Theaflavin photothermal targeted nano-enzyme, and preparation method and application thereof

Abstract

The application discloses a theaflavins photothermal targeted nano-enzyme as well as a preparation method and application thereof. Theaflavins are used as a reducing agent and a capping agent, and the nano-enzyme of cerium oxide can be synthesized without high temperature and high pressure, and is coordinated with iron ions to construct a metal polyphenol network to coat the nano-enzyme of cerium oxide, and the nano-enzyme is further modified by trehalose which has affinity to bacteria, so that a novel photothermal targeted nano-enzyme is developed. The theaflavins photothermal targeted nano-enzyme has good antioxidant, peroxidase-like, photothermal and antibacterial effects, and can be used for the treatment of bacterial infection.

Description

Technical Field

[0001] This invention belongs to the field of medicine, and specifically relates to a theaflavin photothermal targeted nanozyme, its preparation method, and its application. Background Technology

[0002] In the field of antimicrobial therapy, the excessive and irrational use of antibiotics has led to the emergence and rapid evolution of drug-resistant strains, resulting in a significant decline in the efficacy of traditional antibiotics, or even their complete ineffectiveness, seriously threatening human health and safety. Chemidynamic antimicrobial therapy and photothermal antimicrobial therapy, as emerging antimicrobial strategies, show great potential. Chemidynamic antimicrobial therapy effectively destroys bacterial cell membranes and biomolecules by generating reactive oxygen species (ROS), thereby killing bacteria. Photothermal antimicrobial therapy utilizes photothermal reagents to convert light energy into heat energy, raising the local temperature to achieve sterilization. Both possess highly efficient and broad-spectrum antimicrobial properties and are less likely to induce bacterial resistance. However, currently, such drugs are scarce, their preparation processes are complex, and their prices are high, making industrialization difficult.

[0003] Nanozymes, as novel antibacterial agents, have become a research hotspot. Among them, cerium oxide nanozymes, due to the reversible switching of cerium ion valence states, possess multiple enzyme activities such as superoxide dismutase, peroxidase, and oxidase, and can be used for antibacterial applications. Chinese patent application CN120887448A discloses a method for preparing amino-functionalized cerium oxide nanozymes and its application, and CN117085737A discloses a cerium oxide nanozyme derivative and its preparation method, which respectively employ hydrothermal and precipitation methods. However, the preparation process requires high temperature and high pressure, as well as structural modification and other synthetic processes, making the preparation complex and difficult to industrialize. Furthermore, the products are prone to agglomeration, resulting in low and unstable activity. Summary of the Invention

[0004] The primary objective of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a theaflavin photothermal targeted nanoenzyme.

[0005] Another object of the present invention is to provide a method for preparing the theaflavin photothermal targeted nanozyme.

[0006] Another object of the present invention is to provide the application of the theaflavin photothermal targeted nanoenzyme.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] A theaflavin photothermal targeted nanoenzyme is a nanostructured complex formed by using theaflavin as a reducing agent to prepare nano-cerium oxide as the core, a metal polyphenol network constructed from theaflavin and iron ions as the shell, and loading trehalose on the outer surface.

[0009] The theaflavin photothermal targeted nanozyme is preferably prepared by the following method:

[0010] (1) Under stirring conditions, theaflavin aqueous solution was added dropwise to cerium nitrate aqueous solution to carry out the reaction. After the reaction was completed, the precipitate was collected by centrifugation, washed, and dried to obtain cerium oxide nanoparticles (TFs-CeO2 NPs).

[0011] (2) The cerium oxide nanoparticles (TFs-CeO2 NPs) obtained in step (1) were added to water, and then theaflavin aqueous solution and ferric chloride aqueous solution were added dropwise under stirring to carry out the reaction. The reaction was then transferred to ultrasound to continue the reaction. After the reaction was completed, the precipitate was collected by centrifugation, washed, and dried to obtain cerium oxide nanoparticles (TFs / Fe-CeO2 NPs) coated with theaflavin iron-based metal polyphenol network.

[0012] (3) The theaflavin iron-based metal polyphenol network-coated cerium oxide nanoparticles (TFs / Fe-CeO2 NPs) obtained in step (2) were added to water, and then trehalose aqueous solution was added. The reaction was stirred at room temperature. After the reaction was completed, the precipitate was collected by centrifugation, washed, and dried to obtain theaflavin photothermal targeted nanoenzyme (Tre-TFs / Fe-CeO2 NPs).

[0013] Preferably, the water mentioned in steps (1), (2) and (3) is deionized water.

