuged and washed to obtain a hollow spherical single-atom nanoscale enzyme, namely Cu HSAz.

[0082] (5) The hollow spherical single-atom nanoscale enzyme obtained in (4) is freeze-dried, and then resuspended in an ethanol-water solvent for the first time, with a volume ratio of water to ethanol of 1.5:1. 1 mg / mL N,N-di-sec-butyl-N,N-dinitrosopiperazin-6-amine (BNN6) is added under stirring to load the nitric oxide donor molecule in the hollow of the hollow spherical single-atom nanoscale enzyme. After stirring at room temperature for 5 h, the reaction solution is centrifuged at 10,000 rpm for 15 min to obtain a precipitate, which is washed twice with water and then resuspended in 1 mL of PBS buffer with a pH of 7.6 for the second time to obtain a light-controlled nitric oxide releasing hollow single-atom nanoscale enzyme, namely Cu HSAz@BNN6.

[0083] The preparation method of N,N-di-sec-butyl-N,N-dinitrosopiperazin-6-amine, namely BNN6, is as follows: N,N'-di-sec-butyl-p-phenylenediamine, namely BPA, 2.34 mL, 10 mmol, is diluted with ethanol, a constant pressure separatory funnel device is assembled, nitrogen is filled, 6M 20 mL of degassed NaNO2 solution is added, stirring is continued for 30 min, 6M 20 mL of degassed HCl solution is added dropwise using the constant pressure separatory funnel, after reaction for 4 h, the light yellow precipitate is collected by centrifugation at a speed of 10,000 rpm for 30 min, and the excess reactants are removed by repeatedly washing with 50% ethanol aqueous solution. The final product is freeze-dried and stored in the dark at -20°C for further use.

[0084] It should be noted that when the nitric oxide donor molecule is nitroprusside, S-nitrosoglutathione, diazeniumdiolates or N,N'-di-sec-butyl-N,N'-dinitrosopiperazin-6-amine, it can be used to prepare a light-controlled nitric oxide releasing hollow single-atom nanoscale enzyme.

[0085] Experimental Example 1: Structural characterization of the hollow spherical single-atom nanoscale enzyme (Cu HSAz) prepared in Example 1

[0086] The Cu HSAz obtained in Example 1 was observed by transmission electron microscopy.

[0087] The results are shown in Figure 1 The transmission electron microscopy observation results show that the Cu HSAz has a significantly hollow spherical structure, with an average particle size of about 200 nm.

[0088] The Cu HSAz obtained in Example 1 was observed by aberration-corrected high-angle annular dark-field scanning transmission electron microscopy.

[0089] The results are shown in Figure 2As shown, the aberration-corrected high-angle annular dark-field scanning transmission electron microscopy observation can clearly observe the fully exposed dispersed metal monatomic atoms, due to the strong metal support interaction caused by the interface bond brought by the high-temperature pyrolysis, and the metal loses the adsorption ability to small molecules after high-temperature reduction, proving the successful formation of the monatomic structure.

[0090] The particle size distribution of the Cu HSAz obtained in Example 1 was observed.

[0091] The results are shown in Figure 3 The particle size distribution observation shows that the average particle size of the hollow spherical monatomic nanoscale enzyme is 224.91±22.17nm.

[0092] The Cu HSAz obtained in Example 1 was subjected to X-ray powder diffraction and X-ray photoelectron spectroscopy observation.

[0093] The results are shown in Figure 4 , wherein Figure 4 A in Figure 4 is the X-ray diffraction pattern of Cu HSAz, Figure 4 B in Figure 4 is the Cu 2p3 / 2 spectrum of Cu HSAz,

[0094] The X-ray powder diffraction pattern shows that the Cu HSAz has a similar X-ray diffraction pattern to the typical graphene crystal form, which proves the unique existence form of the monatomic copper in the material.

