Light-controlled nitric oxide release hollow monatomic nano-enzyme as well as preparation method and application of light-controlled nitric oxide release hollow monatomic nano-enzyme

Abstract

The invention discloses light-controlled nitric oxide release hollow monatomic nano-enzyme as well as a preparation method and application thereof, and belongs to the technical field of biomedical materials. Spherical silicon dioxide nanoparticles are used as a template, a copper ion-polydopamine coated silicon dioxide nanoparticle structure is formed on the surface of the spherical silicon dioxide nanoparticles through auto-polymerization of dopamine and chelation of copper ions, and the spherical structure is maintained under the support of the silicon dioxide nanoparticles; pyrolyzing the copper ion-polydopamine coated silicon dioxide nanoparticles to obtain a copper-based monatomic nano-enzyme taking carbon as a skeleton, and etching to obtain a hollow spherical monatomic nano-enzyme; and loading nitric oxide donor molecules into the hollow of the hollow spherical monatomic nano-enzyme to obtain the light-controlled nitric oxide release hollow monatomic nano-enzyme. The device can achieve the synergistic treatment effect of photo-thermal treatment, nitric oxide gas treatment and biological catalysis treatment, and is used for efficiently removing bacteria and biological membranes of infected wounds and effectively promoting healing of bacterial infected wounds.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and specifically relates to a light-controlled nitric oxide-releasing hollow single-atom nanozyme, a preparation method and an application thereof. Background Art

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

[0003] In recent years, gas therapy has attracted extensive attention from researchers in biomedical applications such as antibacterial and anti-tumor due to its advantages of being green, safe, and efficient. Nitric oxide (NO) gas is closely related to the healing of bacterial-infected wounds and plays a vital role in regulating inflammatory responses, angiogenesis, and cell metabolism. NO can oxidize intracellular DNA and RNA to make them lose their biological activity, 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 it and achieve effective controlled release, and to maximize its antibacterial properties, has become the key to the current development of NO antibacterial materials. At present, a variety of nanomaterials have been designed and synthesized for NO delivery and controlled release research. These nanosystems can utilize exogenous or endogenous stimulation conditions, such as X-rays, ultrasound, ultraviolet light, near-infrared light, pH, temperature, GSH and H 2 O 2 etc. to achieve controlled release of NO. Compared with endogenous stimulation, exogenous stimulation is more conducive to the controlled release of gas. Among them, light-controlled release nanomaterials with near-infrared response properties are regarded as ideal materials for controlled release of NO. However, single NO therapy is not enough to achieve the ideal antibacterial effect. Summary of the invention

[0004] To solve the above problems, the present invention provides a light-controlled nitric oxide-releasing hollow single-atom nanozyme, a preparation method and an application thereof, so as to improve the antibacterial effect.

[0005] The present invention is achieved through the following technical solutions:

[0006] A light-controlled nitric oxide-releasing hollow single-atom nanozyme uses spherical silica nanoparticles as a template. On its surface, a structure of copper ion-poly dopamine-coated silica nanoparticles is formed through the self-polymerization of dopamine and the chelation of copper ions, and the spherical structure is maintained with the support of silica nanoparticles. The copper ion-poly dopamine-coated silica nanoparticles are pyrolyzed to obtain a carbon-based copper single-atom nanozyme, and after etching, a hollow spherical single-atom nanozyme is obtained. A nitric oxide donor molecule is loaded into the hollow of the hollow spherical single-atom nanozyme to obtain a light-controlled nitric oxide-releasing hollow single-atom nanozyme.

[0007] Preferably, the preparation method of the light-controlled nitric oxide-releasing hollow single-atom nanozyme specifically includes the following steps:

[0008] (1) Mix hydrochloric acid dopamine, spherical silica nanoparticles, and copper dichloride dihydrate for reaction, so that a structure of copper ion-poly dopamine-coated silica nanoparticles is formed on the surface of the spherical silica nanoparticles through the self-polymerization of dopamine and the chelation of copper ions; centrifuge, wash, and collect the precipitate to obtain copper ion-poly dopamine-coated silica nanoparticles; the mass ratio of hydrochloric acid dopamine, spherical silica nanoparticles, and copper dichloride dihydrate is 10-20:3-10:1-5.

[0009] (2) Lyophilize the copper ion-poly dopamine-coated silica nanoparticles obtained in (1), and pyrolyze them in a pyrolysis environment to obtain a carbon-based copper single-atom nanozyme;

[0010] (3) Etch the copper-based single-atom nanozyme obtained in (2) with an etching solution for 12 h to 24 h, and centrifuge and wash to obtain a hollow spherical single-atom nanozyme; the volume-mass ratio of the etching solution to the copper-based single-atom nanozyme is 1-5:10-20.

[0011] (4) Lyophilize the hollow spherical single-atom nanozyme obtained in (3), then resuspend it for the first time, and stir and add a nitric oxide donor molecule so that the nitric oxide donor molecule is loaded into the hollow of the hollow spherical single-atom nanozyme; after centrifugation, washing, and resuspension for the second time, a light-controlled nitric oxide-releasing hollow single-atom nanozyme is obtained.

[0012] Preferably, the conditions of the pyrolysis environment are to first heat up to 340 °C to 360 °C and keep warm for 2.5 h to 3.5 h in a nitrogen atmosphere, and then heat up to 790 °C to 810 °C for calcination pyrolysis for 1.5 h to 2.5 h.

[0013] Preferably, the etching solution is obtained by mixing hydrofluoric acid and ammonium fluoride according to 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; the concentration of the nitric oxide donor is 1 mg / mL to 5 mg / mL.

[0015] Preferably, the conditions for the mixed reaction are room temperature and alkaline conditions; the room temperature and alkaline conditions are provided by a Tris buffer solution with a concentration of 9 mM to 11 mM and a pH of 8.7 to 8.9; the room temperature is 25°C.

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

[0017] Preferably, the preparation method of the spherical silica nanoparticles is as follows:

[0018] Mix absolute ethanol, pure water and ammonia water, add tetraethyl orthosilicate, and react at 45°C to 60°C for 2 h to 3 h, centrifuge and wash, and collect the precipitate to obtain spherical silica nanoparticles; the volume ratio of absolute ethanol, pure water and ammonia water is 90 to 100:10 to 20:2 to 12; the volume ratio of tetraethyl orthosilicate to ammonia water is 1.0 to 2.0:1.0.