[0014] Preferably, the cerium nitrate mentioned in step (1) is cerium nitrate hexahydrate.

[0015] Preferably, the concentration of the cerium nitrate aqueous solution in step (1) is 0.05 to 0.15 mol / L; more preferably, it is 0.1 mol / L.

[0016] Preferably, the concentration of theaflavins in water in step (1) is 0.05 to 0.15 mg / mL; more preferably, it is 0.1 mg / mL.

[0017] Preferably, the volume ratio of the cerium nitrate aqueous solution to the theaflavin aqueous solution in step (1) is 1:(3-6); more preferably, it is 1:6.

[0018] Preferably, the stirring speed in step (1) is 300-600 r / min.

[0019] Preferably, the dripping in step (1) is a slow dripping, with a dripping rate of 5 to 15 mL / min.

[0020] Preferably, the reaction time in step (1) is 10 to 15 hours; more preferably, it is 12 hours.

[0021] Preferably, the centrifugation conditions described in steps (1), (2) and (3) are: centrifugation speed of 8000-12000 r / min and centrifugation time of 5-10 min.

[0022] Preferably, the washing in step (1) is performed using deionized water; more preferably, the washing is performed 2 to 3 times with deionized water, and the amount of deionized water used each time is 1 / 2 to 1 / 3 times the volume of the theaflavins solution.

[0023] Preferably, the drying process described in steps (1), (2) and (3) is freeze drying.

[0024] Preferably, the freeze-drying time is 36 to 48 hours.

[0025] Preferably, the mass ratio of cerium oxide nanoparticles (TFs-CeO2 NPs), theaflavins and ferric chloride in step (2) is 1:(5-10):(5-10); more preferably 1:7.5:7.5.

[0026] Preferably, the amount of cerium oxide nanoparticles used in step (2) is calculated as 50-100 μg of cerium oxide nanoparticles per milliliter of water (the concentration of the cerium oxide nanoparticle suspension is 50-100 μg / mL).

[0027] Preferably, the concentration of the theaflavins aqueous solution in step (2) is 0.5 to 1.5 mg / mL.

[0028] Preferably, the concentration of the ferric chloride aqueous solution in step (2) is 0.5 to 1.5 mg / mL.

[0029] Preferably, the stirring speed in step (2) is 300-600 r / min.

[0030] Preferably, the dropping rate in step (2) is 20 to 40 mL / min.

[0031] Preferably, the power of the ultrasound in step (2) is 200-300W.

[0032] Preferably, the reaction time in step (2) is 20 to 40 minutes.

[0033] Preferably, the reaction time in step (2) is 10 to 20 minutes.

[0034] Preferably, the addition of theaflavins aqueous solution and ferric chloride aqueous solution in step (2) is achieved by the following steps: first, theaflavins aqueous solution is added dropwise at a rate of 20-40 mL / min, and stirring is continued for 10-20 min; then, ferric chloride is added dropwise at a rate of 20-40 mL / min, and stirring is continued for 10-20 min; then, the mixture is transferred to ultrasonic conditions and the reaction continues for 10-20 min, with an ultrasonic power of 200-300 W.

[0035] Preferably, the washing in step (2) is performed using deionized water; more preferably, the washing is performed 2 to 3 times with deionized water, and the amount of deionized water used each time is 1 / 3 to 1 times the volume of the ferric chloride aqueous solution.

[0036] Preferably, the mass ratio of the theaflavin iron-based metal polyphenol network-coated cerium oxide nanoparticles (TFs / Fe-CeO2 NPs) to trehalose in step (3) is 1:(20-40); more preferably 1:30.

[0037] Preferably, the amount of cerium oxide nanoparticles coated with the theaflavin iron-based metal polyphenol network in step (3) is calculated as 40-60 μg of theaflavin iron-based metal polyphenol network coated with cerium oxide nanoparticles per milliliter of water (concentration of 40-60 μg / mL).

[0038] Preferably, the concentration of the trehalose aqueous solution in step (3) is 2-4 mg / mL.

[0039] Preferably, the conditions for the stirring reaction in step (3) are: stirring speed of 400-600 r / min and reaction time of 4-8 h.

[0040] Preferably, the washing in step (3) is performed using deionized water; more preferably, the washing is performed 2 to 3 times with deionized water, and the amount of deionized water used each time is 1 / 3 to 1 times the volume of the trehalose aqueous solution.