[0095] The X-ray photoelectron spectroscopy peak fitting results show that the Cu2p3 / 2 characteristic peak is mainly concentrated between 931ev and 932ev, proving that the valence state of copper element in Cu HSAz is mainly 0~+1, and the lower valence state has superior catalytic performance. By analyzing the peak fitting results of the binding energy of C1s and N1s regions, it is proved that the N element has the existence form of pyrrole nitrogen, pyridine nitrogen, Cu-N and graphite nitrogen; the C element mainly exists in the form of graphite carbon, and these results all verify the formation of the monatomic structure.

[0096] Example 2, the ultraviolet absorption spectrum change of the Cu HSAz obtained in Example 1 before and after loading the nitric oxide donor molecule BNN6 was determined.

[0097] Different samples were dispersed in water to prepare a dispersion solution of 200μg / mL. 800μL of the sample to be tested was placed in a clean two-side light-transmitting quartz cuvette, the detection wavelength was set to 200nm, and the ultraviolet-near-infrared-visible light absorption spectrum of the sample was collected by ultraviolet-spectrophotometer at 25℃.

[0098] Loading rate of BNN6: 5 mg of BNN6 was dissolved in 1 mL of ethanol-water solvent, water: ethanol = 1:1, diluted to concentrations of 0 μg / mL, 10 μg / mL, 15 μg / mL, 25 μg / mL, 50 μg / mL, 75 μg / mL, 100 μg / mL, respectively, and then the ultraviolet absorption curve of BNN6 solution at 200 nm was recorded by ultraviolet spectrophotometer. The absorbance value of BNN6 solution at 214 nm was read, and the concentration-absorbance positive correlation curve was drawn to obtain the concentration-absorbance standard curve of BNN6. After the material was loaded with BNN6, the supernatant was collected by centrifugation, 1 mL of which was taken, and the absorbance value at 214 nm was recorded. According to the standard curve, the content of BNN6 in 1 mL of supernatant was calculated, and the total amount of BNN6 not loaded was known combined with the total volume of supernatant. The loading rate of BNN6 can be obtained by the following formula:

[0099]

[0100] The results are shown in Figure 5 After loading the donor molecule BNN6, the material showed a clear absorption peak at 230 nm, which proved the successful loading of BNN6. The loading rate of BNN6 loaded by the material was further studied by the BNN6 concentration-absorbance standard curve. The concentration of free BNN6 in the supernatant after loading the material was calculated, and it was found that the loading rate of BNN6 in the material was about 38.7%.

[0101] Example 3: Photothermal performance determination of Cu HSAz@BNN6 obtained in Example 1.

[0102] The photothermal performance of Cu HSAz@BNN6 was verified using 1064 nm laser in the near-infrared two region. PBS was used as the control group, Cu HSAz and Cu HSAz@BNN6 (200 μg / mL) as the experimental group, and the photothermal effect of the material under 1064 nm laser (0.5 W / cm 2 ) irradiation for 3 min was detected by a thermal imaging camera.

[0103] Effect of concentration on photothermal performance of the material: The sample to be tested was dispersed in PBS buffer at pH 7.4 to prepare solutions with different concentrations, 0 μg / mL, 100 μg / mL, 200 μg / mL, 300 μg / mL, and 400 μg / mL, and ultrasonically dispersed uniformly. 200 μL of each concentration sample was taken in a 96-well plate, and a thermocouple thermometer probe was inserted. The sample was irradiated with a 1064 nm near-infrared two region laser with a power of 0.5 W / cm 2 at room temperature for 5 min, and the real-time temperature of the solution was recorded every 10 s. The temperature-time curve was plotted with time as the abscissa and temperature as the ordinate, and the photothermal performance of the sample at different concentrations was compared.

[0104] Effect of laser power on the photothermal properties of materials: The sample to be tested was dispersed in PBS at pH 7.4 to prepare a solution of 200 μg / mL, and 200 μL of the sample was taken to a 96-well plate. The sample was irradiated with a 0.3 W / cm 2 , 0.5 W / cm 2 , 0.8 W / cm 2 , and 1 W / cm 2 1064 nm laser at room temperature for 5 min, and the temperature was recorded every 10 s. The temperature-time curve was plotted with time as the abscissa and temperature as the ordinate, and the photothermal properties of the sample under different power laser irradiation were compared.