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

[0020] The application of the photo-controlled nitric oxide-releasing hollow single-atom nanozyme in the preparation of biomedical materials. Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention provides a light-controlled nitric oxide-releasing hollow single-atom nanozyme. Using spherical silica nanoparticles as a template, a structure of copper ion-poly(dopamine) coating silica nanoparticles is formed on its surface through the self-polymerization of dopamine and the chelation of copper ions, and the spherical structure is maintained with the support of silica nanoparticles; the copper ion-poly(dopamine) coating silica nanoparticles are pyrolyzed to obtain a carbon-based copper single-atom nanozyme, and after etching, a hollow spherical single-atom nanozyme is obtained; a nitric oxide donor molecule is loaded into the hollow of the hollow spherical single-atom nanozyme to obtain a light-controlled nitric oxide-releasing hollow single-atom nanozyme. The light-controlled nitric oxide-releasing hollow single-atom nanozyme is loaded with a nitric oxide donor molecule. Due to the excellent photothermal performance of its carbon-based carrier in the second near-infrared region (NIR II), the controllable release of nitric oxide can be achieved under NIR II laser irradiation, and nitric oxide and O 2 ·- can be generated simultaneously under NIR II laser irradiation, and then the two undergo a cascade reaction to generate peroxynitrite anion (ONOO - ), enhancing the antibacterial treatment effect of the nano-system.

[0022] In addition, the hollow spherical single-atom nanozyme used to load the nitric oxide donor molecule in the preparation process of the present invention has excellent peroxidase-like (OXD) activity by itself, and generates O 2 ·- . And, under NIR II laser irradiation, the performance of the hollow spherical single-atom nanozyme in catalyzing the generation of O 2 ·- is greatly improved.

[0023] Therefore, the light-controlled nitric oxide-releasing hollow single-atom nanozyme of the present invention can achieve the synergistic treatment effect of photothermal therapy, nitric oxide gas therapy and biocatalytic therapy, and can be used to efficiently remove bacteria and biofilms at the infected wound site and effectively promote the healing of bacteria-infected wounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0025] Figure 1 It is a transmission electron microscope image of the hollow spherical single-atom nanozyme (Cu HSAz) obtained in Example 1.

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

[0027] Figure 2 Among them, 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 Particle size distribution diagram of Cu HSAz obtained in Example 1.

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

[0030] Figure 4 Among them, A is the X-ray diffraction pattern of Cu HSAz; B is the XPS Cu2p3 / 2 sub-spectrum of Cu HSAz; C is the N1s sub-spectrum of CuHSAz; D is the C1s sub-spectrum of Cu HSAz;

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

[0032] Figure 6 Temperature-time curve of different concentrations of Cu HSAz@BNN6 irradiated with different laser powers.

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

[0034] Among them Figure 6 For B, C, and D in it, the abscissa is time (Time) and the ordinate is temperature (Temperature).

[0035] Figure 7 Is the NO release result diagram of 200 μg / mL Cu HSAz@BNN6 irradiated with a power of 0.5 W / cm 2 Power irradiation.

[0036] Figure 7 Among them, A is the nitric oxide release concentration curve with or without laser irradiation; B is the laser-controlled release NO "switch" curve.

[0037] Where the abscissa is time and the ordinate is the nitric oxide release concentration.

[0038] Figure 8 It is the graph of the release results of superoxide anion (O₂ ·- ) under the conditions of laser irradiation or not for Cu HSAz@BNN6, and the graph of the results of different materials catalyzing the production of peroxynitrite RNS.

[0039] Figure 8 In it, A is the time-concentration curve graph of the release of O₂ ·- by Cu HSAz@BNN6; B is the ESR spectrum of O₂ ·- ; C is the fluorescence intensity graph of different materials catalyzing the production of RNS studied by DHR123 probe.

[0040] Where Figure 8 the abscissa of A in it is time and the ordinate is the superoxide anion concentration (O₂ ·- concentration), Figure 8 the abscissa of B in it is magnetic field and the ordinate is intensity, Figure 8 and the abscissa of C in it is fluorescence wavelength and the ordinate is fluorescent intensity.

[0041] Figure 9 It is the antibacterial effect graph of different materials on Staphylococcus aureus (S. aureus), Escherichia coli (E. coli) and methicillin-resistant Staphylococcus aureus (MRSA).

[0042] Figure 9 In it, A is the antibacterial effect graph of different materials on S. aureus, E. coli and MRSA; B is the bacterial survival rate graph of MRSA; C is the bacterial survival rate graph of E. coli; D is the bacterial survival rate graph of S. aureus.

[0043] Figure 10 It is the crystal violet staining graph of MRSA bacterial biofilms treated with different materials and different concentrations of CuHSAz@BNN6.

[0044] Figure 10Among them, A is the crystal violet staining diagram of MRSA bacterial biofilms treated with different materials; B is the crystal violet staining diagram of MRSA bacterial biofilms treated with different concentrations of CuHSAz@BNN6; C is the crystal violet absorbance analysis diagram of MRSA bacterial biofilms treated with different materials; D is the crystal violet absorbance analysis diagram of MRSA bacterial biofilms treated with different concentrations of CuHSAz@BNN6.

[0045] Among them Figure 10 In C and D, the abscissa represents different materials and different concentrations of CuHSAz@BNN6, and the ordinate represents the crystal violet absorbance (Absorbance).

[0046] Figure 11 It is a statistical chart of the wounds and areas of rats at different days after treatment with different materials.

[0047] Figure 11 Among them, A is the wound diagram of rats at different days after treatment with different materials; B is the statistical chart of the wound area of rats; among them Figure 11 In A and B, the abscissa represents the treatment days (Day), Figure 11 and the ordinate of B represents the relative wound area. Detailed implementation manners

[0048] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below, and preferred embodiments of the present invention are given. However, the present invention can be implemented 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 understanding of the disclosure content of the present invention more thorough and comprehensive.