[0041] The application of the theaflavin photothermal targeted nanozyme in the preparation of antibacterial products, antioxidant products, photothermal materials and / or peroxidase-like products.

[0042] The theaflavin photothermal targeted nanoenzyme has good antioxidant, peroxidase-like, photothermal and antibacterial effects, and can be used to treat bacterial infections.

[0043] Preferably, the bacteria are bacteria; more preferably, Escherichia coli and / or Staphylococcus aureus; and even more preferably, Staphylococcus aureus.

[0044] The principle of this invention:

[0045] Theaflavins (TFs), as natural polyphenols, possess excellent biocompatibility and antibacterial and antioxidant capabilities. As an oxidized polymer of tea polyphenols, they exhibit better stability and unique bioactivity than tea polyphenols. They assemble with metal ions through chelation to form a metallopolyphenol network (MPN), which possesses antibacterial, antioxidant, and photothermal functional properties. Using theaflavins as a reducing agent and end-capping agent, cerium oxide nanozymes can be synthesized without high temperature and pressure. These nanozymes are then coated with iron ions to form a metallopolyphenol network. Further modification of the nanozymes with trehalose, which has an affinity for bacteria, develops a novel photothermal targeted nanozyme, overcoming the shortcomings of traditional antibacterial strategies and providing an innovative solution for the antibacterial field. Under near-infrared light irradiation, the MPN absorbs photoelectrons, generating a photothermal effect and releasing nano-cerium oxide. Both iron ions and nano-cerium oxide can react with hydrogen peroxide produced by bacteria, generating more hydroxyl radicals, thereby efficiently killing bacteria.

[0046] The present invention has the following advantages and effects compared with the prior art:

[0047] (1) This invention utilizes theaflavins to synthesize nano-cerium oxide nanoenzymes in a green manner. The preparation conditions are mild, the process is simple, and it is suitable for industrial production.

[0048] (2) This invention combines a metal polyphenol network with cerium oxide nanozymes to give it excellent photothermal properties and uses trehalose to target bacteria.

[0049] (3) The theaflavin photothermal targeted nanozyme provided by the present invention has excellent antibacterial effect and can be used in the field of antibacterial therapy. Attached Figure Description

[0050] Figure 1 The infrared absorption spectrum of the theaflavin photothermal targeted nanozyme.

[0051] Figure 2 SEM image of the theaflavin photothermal targeted nanozyme.

[0052] Figure 3 This is a graph showing the effect of peroxidase-like activity assay.

[0053] Figure 4 This is a graph showing the photothermal temperature rise under 808nm laser irradiation. Detailed Implementation

[0054] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Test methods in the following embodiments that do not specify specific experimental conditions are generally performed according to conventional experimental conditions or experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the present invention are commercially available.

[0055] The cerium nitrate hexahydrate, theaflavins, ferric chloride, and trehalose mentioned in the examples were all purchased from Guangzhou Reagent Company.

[0056] Example 1

[0057] (1) Accurately weigh 3.2567 g of cerium nitrate hexahydrate, dissolve it in 75 mL of deionized water to obtain a 0.1 mol / L cerium nitrate solution, and stir continuously at 600 r / min at room temperature. Slowly add 450 mL of 0.1 mg / mL theaflavin aqueous solution at a dropping rate of 5 mL / min, and continue stirring for 12 h. After the reaction is completed, centrifuge at 12000 r / min for 5 min, collect the precipitate, wash it three times with 150 mL of deionized water, and freeze-dry for 36 h to obtain 40.2 mg of cerium oxide nanoparticles (TFs-CeO2 NPs).

[0058] (2) Take 20 mg of cerium oxide nanoparticles obtained in step (1), add 300 mL of deionized water, stir continuously at 600 r / min at room temperature, add 150 mL of 1 mg / mL theaflavin aqueous solution at a dropping rate of 30 mL / min, and continue stirring for 10 min. Then slowly add 150 mL of 1 mg / mL ferric chloride aqueous solution at a dropping rate of 20 mL / min. After stirring for 10 min, transfer to ultrasound and continue the reaction for 10 min at an ultrasound power of 300 W. After the reaction is completed, centrifuge at 12000 r / min for 5 min, collect the precipitate, wash it three times with 50 mL of deionized water, and freeze-dry for 36 h to obtain 23.6 mg of theaflavin iron-based metal polyphenol network-coated cerium oxide nanoparticles (TFs / Fe-CeO2 NPs).