[0105] Photothermal stability of the material: The sample to be tested was dispersed in PBS at pH 7.4 to prepare a solution of 200 μg / mL, and 200 μL of the sample was taken to a 96-well plate. The thermocouple probe was inserted into the liquid surface, and the solution surface was irradiated with a 1064 nm laser for 5 min (0.5 W / cm 2 ). Then the laser was turned off to allow the solution to return to the temperature before measurement. The experiment was repeated 4 times, and the temperature was recorded every 10 s during the process. The temperature-time curve was plotted with time as the abscissa and temperature as the ordinate to verify the photothermal stability of the sample.

[0106] The results are shown in Figure 6 , where Figure 6 A is the infrared thermal imaging of Cu HSAz@BNN6, Figure 6 B is the time-temperature curve of Cu HSAz@BNN6 under different power, Figure 6 C is the time-temperature curve of Cu HSAz@BNN6 under different concentrations, Figure 6 and D is the photothermal stability curve of Cu HSAz@BNN6.

[0107] As shown in Figure 6 A, the temperature of Cu HSAz and Cu HSAz@BNN6 rose to 45°C after 3 min of 1064 nm laser irradiation, while the temperature of the control group remained almost unchanged after laser irradiation. At the same time, the effect of different laser powers and different material concentrations on the photothermal properties was further determined. As shown in Figure 6 B, the power of the 1064 nm laser was controlled at 0 W / cm 2 , 0.3 W / cm 2 , 0.5 W / cm 2 , 0.8 W / cm 2 , and 1 W / cm 2, the temperature of Cu HSAz@BNN6 material with a concentration of 200 μg / mL rose to 28℃, 35℃, 44℃, 56℃, 67℃, respectively, after 3 min irradiation; as shown in C of Figure 6 , the power of 1064 nm laser was controlled to be 0.5 W / cm 2 , and the material concentration was 0 μg / mL, 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, respectively, and the temperature rose to 28℃, 34℃, 45℃, 55℃, 58℃, respectively, after 3 min irradiation. These results fully demonstrate that Cu HSAz@BNN6 has good photothermal performance, and the temperature rise is positively correlated with the laser power and the material concentration. Finally, the photothermal stability of Cu HSAz@BNN6 was determined by controlling the laser to intermittently irradiate Cu HSAz@BNN6 to record the temperature change. As shown in D of Figure 6 , the temperature of Cu HSAz@BNN6 (200 μg / mL) rose to 45℃ by 1064 nm laser (0.5 W / cm 2 ), and then the laser was turned off to cool down to room temperature, and the five cycles were repeated, which verified that the material has good photothermal stability, which makes a good preparation for the photothermal control of the release of NO gas.

[0108] Experimental Example 4, the Cu HSAz@BNN6 obtained in Example 1 was subjected to one-way nitrogen oxide generation performance determination.

[0109] The NO generation ability of the material under irradiation and the NIR-II laser controlled release NO ability were detected by Griess method.

[0110] NO release detection: by recording the absorbance change at 540 nm after the reaction of different concentrations of NO, 0 μM, 0.78 μM, 1.56 μM, 3.13 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, and Griess reagent, a standard curve of NO concentration-absorbance was drawn. Two samples with the same concentration (200 μg / mL) were taken and the same volume of Griess reagent was added. One of them was not treated; the other was irradiated by 1064 nm laser with a power of 0.5 W / cm 2 for 3 min; the absorbance change at 540 nm was determined at room temperature by ultraviolet-visible spectrophotometer, and the generated NO concentration was calculated according to the standard curve.

[0111] NIR-II laser controlled release NO performance: the NIR II controlled release ability of NO was determined by intermittently irradiating 1064 nm laser at a power of 0.5 W / cm 2 .

[0112] The results are as follows Figure 7 As shown, Figure 7 A is the nitric oxide release concentration curve with and without laser irradiation (+NIR / -NIR), Figure 7 B is the laser-controlled NO release "switch" curve.