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

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

[0051] At present, a variety of nanomaterials have been designed and synthesized for the research of NO delivery and controlled release. These nanosystems can utilize exogenous or endogenous stimulation conditions, such as X-rays, ultrasound, ultraviolet light, near-infrared light, pH, temperature, GSH, and H 2 O 2 etc. to achieve the controlled release of NO. Compared with endogenous stimulation, exogenous stimulation is more conducive to achieving the controlled release of gases. Among them, the light-controlled release nanomaterials with near-infrared response performance are regarded as ideal materials for the controlled release of NO. However, a single NO therapy is not sufficient to achieve an ideal antibacterial effect.

[0052] Based on this, the present invention provides a light-controlled nitric oxide-releasing hollow single-atom nanozyme. Using spherical silica nanoparticles as a template, a structure of copper ion-poly(dopamine) coated silica nanoparticles is formed on its surface through the self-polymerization of dopamine and the chelation of copper ions, and the spherical structure is maintained with the support of silica nanoparticles; the copper ion-poly(dopamine) coated silica nanoparticles are pyrolyzed to obtain a copper-based single-atom nanozyme with a carbon skeleton, and after etching, a hollow spherical single-atom nanozyme is obtained; a nitric oxide donor molecule is loaded into the hollow of the hollow spherical single-atom nanozyme to obtain a light-controlled nitric oxide-releasing hollow single-atom nanozyme.

[0053] The present invention prepares and synthesizes a copper single-atom nanozyme with a hollow structure. By loading a nitric oxide donor molecule, a light-controlled nitric oxide-releasing hollow single-atom nanozyme with nitric oxide controlled-release performance is obtained for the first time, and gas therapy and biocatalytic therapy are combined for the efficient treatment of bacterial infectious diseases. On the one hand, due to the hollow structure of the copper-based single-atom nanozyme, it can be used for the efficient loading of nitric oxide donor molecules. On the other hand, the hollow spherical single-atom nanozyme has photothermal catalytic performance and generates photothermal performance and catalyzes the production of O 2 ·- performance under 1064 nm laser irradiation. Furthermore, nitric oxide and O 2 ·- undergo a cascade reaction to generate peroxynitrite anion (ONOO - ), further enhancing the antibacterial treatment effect of the nano-system. Using the light-controlled nitric oxide-releasing hollow single-atom nanozyme of the present invention can achieve the synergistic treatment effect of photothermal therapy, nitric oxide gas therapy and biocatalytic therapy, and can be used to efficiently remove bacteria and biofilms at the infected wound site and effectively promote the healing of bacterial-infected wounds.

[0054] The following further describes the present invention in detail with reference to examples and drawings, but the implementation manners of the present invention are not limited thereto.

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

[0056] The purity of the ammonia water is analytical pure (AR), 25% - 28%.

[0057] The purity of tetraethyl orthosilicate is gas chromatography pure (GC) 99% - 100%.

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

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

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

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

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

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

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

[0065] Example 1. A preparation method of a photo-controlled nitric oxide-releasing hollow single-atom nanozyme

[0066] (1) Measure 94.2 mL of absolute ethanol, 16.2 mL of pure water and 4.54 mL of ammonia water in sequence, stir and mix for 10 min, add 5.4 mL of tetraethyl orthosilicate (TEOS), and react in a water bath at 45 °C for 3 h; after the reaction, centrifuge to obtain a milky white precipitate, and wash it twice with a mixed solution of water and ethanol with a volume ratio of 1:1, the rotation speed is 10000 rmp, and the time is 10 min, and collect the precipitate to obtain spherical silica nanoparticles.

[0067] (2) Mix and react the dopamine hydrochloride, the spherical silica nanoparticles obtained in (2) and the copper(II) chloride dihydrate at a mass ratio of 10:3:1 under room temperature alkaline conditions, that is, 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 forms a structure of copper ion-polydopamine-coated silica nanoparticles through the self-polymerization of dopamine and the chelation of copper ions, and then centrifuge to obtain a precipitate, wash it twice with water, the rotation speed is 12000 rmp, and the time is 10 min, and collect the precipitate to obtain copper ion-polydopamine-coated silica nanoparticles, that is, SiO 2 @PDA-Cu.

[0068] (3) After freeze-drying the copper ion-polydopamine-coated silica nanoparticles obtained in (2), perform gradient temperature pyrolysis under a nitrogen atmosphere. First, heat up to 340 °C and keep it warm for 2.5 h, then heat up to 790 °C and calcine and pyrolyze for 1.5 h to obtain a copper-based single-atom nanozyme with carbon as the skeleton, that is, SiO 2 @Cu-SAz.

[0069] (4) Prepare an etching solution by mixing hydrofluoric acid and fluoride at a volume-to-mass ratio of 1 mL:2 mg, and use this etching solution to etch the copper-based single-atom nanozyme obtained in (3) at a ratio of 1 mL:10 mg for 12 h, and then centrifuge and wash to obtain hollow spherical single-atom nanozyme, that is, Cu HSAz.

[0070] (5) The hollow spherical single-atom nanozyme obtained in (4) was freeze-dried and then resuspended in an ethanol-water solvent for the first time. The volume ratio of water to ethanol was 1:1. 1 mg / mL of N,N-di-sec-butyl-N,N-dinitroso-1,4-benzenediamine (BNN6) was added under stirring to load nitric oxide donor molecules into the hollow of the hollow spherical single-atom nanozyme. After stirring at room temperature for 5 h, the reaction solution was centrifuged at 10,000 rpm for 15 min to obtain a precipitate. After washing twice with water, it was resuspended in 1 mL of PBS buffer solution with a pH of 7.2 for the second time to obtain a hollow single-atom nanozyme with light-controlled nitric oxide release, namely Cu HSAz@BNN6.

[0071] Example 2. A preparation method of a hollow single-atom nanozyme with light-controlled nitric oxide release

[0072] (1) 90 mL of absolute ethanol, 10 mL of pure water and 2 mL of ammonia water were successively measured, stirred and mixed for 10 min, and 5.4 mL of tetraethyl orthosilicate (TEOS) was added. The reaction was carried out in a water bath at 48 °C for 2.5 h. After the reaction, a milky white precipitate was obtained by centrifugation and washed twice with a mixed solution of water and ethanol with a volume ratio of 1:1 at a rotation speed of 10,000 rmp for 10 min. The precipitated spherical silica nanoparticles were collected.