[0059] (3) Take 10 mg of the theaflavin iron-based metal polyphenol network-coated cerium oxide nanoparticles obtained in step (2), add 200 mL of deionized water, and stir to disperse. Add 100 mL of 3 mg / mL trehalose aqueous solution, stir at 500 r / min for 4 h at room temperature, and centrifuge at 12000 r / min for 5 min after the reaction is completed. Collect the precipitate, wash it 3 times with 50 mL of deionized water, and freeze-dry for 36 h to obtain 8.7 mg of theaflavin photothermal targeted nanozyme (Tre-TFs / Fe-CeO2 NPs).

[0060] Example 2

[0061] (1) Accurately weigh 4.8850 g of cerium nitrate hexahydrate, dissolve it in 75 mL of deionized water to obtain a 0.15 mol / L cerium nitrate solution, and stir continuously at 300 r / min at room temperature. Slowly add 450 mL of 0.05 mg / mL theaflavin aqueous solution at a dropping rate of 10 mL / min, and continue stirring for 10 h. After the reaction is completed, centrifuge at 10000 r / min for 10 min, collect the precipitate, wash it twice with 150 mL of deionized water, and freeze dry for 42 h to obtain 16.3 mg of cerium oxide nanoparticles (TFs-CeO2 NPs).

[0062] (2) Take 10 mg of cerium oxide nanoparticles obtained in step (1), add 100 mL of deionized water, stir continuously at 500 r / min at room temperature, add 200 mL of 0.5 mg / mL theaflavin aqueous solution at a dropping rate of 40 mL / min, and continue stirring for 20 min. Then slowly add 33 mL of 1.5 mg / mL ferric chloride aqueous solution at a dropping rate of 20 mL / min. After stirring for 15 min, transfer to ultrasound and continue the reaction for 20 min at an ultrasound power of 200 W. After the reaction is completed, centrifuge at 8000 r / min for 8 min, collect the precipitate, wash it three times with 33 mL of deionized water, and freeze dry for 48 h to obtain 11.2 mg of theaflavin iron-based metal polyphenol network-coated cerium oxide nanoparticles (TFs / Fe-CeO2 NPs).

[0063] (3) Take 8 mg of the theaflavin iron-based metal polyphenol network-coated cerium oxide nanoparticles obtained in step (2), add 200 mL of deionized water, and stir to disperse. Add 160 mL of 2 mg / mL trehalose aqueous solution, stir at 600 r / min for 8 h at room temperature, and centrifuge at 10000 r / min for 8 min after the reaction is completed. Collect the precipitate, wash twice with 80 mL of deionized water, and freeze-dry for 48 h to obtain 5.4 mg of theaflavin photothermal targeted nanozyme (Tre-TFs / Fe-CeO2 NPs).

[0064] Example 3

[0065] (1) Accurately weigh 3.2567 g of cerium nitrate hexahydrate, dissolve it in 150 mL of deionized water to obtain a 0.05 mol / L cerium nitrate solution, and stir continuously at 500 r / min at room temperature. Slowly add 500 mL of 0.15 mg / mL theaflavins aqueous solution at a dropping rate of 15 mL / min, and continue stirring for 15 h. After the reaction is completed, centrifuge at 8000 r / min for 8 min, collect the precipitate, wash it three times with 250 mL of deionized water, and freeze-dry for 48 h to obtain 38.7 mg of cerium oxide nanoparticles (TFs-CeO2 NPs).

[0066] (2) Take 20 mg of cerium oxide nanoparticles obtained in step (1), add 400 mL of deionized water, stir continuously at 300 r / min at room temperature, add 67 mL of 1.5 mg / mL theaflavin aqueous solution at a dropping rate of 20 L / min, and continue stirring for 15 min. Then add 400 mL of 0.5 mg / mL ferric chloride aqueous solution at a dropping rate of 40 mL / min. After stirring for 20 min, transfer to ultrasound and continue the reaction for 15 min at an ultrasound power of 250 W. After the reaction is completed, centrifuge at 10000 r / min for 10 min, collect the precipitate, wash twice with 200 mL of deionized water, and freeze dry for 42 h to obtain 21.8 mg of theaflavin iron-based metal polyphenol network-coated cerium oxide nanoparticles (TFs / Fe-CeO2 NPs).

[0067] (3) Take 12 mg of the theaflavin iron-based metal polyphenol network-coated cerium oxide nanoparticles obtained in step (2), add 200 mL of deionized water, and stir to disperse. Add 60 mL of 4 mg / mL trehalose aqueous solution, stir at 400 r / min for 6 h at room temperature, and centrifuge at 8000 r / min for 10 min after the reaction is completed. Collect the precipitate, wash it 3 times with 60 mL of deionized water, and freeze-dry for 42 h to obtain 9.1 mg of theaflavin photothermal targeted nanozyme (Tre-TFs / Fe-CeO2 NPs).