[0113] like Figure 7 As shown in A, a 1064 nm laser (0.5 W / cm 2 After irradiation of CuHSAz@BNN6 (200 μg / mL), the material released a large amount of NO, causing the Griess reagent to appear red. The concentration of NO gradually increased with laser irradiation, reaching 25 μM after 20 minutes. The control group without laser irradiation only released a small amount of NO, with a concentration of 4.5 μM after 20 minutes. Figure 7 As shown in Figure B, when laser irradiated, the material was able to respond to photothermal release of a large amount of NO, while without laser irradiation, the rate of NO release slowed down significantly. This result remained consistent over multiple cycles, forming a "switching curve" for the light-controlled NO release of Cu HSAz@BNN6.

[0114] Experimental Example 5: The Cu HSAz@BNN6 obtained in Example 1 was subjected to superoxide anion O2 ·- and peroxynitrite RNS generation performance determination.

[0115] The superoxide anion production performance of the material was characterized using a superoxide anion content assay kit and a cis-electron spin resonance spectrometer (EMS Plus). The superoxide anion content assay kit evaluated the time-dependent superoxide anion production of the sample (200 μg / mL) over 30 minutes. Characteristic superoxide anion spectra were obtained using cis-electron spin resonance spectrometry using DMPO as a probe.

[0116] The ability of a material to generate RNS is determined by measuring the change in fluorescence intensity between 500 nm and 600 nm under 488 nm excitation after DHR123 binds to peroxynitroso RNS. A 200 μL sample of 200 μg / mL material was added to a 96-well plate, followed by the addition of 10 μM DHR123 fluorescent probe. After incubation for half an hour in the dark, the fluorescence intensity change between 500 nm and 600 nm under 488 nm excitation was measured using a microplate reader to assess the material's RNS-generating ability.

[0117] The results are as follows Figure 8 As shown, Figure 8 A in the figure represents Cu HSAz@BNN6 releasing O2 ·- The time-concentration curve of Figure 8 B in the equation is O2 ·- The ESR spectrum of Figure 8C is the result of DHR123 probe to study the fluorescence intensity of RNS generated by different materials catalysis.

[0118] Figure 8 A in the results show that 1064 nm laser irradiation can enhance the production of O2 ·- . Figure 8 B in the results of the cis electron spin spectrum show the specific production of superoxide anion, while further proving that 1064 nm laser irradiation promotes the generation of O2 ·- , consistent with the results of A in Figure 8 .

[0119] Superoxide anion can combine with NO to produce more active peroxynitroso RNS, and the specific fluorescence probe dihydrorhodamine 123, namely DHR123, will have obvious fluorescence intensity at 530 nm after reacting with RNS, as shown in C in Figure 9 , only the final group Cu HSAz@BNN6+NIR can obviously produce a large amount of RNS, proving that Cu HSAz@BNN6 has good catalytic effect under 1064 nm laser irradiation.

[0120] Example 6, in vitro antibacterial performance determination of Cu HSAz@BNN6 obtained in Example 1.

[0121] After the bacteria were recovered and cultured to the logarithmic phase state, the absorbance value OD 600 =1.0 at 600 nm was detected by ultraviolet spectrophotometer, 1 mL of bacterial suspension was centrifuged and washed with sterile PBS for multiple times to completely remove the culture medium components and adjust the bacterial density to 1×10 8 CFU / mL. The bacteria were inoculated into a 96-well plate, with PBS as the Control group, Cu HSAz, Cu HSAz@BNN6, Cu HSAz+NIR, HCN@BNN6+NIR, Cu HSAz@BNN6+NIR as the experimental groups, HCN as the carbon skeleton without loading copper metal, and the 1064 nm laser power used was 0.5 W / cm 2 , and the irradiation time was 3 min. The treated bacteria in each group were placed in a 37°C constant temperature incubator for shaking incubation for 4 h, then washed with PBS to remove excess materials, and the bacterial suspension was diluted and spread on sterilized LB solid agar plates, which were placed in a 37°C biochemical incubator for culture for 12 h. A digital camera was used to take pictures to record the growth state of the bacteria, and Image J software was used to calculate the number of bacteria, to determine the antibacterial effect after material treatment.