[0073] (2) Dopamine hydrochloride, the spherical silica nanoparticles obtained in (2), and copper(II) chloride dihydrate were mixed and reacted at a mass ratio of 10:3:1 under alkaline conditions at room temperature, that is, in a Tris buffer solution with a concentration of 10 mM and a pH of 8.8, for 24 h, so that a structure of copper ion-poly(dopamine) coated silica nanoparticles was formed on the surface of the spherical silica nanoparticles through the self-polymerization of dopamine and the chelation of copper ions. Then, a precipitate was obtained by centrifugation and washed twice with water at a rotation speed of 12,000 rmp for 10 min. The precipitate of copper ion-poly(dopamine) coated silica nanoparticles, namely SiO 2 @PDA-Cu, was collected.

[0074] (3) After the copper ion-poly(dopamine) coated silica nanoparticles obtained in (2) were freeze-dried, gradient temperature pyrolysis was carried out under a nitrogen atmosphere. First, it was heated to 350 °C and kept warm for 3 h, and then heated to 800 °C for calcination pyrolysis for 2 h to obtain a copper-based single-atom nanozyme with carbon as the skeleton, namely SiO 2 @Cu-SAz.

[0075] (4) An etching solution was prepared by mixing hydrofluoric acid and hydrogen fluoride at a volume-mass ratio of 3 mL:6 mg. The etching solution was used to etch the copper-based single-atom nanozyme obtained in (3) at a ratio of 1 mL:10 mg for 12 h, and then centrifuged and washed to obtain hollow spherical single-atom nanozyme, namely Cu HSAz.

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

[0077] Example 3. A preparation method of a photo-controlled nitric oxide-releasing hollow single-atom nanozyme

[0078] (1) Measure 100 mL of absolute ethanol, 20 mL of pure water, and 12 mL of ammonia water in sequence, stir and mix for 10 min, add 5.4 mL of tetraethyl orthosilicate (TEOS), and react in a water bath at 60 °C for 3 h; after the reaction, centrifuge to obtain a milky white precipitate, and wash it twice with a mixed solution of water and ethanol with a volume ratio of 1:1, the rotation speed is 10,000 rmp, and the time is 10 min. Collect the precipitate to obtain spherical silica nanoparticles.

[0079] (2) Mix and react the hydrochloride dopamine, the spherical silica nanoparticles obtained in (2), and copper(II) chloride dihydrate at a mass ratio of 20:10:5 under alkaline conditions at room temperature, that is, in a Tris buffer solution with a concentration of 11 mM and a pH of 8.9, for 24 h, so that a structure of copper ion-poly dopamine-coated silica nanoparticles is formed on the surface of the spherical silica nanoparticles through the self-polymerization of dopamine and the chelation of copper ions. Then centrifuge to obtain a precipitate, wash it twice with water, the rotation speed is 12,000 rmp, and the time is 10 min. Collect the precipitate to obtain copper ion-poly dopamine-coated silica nanoparticles, namely SiO 2 @PDA-Cu.

[0080] (3) After freeze-drying the copper ion-poly dopamine-coated silica nanoparticles obtained in (2), perform gradient heating pyrolysis under a nitrogen atmosphere. First, heat up to 360 °C and keep it warm for 3.5 h, and then heat up to 810 °C and calcine and pyrolyze for 2.5 h to obtain a carbon-based copper-based single-atom nanozyme, namely SiO 2 @Cu-SAz.

[0081] (4) Prepare an etching solution by mixing hydrofluoric acid and fluoride in a volume-to-mass ratio of 5 mL:10 mg. Use this etching solution to etch the copper-based single-atom nanozyme obtained in (3) in a ratio of 5 mL:2 mg for 24 h, and then centrifuge and wash to obtain hollow spherical single-atom nanozyme, namely Cu HSAz.

[0082] (5) After freeze-drying the hollow spherical single-atom nanozyme obtained in (4), resuspend it for the first time in an ethanol-water solvent with a volume ratio of water to ethanol of 1.5:1. Add 1 mg / mL N,N-di-sec-butyl-N,N-dinitroso-1,4-benzenediamine (BNN6) under stirring to load the nitric oxide donor molecule into the hollow of the hollow spherical single-atom nanozyme. After stirring at room temperature for 5 h, centrifuge the reaction solution at 10000 rpm for 15 min to obtain a precipitate. Wash it twice with water and then resuspend it for the second time in 1 mL of PBS buffer solution with a pH of 7.6 to obtain a photo-controlled nitric oxide-releasing hollow single-atom nanozyme, namely Cu HSAz@BNN6.

[0083] The preparation method of N,N-di-sec-butyl-N,N-dinitroso-1,4-benzenediamine, namely BNN6, is as follows: Dilute N,N'-di-sec-butyl-p-phenylenediamine, namely BPA, 2.34 mL, 10 mmol to 18 mL with ethanol. Set up a constant-pressure dropping funnel device, fill it with nitrogen, and add 20 mL of degassed 6M NaNO 2 solution. Continue stirring for 30 min, and then use the constant-pressure dropping funnel to drop 20 mL of degassed 6M HCl solution. After reacting for 4 h, centrifuge at a speed of 10000 rpm for 30 min to collect the light yellow precipitate. Wash it repeatedly with 50% ethanol aqueous solution to remove the excess reactants. Freeze-dry the final product and store it in the dark at -20 °C for further use.

[0084] It should be noted that when the nitric oxide donor molecule is sodium nitroprusside, S-nitrosoglutathione, glycol dinitrenium, or N,N'-di-sec-butyl-N,N'-dinitroso-1,4-benzenediamine, it can be used to prepare the photo-controlled nitric oxide-releasing hollow single-atom nanozyme.

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

[0086] Perform transmission electron microscopy observation on the Cu HSAz obtained in Example 1.

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

[0088] The aberration-corrected high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) was used to observe the Cu HSAz obtained in Example 1.