[0068] Compare with Example 1

[0069] The results are basically the same as in Example 1, except that the amount of theaflavins in step (1) is 150 mL, and 4.6 mg of cerium oxide nanoparticles are obtained. The yield of nanoparticles is significantly reduced, indicating that if the amount of theaflavins used in this invention is too low, it will be difficult to produce cerium oxide nanoparticles. The amount of theaflavins used in this invention is within a better range.

[0070] Compare with Example 2

[0071] The reaction was basically the same as in Example 1, except that the concentration of cerium nitrate in step (1) was 0.3 mol / L and the concentration of theaflavins was 0.03 mg / mL. The solution did not become turbid during the reaction and no cerium oxide nanoparticles were produced, indicating that cerium nitrate and theaflavins must be at a certain concentration to react. The concentration of the raw materials in this invention is within a better range.

[0072] Compare with Example 3

[0073] The results are basically the same as in Example 1, except that the concentration of the ferric chloride aqueous solution in step (2) is 5 mg / mL, and 17.4 mg of cerium oxide nanoparticles (TFs / Fe-CeO2 NPs) coated with theaflavin iron-based metal polyphenol network are finally obtained. This shows that the increase in iron ion content hinders the formation of metal polyphenol network and leads to a significant reduction in yield. The concentration of ferric chloride in this invention is within the optimal range.

[0074] Compare with Example 4

[0075] The process was essentially the same as in Example 1, except that commercially available tea polyphenols (95% content) were used instead of theaflavins in steps (1) and (2) to prepare 10.1 mg of cerium oxide nanoparticles (TPs / Fe-CeO2NPs) coated with an iron-based metal polyphenol network and 4.6 mg of cerium dioxide nanoparticles (Tre-TPs / Fe-CeO2NPs). The yield was significantly reduced because the complex formed by tea polyphenols and iron ions has good water solubility and is lost through washing.

[0076] Effect Example

[0077] Test 1: Infrared absorption spectroscopy analysis of the theaflavin photothermal targeted nanozyme obtained in Example 1.

[0078] Methods: Fourier transform infrared spectroscopy was used to determine theaflavins (TFs), trehalose (Tre), and theaflavin photothermal targeted nanozymes (Tre-TFs / Fe-CeO2 NPs).

[0079] result: Figure 1 Infrared absorption spectra of theaflavins, trehalose, and theaflavins photothermal targeted nanozymes. 410-400 cm⁻¹ in Tre-TFs / Fe-CeO₂ NPs. -1 The absorption peak is the O-Ce-O tensile vibration absorption peak, 3400 cm⁻¹. -1 The absorption peaks are caused by the stretching vibration of -OH, corresponding to 3250 cm⁻¹ for TFs and Tre, respectively. -1 3364cm -1 This wavenumber change is due to the coordination of hydroxyl groups with cerium and iron ions. 2927cm -1 Corresponding to 2947cm in Tre -1 CH stretching vibration absorption, 1629cm -1 and 1384cm -1 This corresponds to 1616cm in TFBoys. -1 and 1450cm -1 The characteristic absorption of the benzene ring skeleton at 995 cm⁻¹. -1 This corresponds to 1002cm in Tre. -1The infrared spectroscopy results indicate the successful preparation of theaflavin photothermal targeted nanozyme. (The absorption peak at COC of the glycosidic bond is visible.)

[0080] Test 2: Scanning electron microscopy observation of the theaflavin photothermal targeted nanozyme obtained in Example 1.

[0081] Methods: A suspension of theaflavin photothermal targeted nanozymes (Tre-TFs / Fe-CeO2 NPs) prepared in Example 1 was prepared with 100 μg / mL of deionized water, sonicated to disperse it evenly, and diluted with anhydrous ethanol to 5 μg / mL. 10 μL was dropped onto a silicon wafer, sputtered with gold, and observed under a scanning electron microscope (SEM).

[0082] result: Figure 2 The image shows a SEM image of the theaflavin photothermal targeted nanozyme. It can be clearly observed that the nanozyme has a spherical particle morphology with a relatively uniform size, between 20-30 nm, and no obvious aggregation or adhesion, indicating that the theaflavin photothermal targeted nanozyme has good dispersibility.