[0122] The results are shown in Figure 9 , wherein Figure 9A in the table shows the antibacterial effect of Control, Cu HSAz, Cu HSAz@BNN6 material group on MRSA, E. coli and S. aureus after laser irradiation, Figure 9 B, C, D in the table shows the bacterial survival rate of MRSA, E. coli and S. aureus respectively.

[0123] Figure 9 A in the table shows that the removal effect of Control, Cu HSAz, Cu HSAz@BNN6 material group on three kinds of bacteria is negligible without laser irradiation, which shows that the O2 ·- released by the material has relatively limited antibacterial effect. Cu HSAz, HCN@BNN6 material group after laser irradiation shows certain antibacterial effect, which may be due to the synergistic effect of photothermal and O2 ·- or NO. Cu HSAz@BNN6 group after laser irradiation almost completely removes the bacteria on the agar plate, which is due to the synergistic effect of photothermal and RNS. As shown in B-D in the table, Figure 10 by counting and analyzing the bacteria on the agar plate, Cu HSAz+NIR, HCN@BNN6+NIR group has about 50% antibacterial rate on MRSA, E. coli and S. aureus, which shows that the temperature rise and O2 ·- or NO has a certain removal effect on bacteria. Cu HSAz@BNN6 after NIR laser irradiation shows the best antibacterial performance on three kinds of bacteria, and the bacteria on the agar plate are almost completely removed, and the counting shows that the antibacterial rate is 99.89%, 99.78% and 99.91% respectively, which shows that the synergistic effect of photothermal and RNS has the best antibacterial effect.

[0124] Example 7, the biofilm formation inhibition performance of Cu HSAz@BNN6 obtained in Example 1 was determined.

[0125] Crystal violet dye can stain the bacterial biofilm after it is fixed, and the ability of Cu HSAz@BNN6 to promote the dispersion of bacterial biofilm was determined by staining the biofilm after different experimental groups, Control, Cu HSAz, Cu HSAz@BNN6, Cu HSAz+NIR, Cu HSAz@BNN6+NIR treatment. The bacteria were recovered and cultured to the logarithmic phase state, OD 600 =1.0, inoculated into 96-well plates, and placed in a 37℃ constant temperature biochemical incubator for incubation for 48h, and the fresh medium was replaced every 12h during the incubation to make it proliferate to form a dense bacterial biofilm.

[0126] Different treatments were carried out in different wells with formed bacterial biofilm, and the 1064nm laser power used was 0.5W / cm2 The biofilm was left to stand for 12 hours in a 37 °C incubator after different treatments. After the incubation, the free materials and floating bacteria were washed with sterile PBS, and 200 μL of anhydrous methanol was added to fix the biofilm for 15 min. Then 1% crystal violet was added for staining and continued to stand in a 37 °C incubator for 30 min. The free dye was removed by washing with sterile PBS several times, and a digital camera was used to take pictures to record the dissipation of the biofilm. 200 μL of anhydrous ethanol was added to the well, and after the crystal violet was completely dissolved, the absorbance at 570 nm was measured by a multifunctional enzyme label instrument to determine the effect of different experimental groups on promoting the dissipation of the biofilm.

[0127] The results are shown in Figure 10 , where Figure 10 A is the crystal violet staining of MRSA bacterial biofilm treated with different materials, Figure 10 B is the crystal violet staining of MRSA bacterial biofilm treated with different concentrations of CuHSAz@BNN6, Figure 10 C is the crystal violet absorbance analysis of MRSA bacterial biofilm treated with different materials, Figure 10 D is the crystal violet absorbance analysis of MRSA bacterial biofilm treated with different concentrations of CuHSAz@BNN6.