[0089] The results are shown in Figure 2 As shown, the aberration-corrected HAADF-STEM observation can clearly observe the well-exposed and dispersed single metal atoms. This is due to the strong metal-support interaction generated by the interfacial bonds brought about by high-temperature pyrolysis. After high-temperature reduction, the metal loses its adsorption ability for small molecules, which proves the successful formation of the single-atom structure.

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

[0091] The results are shown in Figure 3 As shown, the particle size distribution observation shows that the average particle size of the hollow spherical single-atom nanozyme is 224.91 ± 22.17 nm.

[0092] The X-ray powder diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) of the Cu HSAz obtained in Example 1 were observed.

[0093] The results are shown in Figure 4 As shown, where Figure 4 A in is the XRD pattern of Cu HSAz, Figure 4 B in is the Cu 2p3 / 2 sub-spectrum of the XPS of Cu HSAz, Figure 4 C in is the N1s sub-spectrum of Cu HSAz, Figure 4 D in is the C1s sub-spectrum of Cu HSAz.

[0094] The XRD pattern shows that Cu HSAz has an XRD pattern similar to that of typical graphene crystal form, which proves the unique existence form of single-atom copper in the material.

[0095] The peak fitting results of the XPS show that the Cu 2p3 / 2 characteristic peaks are mainly concentrated between 931 eV and 932 eV, which proves that the valence state of copper in Cu HSAz is mainly 0 to +1, and the lower valence state has excellent catalytic performance. By analyzing the peak fitting results of the binding energy in the C1s and N1s regions, it is proved that the existing forms of N element are pyrrolic nitrogen, pyridinic nitrogen, Cu-N, and graphitic nitrogen; the C element mainly exists in the form of graphitic carbon. These results all verify the formation of the single-atom structure.

[0096] In Experimental Example 2, the changes in the ultraviolet absorption spectra of the Cu HSAz obtained in Example 1 before and after loading the nitric oxide donor molecule BNN6 were measured.

[0097] Disperse different samples in water to prepare a dispersion solution with a concentration of 200 μg / mL. Place 800 μL of the sample to be tested in a clean quartz cuvette with light transmission on both sides. Set the detection wavelength at 200 nm, and collect the ultraviolet-near-infrared-visible light absorption spectrum of the sample at 25 °C using a UV-spectrophotometer.

[0098] Loading rate of BNN6: Dissolve 5 mg of BNN6 in 1 mL of ethanol-water solvent (water:ethanol = 1:1), and dilute it to concentrations of 0 μg / mL, 10 μg / mL, 15 μg / mL, 25 μg / mL, 50 μg / mL, 75 μg / mL, and 100 μg / mL respectively. Then, use a UV-spectrophotometer to record the ultraviolet absorption curves of BNN6 solutions with different concentrations at 200 nm. Read the absorbance values of BNN6 solutions with different concentrations at 214 nm, plot a positive correlation curve of concentration-absorbance value, and obtain the concentration-absorbance value standard curve of BNN6. After the material is loaded with BNN6, centrifuge to collect 1 mL of the supernatant, record its absorbance value at 214 nm, calculate the content of BNN6 in 1 mL of the supernatant according to the standard curve, and combine the total volume of the supernatant to know the total amount of unloaded BNN6. The loading rate of BNN6 can be obtained through the following formula:

[0099]

[0100] The results are as Figure 5 shown. After loading the donor molecule BNN6, an obvious absorption peak appears at 230 nm for the material, which proves the successful loading of BNN6. The loading rate of BNN6 on the material was further studied through the BNN6 concentration-absorbance standard curve. Calculate the concentration of free BNN6 in the supernatant after the material is loaded, and it can be known that the loading rate of BNN6 in the material is about 38.7%.

[0101] Experimental Example 3: Measure the photothermal performance of Cu HSAz@BNN6 obtained in Example 1.

[0102] Use a 1064 nm laser in the second near-infrared region to verify the photothermal performance of Cu HSAz@BNN6. Take PBS as the control group, and Cu HSAz and Cu HSAz@BNN6 (200 μg / mL) as the experimental groups. Detect the photothermal effect of the materials irradiated with a 1064 nm laser (0.5 W / cm 2 ) for 3 min through an infrared thermal imaging camera.

[0103] Effect of Concentration on the Photothermal Performance of the Material: The sample to be tested was dispersed in PBS buffer solution with pH 7.4 to prepare solutions with different concentrations, namely 0 μg / mL, 100 μg / mL, 200 μg / mL, 300 μg / mL, and 400 μg / mL, and ultrasonically dispersed evenly. Take 200 μL of each concentration sample into a 96-well plate, insert the thermocouple thermometer probe, and irradiate the sample with a 1064 nm near-infrared second-region laser with a power of 0.5 W / cm 2 for 5 min at room temperature, and record the real-time temperature of the solution every 10 s. Plot a temperature-time curve with time as the abscissa and temperature as the ordinate to compare the photothermal performance of the samples at different concentrations.

[0104] Effect of Laser Power on the Photothermal Performance of the Material: The sample to be tested was dispersed in PBS with pH 7.4 to prepare a 200 μg / mL solution. Take 200 μL of the sample into a 96-well plate, and irradiate the sample with 1064 nm lasers with powers of 0.3 W / cm 2 , 0.5 W / cm 2 , 0.8 W / cm 2 , and 1 W / cm 2 at room temperature for 5 min, and record once every 10 s. Plot a temperature-time curve with time as the abscissa and temperature as the ordinate to compare the photothermal performance of the samples irradiated with lasers of different powers.

[0105] Photothermal Stability of the Material: The sample to be tested was dispersed in PBS with pH 7.4 to prepare a 200 μg / mL solution. Take 200 μL of the sample into a 96-well plate, insert the thermocouple thermometer probe into the liquid surface, and irradiate the liquid surface of the solution with a 1064 nm laser for 5 min (0.5 W / cm 2 ). Then turn off the laser to let the solution return to the temperature before measurement, repeat this experimental step 4 times, and record the temperature every 10 s during this process. Plot a temperature-time curve with time as the abscissa and temperature as the ordinate to verify the photothermal stability of the sample.