[0083] Test 3: Antioxidant activity test of the theaflavin photothermal nanoenzyme prepared in Example 1.

[0084] Method: Prepare 7 mM ABTS (2,2'-adiazonium salt of 2,2'-diammonium salt of 3-ethylbenzothiazoline-6-sulfonic acid) and 2.45 mM potassium persulfate solution with deionized water, respectively. Mix 1 mL of each solution and react in the dark for 16 h. Adjust the absorbance at 734 nm wavelength with water to obtain 0.7 ± 0.02 to obtain ABTS working solution. A 100 μg / mL solution of commercially available cerium oxide nanoparticles (S-CeO2NPs; purchased from Beijing Deco Island Gold Technology Co., Ltd.), cerium oxide nanoparticles (TFs-CeO2 NPs) prepared in step (1) of Example 1, cerium oxide nanoparticles coated with theaflavins and iron-based metal polyphenols network (TFs / Fe-CeO2 NPs) prepared in step (2) of Example 1, theaflavins photothermal nanoenzymes (Tre-TFs / Fe-CeO2NPs) prepared in step (3) of Example 1, tea polyphenol cerium dioxide nanoparticles (Tre-TPs / Fe-CeO2 NPs) prepared in control Example 4, and control vitamin C (VC) solution were prepared. 0.5 mL of each sample solution was taken, and 0.5 mL of ABTS working solution was added to each. After mixing thoroughly, the mixture was reacted in the dark for 20 min. A blank control group was set up using deionized water instead of the sample solution, and a sample blank group was set up using deionized water instead of ABTS. Using water as a reference, the absorbance of the solution after the reaction was measured at a wavelength of 734 nm, and the ABTS radical scavenging rate of each sample was calculated according to formula (1). The experiment was repeated three times.

[0085] (1)

[0086] Where: A1 is the absorbance value of the blank control group; A2 is the absorbance value of the sample group; A s This represents the absorbance value of the blank sample group.

[0087] Results: Table 1 shows the ABTS radical scavenging rate of the theaflavin photothermal nanozyme prepared in Example 1. Commercially available nano-cerium oxide showed virtually no ABTS radical scavenging effect, while TFs-CeO2 NPs, TFs / Fe-CeO2 NPs, and Tre-TFs / Fe-CeO2 NPs exhibited nearly 100% ABTS radical scavenging rate, superior to vitamin C, demonstrating strong ABTS radical scavenging activity and excellent antioxidant activity. The free radical scavenging activity of Tre-TPs / Fe-CeO2 NPs obtained by replacing theaflavins with tea polyphenols was significantly reduced, possibly because the iron complex of tea polyphenols is water-soluble and thus washed away, leading to a decrease in the proportion of polyphenolic iron in the nanoparticles.

[0088] Table 1 ABTS free radical scavenging rate

[0089]

[0090] Test 4: Peroxidase-like activity test of the theaflavin photothermal targeted nanozyme prepared in Example 1.

[0091] Methods: 20 mM of 3,3',5,5'-tetramethylbenzidine (TMB) was prepared using dimethyl sulfoxide (DMSO), 10 mM of H2O2 solution was prepared using water, and 100 μg / mL of commercially available nano-cerium oxide (S-CeO2 NPs; purchased from Beijing Deco Island Gold Technology Co., Ltd.) was prepared using deionized water. The following nanoparticles were also prepared: cerium oxide nanoparticles (TFs-CeO2 NPs) prepared in step (1) of Example 1, cerium oxide nanoparticles (TFs / Fe-CeO2 NPs) coated with theaflavins iron-based metal polyphenol network prepared in step (2) of Example 1, theaflavin photothermal nanoenzymes (Tre-TFs / Fe-CeO2 NPs) prepared in step (3) of Example 1, tea polyphenol cerium dioxide nanoparticles (Tre-TPs / Fe-CeO2 NPs) prepared in control Example 4, and control vitamin C (VC) solution. Take 0.7 mL of acetate buffer solution (pH=4) and add 0.1 mL each of the sample solution or water, TMB solution and H2O2 solution in sequence. React for 5 min and measure the absorbance of each solution at 652 nm. The experiment was repeated in 3 replicates.