[0128] As shown in Figure 10 A, the Control group and the bacterial biofilm treated with CuHSAz and CuHSAz@BNN6 materials without laser irradiation are relatively complete, and the crystal violet color is deep. The CuHSAz+NIR photothermal treatment group alone can effectively promote the dissipation of the biofilm, and the crystal violet color is light. After the CuHSAz@BNN6 material group is treated with laser irradiation, the bacterial biofilm is almost completely dissipated, and the crystal violet color tends to be colorless. As shown in Figure 10 C, the Control group and the material group without laser irradiation have high absorbance values, indicating that the biofilm is relatively complete. The CuHSAz@BNN6 treatment group after laser irradiation has a low absorbance value, indicating that the biofilm has almost completely dissipated.

[0129] The ability of different concentrations of CuHSAz@BNN6 to promote the dissipation of bacterial biofilm was further determined. As shown in Figure 10 B, the MRSA bacterial biofilm treated with low concentration of CuHSAz@BNN6 is relatively complete, and the crystal violet color is deep. When the concentration of CuHSAz@BNN6 reaches 200 μg / mL, after laser irradiation treatment, the MRSA bacterial biofilm is almost completely dissipated, and the crystal violet color tends to be transparent. As shown in Figure 11As shown in D of FIG. 12, the absorbance of crystal violet decreased significantly with the increase of the concentration of Cu HSAz@BNN6, indicating that the increase of the concentration of Cu HSAz@BNN6 can effectively promote the dispersion of MRSA bacterial biofilm.

[0130] The crystal violet staining experiment shows that Cu HSAz@BNN6 can promote the dispersion of bacterial biofilm in the bacterial biofilm environment through photothermal and RNS release, further verifying the good in vitro antibacterial effect of Cu HSAz@BNN6.

[0131] Example 8: Promoting wound healing performance determination of Cu HSAz@BNN6 obtained in Example 1.

[0132] After the MRSA infected wound model was constructed, the divided five groups of animals were treated with different dosages every two days, with PBS as the Control group, Cu HSAz, Cu HSAz@BNN6, Cu HSAz+NIR, Cu HSAz@BNN6+NIR as the experimental groups. The materials were dispersed in sterile PBS with a concentration of 200 μg / mL. The same operation was performed on each group of animals, and on the 0th day, 3rd day, 5th day, 7th day and 10th day after administration, the wound diameter of each group of rats was measured by a precision steel ruler and photographed by a digital camera, and the wound area of each group of rats was calculated using Image J software, and a wound area change graph was drawn.

[0133] The results are shown in Figure 11 , wherein Figure 11 A of Figure 11 is a picture of the wound of the rat after treatment with different materials, B is a statistical diagram of the wound area of the rat.

[0134] As shown in A of Figure 11 , in the Control, Cu HSAz and Cu HSAz@BNN6 groups, the wounds of the rats were still not closed after treatment, and the healing was relatively slow. The wound of the rat in the Cu HSAz+NIR group was relatively close to closure, which may be due to the photothermal effect clearing part of the bacteria, accelerating wound healing. The wound of the rat in the Cu HSAz@BNN6+NIR group was completely closed, and the wound healing rate was greatly increased, which was attributed to the synergistic effect of photothermal and RNS on bacterial clearance. By statistical analysis of the wound area of the rat after treatment with different materials, as shown in ​As shown in B, due to the effect of photothermal, the wound closure rate of the rats in the Cu HSAz+NIR group reached 60% after 7 days, and reached 70% after 10 days. However, the synergistic effect of photothermal and RNS brought about by the Cu HSAz@BNN6+NIR group significantly improved the wound closure rate of the rats, which reached 96% after 10 days, indicating that the synergistic effect of photothermal and RNS helps to promote the healing of infected wounds.

[0135] Each of the technical features of the above-described embodiments can be combined arbitrarily. In order to make the description simple, each of the technical features in the above-described embodiments is not described in all possible combinations, however, as long as the combination of the technical features does not exist contradictory, it should be considered as the scope of the present disclosure.

[0136] The above-described embodiments only express several embodiments of the present application, and the description is more specific and detailed, but it should not be understood as a limitation on the scope of the patent. It should be pointed out that for ordinary skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are within the scope of the present application. Therefore, the scope of protection of the present application should be subject to the appended claims.