[0106] The results are as Figure 6 shown, where Figure 6 A in Figure 6 is the infrared thermal imaging diagram of Cu HSAz@BNN6, Figure 6 B in Figure 6 is the time-temperature curve of Cu HSAz@BNN6 with different powers,

[0107] C in Figure 6As shown in A, the temperature of Cu HSAz and Cu HSAz@BNN6 rose to 45 °C after being irradiated with a 1064 nm laser for 3 min. In contrast, the temperature of the control group remained almost unchanged after laser irradiation. At the same time, the effects of different laser powers and different material concentrations on the photothermal performance were further measured. As Figure 6 shown in B of 2 , the power of the 1064 nm laser was controlled to be 0 W / cm 2 , 0.3 W / cm 2 , 0.5 W / cm 2 , 0.8 W / cm 2 , and the concentration of the Cu HSAz@BNN6 material was 200 μg / mL. After irradiation for 3 min, the temperatures rose to 28 °C, 35 °C, 44 °C, 56 °C, and 67 °C respectively; as Figure 6 shown in C of 2 , the power of the 1064 nm laser was controlled to be 0.5 W / cm 2 , and the material concentrations were 0 μg / mL, 100 μg / mL, 200 μg / mL, 300 μg / mL, and 400 μg / mL respectively. After irradiation for 3 min, the temperatures rose to 28 °C, 34 °C, 45 °C, 55 °C, and 58 °C respectively. These results fully illustrate that Cu HSAz@BNN6 has good photothermal performance, and the temperature rise is affected by the laser power and the material concentration and shows a positive correlation. Finally, the photothermal stability of Cu HSAz@BNN6 was determined by controlling the intermittent irradiation of the laser on Cu HSAz@BNN6 and recording the temperature change. As Figure 6 shown in D of 2 , irradiating Cu HSAz@BNN6 (200 μg / mL) with a 1064 nm laser (0.5 W / cm 2 ) caused the temperature to rise to 45 °C, and then the laser was turned off to cool it down to room temperature. Five cycles were repeated, verifying that the material has good photothermal stability, which makes a good foundation for the photothermal control of the release of NO gas by the donor molecule.

[0108] Experimental Example 4: The nitric oxide generation performance of the obtained Cu HSAz@BNN6 in Example 1 was measured.

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

[0110] NO Release Detection: By recording the absorbance changes at 540 nm after the reaction of NO at different concentrations, namely 0 μM, 0.78 μM, 1.56 μM, 3.13 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, and 100 μM, with Griess reagent, a standard curve of NO concentration - absorbance was plotted. Take two samples with the same concentration (200 μg / mL) and add the same volume of Griess reagent. Among them, one was not treated; the other was irradiated with a 1064 nm laser at a power of 0.5 W / cm 2 , for 3 min; the absorbance changes at 540 nm at the corresponding time points were measured with a UV - visible spectrophotometer at room temperature, and the generated NO concentration was calculated according to the standard curve.

[0111] NIR - II Laser - Controlled NO Release Performance: The ability of NO NIRⅡ controlled release was measured by intermittently irradiating with a 1064 nm laser at a power of 0.5 W / cm 2 .

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

[0113] As shown in Figure 7 A of 2 , after irradiating CuHSAz@BNN6 (200 μg / mL) with a 1064 nm laser (0.5 W / cm 2 ), the material released a large amount of NO, making the Griess reagent turn red. Moreover, with the laser irradiation, the concentration of NO gradually increased and reached 25 μM at 20 min. While the control group without laser irradiation only released a trace amount of NO, and the concentration was 4.5 μM after 20 min. As shown in Figure 7 B of Figure 7 , when irradiated with a laser, the material could respond to photothermal and release a large amount of NO. When there was no laser irradiation, the rate of NO release slowed down significantly, and this result remained consistent in multiple cycles, forming a "switch curve" for photo - controlled NO release from Cu HSAz@BNN6.

[0114] Experimental Example 5: The performance of superoxide anion O 2 ·- and peroxynitrite RNS generation of the Cu HSAz@BNN6 obtained in Example 1 was measured.

[0115] The performance of the material in generating superoxide anions was characterized by a superoxide anion content detection kit and a cis - electron spin resonance spectrometer, namely EMS·Plus. The change in the content of superoxide anions generated by the sample (200 μg / mL) over time within 30 min was evaluated using the superoxide anion content detection kit. Using DMPO as a probe, the characteristic spectrogram of superoxide anions was obtained by the cis - electron spin resonance spectrometer.

[0116] The ability of the material to generate RNS was detected by the change in fluorescence intensity at 500 nm - 600 nm under 488 nm excitation after the combination of DHR123 and peroxynitrite RNS. 200 μL of the material sample at 200 μg / mL was added to a 96 - well plate, and then 10 μM of the DHR123 fluorescent probe was added. After incubating in the dark for half an hour, the change in fluorescence intensity at 500 nm - 600 nm under 488 nm excitation was detected by a microplate reader to detect the ability of the material to generate RNS.

[0117] The results are as Figure 8 shown, where Figure 8 A in 2 ·- is the time - concentration curve of Cu HSAz@BNN6 releasing O Figure 8 B in 2 ·- is the ESR spectrogram of O Figure 8 C in

[0118] Figure 8 The result of A in 2 ·- shows that the irradiation with a 1064 - nm laser can enhance the generation of O Figure 8 The result of the B cis - electron spin spectrum in 2 ·- shows the specific generation of superoxide anions, and further proves that the irradiation with a 1064 - nm laser promotes the generation of O Figure 8 which is consistent with the result of A in

[0119] Superoxide anions can combine with NO to produce more reactive peroxynitrite RNS. The peroxynitrite - specific fluorescent probe dihydro - rhodamine 123, namely DHR123, will have an obvious fluorescence intensity at 530 nm after reacting with RNS. As shown in Figure 8 C, only the final group Cu HSAz@BNN6 + NIR can significantly generate a large amount of RNS, proving that Cu HSAz@BNN6 has a good catalytic effect under 1064 - nm laser irradiation.

[0120] Experimental Example 6: The in - vitro antibacterial performance of the obtained Cu HSAz@BNN6 in Example 1 was measured.