[0092] Results: In the presence of peroxidase, H2O2 can be decomposed to produce ∙OH, which oxidizes colorless TMB to blue ox-TMB, with an absorption peak at 652 nm. Figure 3To assess the peroxidase-like activity of each sample, vitamin C lacks peroxidase activity. TFs-CeO2 NPs, commercially available cerium oxide nanoparticles (S-CeO2 NPs) and Tre-TPs / Fe-CeO2 NPs exhibit weak peroxidase activity. Although TFs-CeO2 has very low peroxidase activity, after introducing theaflavin metal polyphenol network, TFs / Fe-CeO2 NPs and Tre-TFs / Fe-CeO2 NPs both showed excellent peroxidase-like activity, catalyzing the decomposition of H2O2 to produce ∙OH, oxidizing colorless TMB to blue ox-TMB. This indicates that the theaflavin metal polyphenol network plays an important role, while the cerium dioxide nanoparticles of tea polyphenols are difficult to form an effective network due to their good water solubility. Therefore, the theaflavin photothermal targeted nanozyme of this invention can decompose H2O2 to produce ∙OH in the bacterial infection microenvironment, exerting a chemodynamic antibacterial effect.

[0093] The photothermal performance of the theaflavin photothermal targeted nanozyme prepared in Example 1 was tested.

[0094] Method: Prepare 100 μg / mL suspensions of each sample (same as test 4 above) using deionized water. Then, take 0.5 mL of each 100 μg / mL sample suspension and apply it to an 808 nm near-infrared laser at a power density of 2 W / cm². 2 The sample was irradiated for 10 minutes, and the temperature was recorded every 30 seconds using a thermal infrared imager. The experiment was repeated three times.

[0095] result: Figure 4 These are the heating curves of each sample under 808nm laser irradiation. TFs-CeO2 NPs showed poorer photothermal heating effects compared to commercially available nano-cerium oxide, but the introduction of the theaflavin-metal polyphenol network demonstrated better photothermal performance. Although trehalose modification slightly reduced its photothermal properties, it was still significantly higher than Tre-TPs / Fe-CeO2 NPs prepared by replacing theaflavins with tea polyphenols. This indicates that the present invention significantly improves the photothermal performance of nano-cerium oxide through the formation of the theaflavin-metal polyphenol network, an effect that tea polyphenols struggle to achieve.

[0096] Test 6: Antibacterial performance of the theaflavin photothermal targeted nanozyme prepared in Example 1.

[0097] Methods: The antibacterial activity of theaflavin photothermal targeted nanozyme was determined using the plate count method. Single colonies of Escherichia coli (ATCC35218) and Staphylococcus aureus (ATCC6538) were selected and placed in sterile physiological saline to adjust the colony concentration to 10. 5CFU / mL, ready for use. Take 200 μL of each sample suspension (same as test 4 above) or physiological saline, add 25 μL of bacterial culture, and then add 25 μL of acetate buffer solution or acetate buffer solution containing H2O2 (10 mM) (pH=4). Mix well and treat for 3 h. The light group is treated under 808 nm laser (2 W / cm²). 2 Irradiate for 10 minutes, and perform three copies of each group. Finally, take 100 μL of each group and spread it on a nutrient agar plate. Incubate in a 37℃ incubator for 24 hours, count the number of colonies and calculate the inhibition rate.

[0098] Results: Table 2 records the inhibition rates of each sample against *Escherichia coli* and *Staphylococcus aureus*. Compared to TFs-CeO2NPs, TFs / Fe-CeO2 NPs with the introduction of a metal polyphenol network significantly improved the inhibition rates against both bacteria. Furthermore, after the introduction of trehalose, the theaflavin photothermal targeted nanozyme Tre-TFs / Fe-CeO2 NPs showed a higher inhibition rate against *Escherichia coli* than TFs / Fe-CeO2 NPs, indicating a targeted antibacterial effect.

[0099] In the group with hydrogen peroxide added alone, the inhibition rate against *E. coli* was significantly improved compared to the group without hydrogen peroxide, indicating that the theaflavin photothermal targeted nanozyme can catalyze the decomposition of hydrogen peroxide, generating hydroxyl radicals to kill bacteria. After the introduction of light, the sterilization rate reached 100%, demonstrating a significant photothermal sterilization effect. However, the sterilization effect of Tre-TPs / Fe-CeO2 NPs prepared by replacing theaflavins with tea polyphenols was significantly worse than that of the theaflavin photothermal targeted nanozyme, and neither light exposure nor the addition of hydrogen peroxide significantly improved the antibacterial activity. Therefore, the theaflavin photothermal nanozyme prepared in this invention has excellent antibacterial activity, and its inhibitory effect on *Staphylococcus aureus* is significantly stronger than that on *Escherichia coli*.