Claims

1. A light-controlled nitric oxide releasing hollow single-atom nanoszyme, characterized in that, The application discloses a preparation method of a hollow single-atom nanoscale enzyme for releasing nitric oxide under light control. The preparation method of the hollow single-atom nanoscale enzyme for releasing nitric oxide under light control specifically comprises the following steps: (1) mixing hydrochloric acid dopamine, spherical silica nanoparticles and copper chloride dihydrate to form a copper ion-polydopamine coated silica nanoparticle structure on the surface of the spherical silica nanoparticles through self-polymerization of dopamine and chelation of copper ions; centrifuging, washing and collecting the precipitate to obtain the copper ion-polydopamine coated silica nanoparticles; the mass ratio of the hydrochloric acid dopamine, the spherical silica nanoparticles and the copper chloride dihydrate is 10-20:3-10:1-5; (2) freeze-drying the copper ion-polydopamine coated silica nanoparticles obtained in the step (1) and pyrolyzing the same in a pyrolysis environment to obtain a carbon skeleton copper-based single-atom nanoscale enzyme; (3) etching the copper-based single-atom nanoscale enzyme obtained in the step (2) for 12-24 hours using an etching solution, and centrifuging and washing the same to obtain a hollow spherical single-atom nanoscale enzyme; the volume-to-mass ratio of the etching solution to the copper-based single-atom nanoscale enzyme is 1-5 mL:10-20 mg; (4) freeze-drying the hollow spherical single-atom nanoscale enzyme obtained in the step (3) and resuspending the same for the first time, and then adding a nitric oxide donor molecule to the hollow spherical single-atom nanoscale enzyme to load the nitric oxide donor molecule in the hollow of the hollow spherical single-atom nanoscale enzyme; The hollow single-atom nanoscale enzyme for releasing nitric oxide under light control is obtained after centrifugation, washing and resuspension for the second time.

2. The light-controlled nitric oxide releasing hollow single-atom nanoszyme of claim 1, wherein, The pyrolysis environment is under a nitrogen atmosphere, and the temperature is first increased to 340-360 DEG C and kept for 2.5-3.5 hours, and then increased to 790-810 DEG C and calcined and pyrolyzed for 1.5-2.5 hours.

3. The light-controlled nitric oxide releasing hollow single-atom nanoszyme of claim 1, wherein, The etching solution is obtained by mixing hydrofluoric acid and ammonium fluoride in a volume-to-mass ratio of 1-5 mL:2-10 mg.

4. The light-controlled nitric oxide releasing hollow single-atom nanoszyme of claim 1, wherein, The mixing reaction is under room temperature alkaline conditions provided by a Tris buffer with a concentration of 9-11 mM and a pH of 8.7-8.

9.

5. The light-controlled nitric oxide releasing hollow single-atom nanoszyme of claim 1, wherein, The first resuspension reagent is a mixed solution of pure water and anhydrous ethanol, and the volume ratio of the pure water to the anhydrous ethanol is 1-1.5:1; the second resuspension reagent is a PBS buffer with a pH of 7.2-7.

6.

6. The light-controlled nitric oxide releasing hollow single-atom nanoszyme of claim 1, wherein, The preparation method of the spherical silica nanoparticles is as follows: Mixing anhydrous ethanol, pure water and ammonia water, adding tetraethyl silicate, reacting at 45-60 DEG C for 2-3 hours, centrifugal washing, collecting the precipitate to obtain spherical silica nanoparticles; the volume ratio of the anhydrous ethanol, pure water and ammonia water is 90-100: 10-20: 2-12; the volume ratio of the tetraethyl silicate and ammonia water is 1.0-2.0: 1.

0.

7. The light-controlled nitric oxide releasing hollow single-atom nanoszyme of claim 1, wherein, The nitric oxide donor molecule includes at least one of nitroprusside, S-nitrosoglutathione, diol diazeniumdioxide and N, N'-di-sec-butyl-N, N'-dinitroso-1, 4-benzenediamine.

8. Use of the light-controlled nitric oxide releasing hollow monoatomic nanoszyme of claim 1 in the preparation of biomedical materials.

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