[0121] After resuscitating the bacteria and culturing them to the logarithmic phase, the absorbance OD at 600 nm was detected using an ultraviolet spectrophotometer. 600 When OD = 1.0, 1 mL of the bacterial suspension was centrifuged and washed multiple times with sterile PBS to thoroughly 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. PBS was used as the Control group, and Cu HSAz, Cu HSAz@BNN6, CuHSAz+NIR, HCN@BNN6+NIR, and Cu HSAz@BNN6+NIR were used as the experimental groups. HCN is a carbon skeleton without loaded copper metal. The power of the 1064 nm laser used was 0.5 W / cm 2 and the irradiation time was 3 min. The treated bacteria in each group were placed in a constant temperature shaker at 37 °C and incubated with shaking for 4 h. Subsequently, they were washed with PBS to remove the excess materials. The bacterial suspension was diluted and spread on a sterilized LB solid agar plate, which was then placed in a biochemical incubator at 37 °C for 12 h. A digital camera was used to take pictures to record the bacterial growth status, and the number of bacterial colonies was calculated using Image J software to determine the antibacterial effect after the material treatment.

[0122] The results are as Figure 9 shown, where Figure 9 A in it is the antibacterial effect of different materials on methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli (E. coli), and Staphylococcus aureus (S. aureus) after treatment, Figure 9 and B, C, and D in it are the bacterial survival rates of MRSA, E. coli, and S. aureus, respectively.

[0123] Figure 9 A in it shows that the clearance effects of the Control, Cu HSAz, and Cu HSAz@BNN6 material groups on the three bacteria without laser irradiation are negligible, indicating that the O released by the materials 2 ·- has relatively limited antibacterial effects. The Cu HSAz and HCN@BNN6 material groups showed certain antibacterial effects after laser irradiation, which may be due to the synergistic effects of photothermal and O 2 ·- or NO. The Cu HSAz@BNN6 group almost completely cleared the bacteria on the agar plate after laser irradiation, which is due to the synergistic effects of photothermal and RNS. As Figure 9 shown by B - D in it, through the counting analysis of the bacteria on the agar plate, the bacteriostatic rates of the Cu HSAz+NIR and HCN@BNN6+NIR groups against MRSA, E. coli, and S. aureus reached about 50%, indicating that the increase in temperature and O 2 ·-Or NO has a certain bactericidal effect. After Cu HSAz@BNN6 is irradiated by NIR laser, it shows the best antibacterial performance against three kinds of bacteria. Almost all the bacteria on the agar plate are completely removed. After counting, the antibacterial rates are found to be 99.89%, 99.78% and 99.91% respectively, indicating that the synergistic effect of photothermal and RNS has the best antibacterial effect.

[0124] Experimental Example 7: The Cu HSAz@BNN6 obtained in Example 1 was measured for its inhibitory performance on biofilm formation.

[0125] Crystal violet dye can stain bacteria after biofilm fixation. By measuring the staining of biofilms treated with different experimental groups, Control, Cu HSAz, Cu HSAz@BNN6, Cu HSAz+NIR, Cu HSAz@BNN6+NIR, the ability of Cu HSAz@BNN6 to promote the dissipation of bacterial biofilms was determined. The bacteria were resuscitated and cultured to the logarithmic phase state, OD 600 =1.0, inoculated into 96-well plates, and placed in a constant temperature biochemical incubator at 37°C for static culture for 48 h. During this period, the fresh medium was replaced every 12 h to allow them to multiply and form a dense bacterial biofilm.

[0126] Different treatments were carried out in different wells where bacterial biofilms had already formed. The power of the 1064 nm laser used was 0.5 W / cm 2 , and the irradiation time was 3 min. After the biofilms were treated differently, they were statically cultured in an incubator at 37°C for 12 hours. After the culture was completed, 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. Subsequently, 1% crystal violet was added for staining and continued to be placed in a constant temperature incubator at 37°C in the dark for incubation. After 30 min, it was washed repeatedly with sterile PBS to remove the free dye, and the dissipation of the biofilm was photographed with a digital camera. 200 μL of anhydrous ethanol was added to the wells. After the crystal violet was completely dissolved, the absorbance at 570 nm was measured by a multifunctional microplate reader to determine the effect of different experimental groups on promoting biofilm dissipation.

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

[0128] As shown Figure 10 in A of [reference], the bacterial biofilms treated with the Control group and Cu HSAz and Cu HSAz@BNN6 materials without laser irradiation were relatively intact, and the crystal violet color was relatively deep; the photothermal therapy group alone, Cu HSAz+NIR, could effectively promote the dissipation of the biofilm, and the crystal violet color was relatively light; after treatment with the Cu HSAz@BNN6 material group irradiated by laser, the bacterial biofilm almost completely dissipated, and the crystal violet color tended to be colorless. As shown Figure 10 in C of [reference], the absorbance values of the Control group and the material group without laser irradiation were relatively high, proving that the biofilm was relatively intact. The absorbance value of the CuHSAz@BNN6 treatment group after laser irradiation was relatively low, proving that the biofilm had almost completely dissipated.

[0129] The ability of Cu HSAz@BNN6 at different concentrations to promote the dissipation of bacterial biofilms was further measured. As shown Figure 10 in B of [reference], the MRSA bacterial biofilm treated with low concentration of Cu HSAz@BNN6 was relatively intact, and the crystal violet color was relatively deep; when the concentration of Cu HSAz@BNN6 reached 200 μg / mL and was treated with laser irradiation, the MRSA bacterial biofilm almost completely dissipated, and the crystal violet color tended to be transparent. As shown Figure 10 in D of [reference], the absorbance of crystal violet decreased significantly with the increase of the concentration of Cu HSAz@BNN6, indicating that increasing the concentration of Cu HSAz@BNN6 could effectively improve the promotion of the dissipation of the MRSA bacterial biofilm.

[0130] The crystal violet staining experiment showed that Cu HSAz@BNN6 could jointly promote the dissipation of bacterial biofilms through photothermal effect and the release of RNS in the bacterial biofilm environment, further confirming the good in vitro antibacterial effect of Cu HSAz@BNN6.

[0131] Experimental Example 8: The wound healing promoting performance of the obtained Cu HSAz@BNN6 in Example 1 was measured.