[0100] Table 2 Inhibition rates of different sample treatments on bacteria

[0101]

[0102] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A theaflavin photothermal targeted nanoenzyme, characterized in that: It is a nanostructured composite formed by using theaflavins as a reducing agent to prepare nano-cerium oxide as the core, the theaflavins and iron ions to construct a metal polyphenol network as the shell, and trehalose loaded on the outer surface.

2. The theaflavin photothermal targeted nanoenzyme according to claim 1, characterized in that: The theaflavin photothermal targeted nanozyme was prepared by the following method: (1) Under stirring conditions, theaflavin aqueous solution was added dropwise to cerium nitrate aqueous solution to carry out the reaction. After the reaction was completed, the precipitate was collected by centrifugation, washed, and dried to obtain cerium oxide nanoparticles. (2) Add the cerium oxide nanoparticles obtained in step (1) to water, and then add theaflavin aqueous solution and ferric chloride aqueous solution dropwise under stirring conditions to react. Then transfer to ultrasonic conditions to continue the reaction. After the reaction is completed, centrifuge to collect the precipitate, wash and dry it to obtain cerium oxide nanoparticles coated with theaflavin iron-based metal polyphenol network. (3) Add the cerium oxide nanoparticles coated with the theaflavin iron-based metal polyphenol network obtained in step (2) to water, then add trehalose aqueous solution, stir the reaction at room temperature, and after the reaction is completed, centrifuge to collect the precipitate, wash and dry it to obtain the theaflavin photothermal targeted nanozyme.

3. The theaflavin photothermal targeted nanoenzyme according to claim 2, characterized in that: The mass ratio of cerium oxide nanoparticles, theaflavins and ferric chloride mentioned in step (2) is 1:5-10:5-10; The mass ratio of the cerium oxide nanoparticles coated with the theaflavin iron-based metal polyphenol network in step (3) to trehalose is 1:20-40.

4. The theaflavin photothermal targeted nanoenzyme according to claim 3, characterized in that: The mass ratio of cerium oxide nanoparticles, theaflavins, and ferric chloride mentioned in step (2) is 1:7.5:7.5; The mass ratio of the cerium oxide nanoparticles coated with the theaflavin iron-based metal polyphenol network in step (3) to trehalose is 1:

30.

5. The theaflavin photothermal targeted nanoenzyme according to claim 2, characterized in that: The concentration of the cerium nitrate aqueous solution mentioned in step (1) is 0.05–0.15 mol / L; The concentration of theaflavins in water mentioned in step (1) is 0.05–0.15 mg / mL; The volume ratio of the cerium nitrate aqueous solution to the theaflavin aqueous solution in step (1) is 1:3 to 6.

6. The theaflavin photothermal targeted nanoenzyme according to claim 5, characterized in that: The concentration of the cerium nitrate aqueous solution mentioned in step (1) is 0.1 mol / L; The theaflavin concentration in step (1) is 0.1 mg / mL; The volume ratio of the cerium nitrate aqueous solution to the theaflavin aqueous solution in step (1) is 1:

6.

7. The theaflavin photothermal targeted nanoenzyme according to claim 1, characterized in that: The concentration of the theaflavins aqueous solution mentioned in step (2) is 0.5–1.5 mg / mL; The concentration of the ferric chloride aqueous solution mentioned in step (2) is 0.5–1.5 mg / mL; The concentration of the trehalose aqueous solution mentioned in step (3) is 2-4 mg / mL.

8. The theaflavin photothermal targeted nanoenzyme according to claim 1, characterized in that: The stirring speed mentioned in step (1) is 300-600 r / min; The dropping rate described in step (1) is 5–15 mL / min; The reaction time described in step (1) is 10–15 h; The washing described in steps (1), (2) and (3) is performed using deionized water; The drying process described in steps (1), (2), and (3) is freeze drying; The stirring speed mentioned in step (2) is 300-600 r / min; The dropping rate described in step (2) is 20–40 mL / min; The ultrasonic power mentioned in step (2) is 200-300W; The reaction time described in step (2) is 20–40 min; The reaction time described in step (2) is 10–20 min; The conditions for the stirring reaction in step (3) are: stirring speed of 400-600 r / min and reaction time of 4-8 h.

9. The use of the theaflavin photothermal targeted nanozyme according to any one of claims 1 to 8 in the preparation of antibacterial products, antioxidant products, photothermal materials and / or peroxidase-like products.

10. The application according to claim 9, characterized in that: The bacteria are bacteria; more specifically, Escherichia coli and / or Staphylococcus aureus.

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