[0132] After constructing the MRSA-infected wound model, the five groups of animals were given different drug treatments every two days. The PBS was used as the Control group, and Cu HSAz, Cu HSAz@BNN6, Cu HSAz+NIR, and Cu HSAz@BNN6+NIR were used as the experimental groups. The materials were dispersed in sterile PBS at a concentration of 200 μg / mL. The same operations were performed on each group of animals. On the 0th, 3rd, 5th, 7th, and 10th days after drug administration, the wound diameters on the backs of the rats in each group were measured with a precision steel ruler and photographed with a digital camera. The wound areas of the rats in each group were statistically analyzed using Image J software, and the wound area change diagrams were drawn.

[0133] The results are as Figure 11 shown, where Figure 11 A in Figure 11 is the picture of the wound of rats at different days after treatment with different materials,

[0134] As Figure 11 shown in A in Figure 11 , in the Control, Cu HSAz, and Cu HSAz@BNN6 groups, the wounds of rats were still not closed after treatment and the healing was relatively slow. The wounds of rats in the CuHSAz+NIR group were relatively close to closing, which may be due to the photothermal effect removing some bacteria and accelerating wound healing. While the wounds of rats in the Cu HSAz@BNN6+NIR group were completely closed and the wound healing speed increased significantly. This result is attributed to the synergistic bactericidal effect of photothermal and RNS. By statistically analyzing the area of the wounds of rats after treatment with different materials, as

[0135] shown in B in

[0136] , due to the photothermal effect, the wound closure rate of rats in the Cu HSAz+NIR group reached 60% after 7 days and 70% after 10 days. While the wound closure speed of rats in the Cu HSAz@BNN6+NIR group brought by the synergistic effect of photothermal and RNS was significantly improved, and the wound closure rate of the Cu HSAz@BNN6+NIR group reached 96% after 10 days, indicating that the synergistic effect of photothermal and RNS helps to promote the healing of infected wounds.

[0135] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope recorded in this specification.

[0136] The above-described embodiments only represent several implementation manners of the present invention. Their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several deformations and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent should be subject to the appended claims.

Claims

1. A light-controlled nitric oxide-releasing hollow single-atom nanozyme, characterized in that: Spherical silica nanoparticles are used as templates, and a structure of copper ions-polydopamine coated silica nanoparticles is formed on the surface through the self-polymerization of dopamine and the 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 nanozyme with a carbon skeleton, and a hollow spherical single-atom nanozyme is obtained after etching; a nitric oxide donor molecule is loaded into the hollow core of the hollow spherical single-atom nanozyme to obtain a light-controlled nitric oxide releasing hollow single-atom nanozyme.

2. The method for preparing a light-controlled nitric oxide-releasing hollow single-atom nanozyme according to claim 1, characterized in that: The specific steps include: (1) mixing dopamine hydrochloride, spherical silica nanoparticles and copper chloride dihydrate to react, so that the surface of the spherical silica nanoparticles forms a structure of copper ions-polydopamine wrapped silica nanoparticles through self-polymerization of dopamine and chelation of copper ions; centrifuging, washing, and collecting the precipitate to obtain copper ions-polydopamine wrapped silica nanoparticles; the mass ratio of dopamine hydrochloride, spherical silica nanoparticles and copper chloride dihydrate is 10-20:3-10:1-5; (2) freeze-drying the copper ion-polydopamine-encapsulated silica nanoparticles obtained in (1), and pyrolyzing them under a pyrolysis environment to obtain a copper-based single-atom nanozyme with a carbon skeleton; (3) using an etching solution to etch the copper-based single-atom nanozyme obtained in (2) for 12 h to 24 h, and centrifugally washing to obtain a hollow spherical single-atom nanozyme; the volume mass ratio of the etching solution to the copper-based single-atom nanozyme is 1 to 5:10 to 20; (4) freeze-drying the hollow spherical single-atom nanozyme obtained in (3) and resuspending it for the first time, stirring and adding a nitric oxide donor molecule so that the hollow core of the hollow spherical single-atom nanozyme is loaded with the nitric oxide donor molecule; After centrifugation, washing, and a second resuspension, a light-controlled nitric oxide-releasing hollow single-atom nanozyme was obtained.

3. The preparation method according to claim 2, characterized in that: The pyrolysis environment conditions are: firstly heating to 340°C-360°C in a nitrogen atmosphere and keeping the temperature for 2.5h-3.5h, then heating to 790°C-810°C and calcining and pyrolysis for 1.5h-2.5h.

4. The preparation method according to claim 2, characterized in that: The etching solution is obtained by mixing hydrofluoric acid and ammonium fluoride in a volume mass ratio of 1-5:2-10.

5. The preparation method according to claim 2, characterized in that: 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; The concentration of the nitric oxide donor is 1 mg / mL to 5 mg / mL.

6. The preparation method according to claim 2, characterized in that: The mixed reaction condition is alkaline condition at room temperature; the alkaline condition at room temperature is provided by Tris buffer with a concentration of 9mM to 11mM and a pH of 8.7 to 8.

9.

7. The preparation method according to claim 2, characterized in that: The reagent for the first resuspension is a mixed solution of pure water and anhydrous ethanol; the volume ratio of the pure water to the anhydrous ethanol is 1-1.5:1; the reagent for the second resuspension is a PBS buffer solution with a pH of 7.2-7.

6.

8. The preparation method according to claim 2, characterized in that: The preparation method of the spherical silicon dioxide nanoparticles is as follows: Anhydrous ethanol, pure water and ammonia water are mixed, tetraethyl silicate is added, and the mixture is reacted at 45°C to 60°C for 2h to 3h, and the mixture is centrifuged and washed, and the precipitate is collected to obtain 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 silicate to ammonia water is 1.0 to 2.0:1.

0.

9. The preparation method according to claim 2, characterized in that: The nitric oxide donor molecule comprises at least one of sodium nitroprusside, S-nitrosoglutathione, diazeniumdiolate and N,N'-di-sec-butyl-N,N'-dinitroso-1,4-phenylenediamine.

10. Use of the light-controlled nitric oxide-releasing hollow single-atom nanozyme according to claim 1 in the preparation of biomedical materials.

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