Transition metal-based nano-enzyme with multiple enzyme activities as well as preparation method and application of transition metal-based nano-enzyme

Abstract

The invention relates to the technical field of nano-enzymes, and discloses a transition metal-based nano-enzyme with multiple enzyme activities as well as a preparation method and application of the transition metal-based nano-enzyme. The preparation method comprises the following steps: preparing an organic compound I; the organic compound I and metal salt are subjected to a reaction through a solvent stirring method, a liquid-liquid interface method or a solvothermal method, and the transition metal-based nano-enzyme with multiple enzyme activities is obtained. The preparation method is high in safety, high-temperature and high-pressure equipment is not needed, and batch production is facilitated; the transition metal-based nano-enzyme with the synergistic effect of photodynamic therapy and photothermal therapy can be prepared without a photosensitizer, and the preparation process is greatly simplified. The transition metal-based nano enzyme has photo-thermal, photodynamic and multi-enzyme activities, can be used as a peroxidase-like catalyst, a superoxide dismutase catalyst, a catalase catalyst, a halogen peroxidase catalyst and a glutathione peroxidase catalyst, and can be combined with photo-thermal and photodynamic efficient sterilization.

Description

Technical Field

[0001] The present application relates to the field of nanozyme technology, and specifically to a transition metal-based nanozyme with multiple enzyme activities, a preparation method thereof, and an application thereof. Background Art

[0002] Phototherapy includes photodynamic therapy (PDT) and photothermal therapy (PTT). Compared with traditional chemotherapy, phototherapy is one of the least invasive treatment methods. Many studies have shown that the combination of PDT and PTT can produce a strong synergistic antibacterial effect and minimize the side effects during treatment.

[0003] Nanozymes have the advantages of good stability, adjustable structure and composition, and adjustable catalytic activity. Their enzyme activity types include peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD), which have been widely used in medicine. Coordination polymer (CPs) materials have porous structures and hybrid physical and chemical properties. Many CPs have been found to have the characteristics of nanozymes and are used in the field of nanozymes. Porphyrin derivatives have efficient photodynamic and photothermal conversion properties. It is of great significance to prepare new transition metal-based nanozymes with good enzyme activity, photothermal and photodynamic performance synergy by combining porphyrins, coordination polymers and transition metals for antibacterial treatment.

[0004] Currently, the preparation methods of transition metal-based nanozymes include hydrothermal method, solvothermal method, chemical reduction method, biomimetic mineralization method and sol-gel method. For example, the patent application with publication number CN115845086A discloses a photothermal-Fenton-like reaction artificial nanozyme and a preparation method thereof, which is formed by high-temperature carbonization at 800°C under the condition of inert gas N2, and the nanozyme has a single POD enzyme activity and photothermal performance; the patent application with publication number CN 114100586A discloses a composite material nanozyme and a preparation method and application thereof, which requires high-temperature calcination synthesis using a tubular furnace under N2 or Ar2 atmosphere, and the nanozyme has POD enzyme and OXD enzyme activities; the patent application with publication number CN 115368579A discloses a preparation method and application of a nanozyme with manganese porphyrin as a metal organic framework, which has two SOD and CAT enzyme activities, but no photothermal and photodynamic performance; the patent application with publication number CN 118185052A discloses a zirconium porphyrin metal organic framework nanozyme and a preparation method and application thereof, and the nanozyme is synthesized by a two-step method, and it has POD enzyme activity.

[0005] However, the current transition metal-based nanozymes not only have a complicated preparation process, but most of them only have one or two enzyme-like activities and cannot simultaneously have photothermal, photodynamic and multiple enzyme activities. When integrating the synergistic effects of photodynamic therapy and photothermal therapy for antibacterial purposes, two photosensitizers with different absorption wavelengths are usually required, which greatly increases the complexity of the operation. Summary of the invention

[0006] The present application provides a transition metal-based nanozyme with multiple enzyme activities and a preparation method and application thereof, aiming to solve the technical problem that existing transition metal-based nanozymes cannot simultaneously have photothermal, photodynamic and multiple enzyme activities.

[0007] In order to achieve the above objectives, the present application adopts the following technical solutions.

[0008] In a first aspect of the present application, a method for preparing a transition metal-based nanozyme having multiple enzyme activities is provided, comprising:

[0009] Prepare organic compound I, whose chemical structure is shown in formula (1):

[0010]

[0011] The organic compound I and the metal salt are reacted by a solvent stirring method, a liquid-liquid interface method or a solvent thermal method to obtain a transition metal-based nanozyme with multiple enzyme activities.

[0012] Preferably, the metal salt comprises chloride, nitrate or acetate of cobalt, nickel, iron, zinc, manganese, copper or ruthenium.

[0013] Further preferably, the metal salt is Ru(DMSO)4Cl2.

[0014] Preferably, the molar ratio of the organic compound I to the metal salt is 0.1:1.

[0015] Preferably, the solvent stirring method specifically comprises:

[0016] dissolving an organic compound I in a first solvent to obtain an organic solution; dissolving a metal salt in a second solvent to obtain a metal salt solution;

[0017] The metal salt solution is slowly added dropwise to the organic solution, stirred at 50-120°C for 8-24 hours, and after the reaction, the reaction solution is centrifuged to collect the solid phase product, washed, dried and activated to obtain a transition metal-based nanozyme with multiple enzyme activities;

[0018] Wherein, the first solvent is at least one of DMF, methanol, chloroform or dichloromethane; and the second solvent is DMF or methanol.

[0019] Preferably, the liquid-liquid interface method is specifically:

[0020] The metal salt aqueous solution is slowly added to the chloroform solution of the organic compound I, and the mixture is allowed to stand for 72 to 84 hours. The reaction solution is centrifuged to collect the solid phase product, which is then washed, dried and activated to obtain a transition metal-based nanozyme with multiple enzyme activities.

[0021] Preferably, the solvent thermal method is specifically:

[0022] The organic compound I and the metal salt are dissolved in DMF, ultrasonically dissolved, heated to 110-130°C for reaction for 6-10 hours, the reaction solution is centrifuged to collect the solid phase product, washed, dried and activated to obtain a transition metal-based nanozyme with multiple enzyme activities.

[0023] Preferably, the organic compound I is prepared by the following method:

[0024] Dissolve 4-(4-formylphenyl)-2,2':6',2"-terpyridine in propionic acid, heat to 140-160°C and reflux; then add pyrrole solution dropwise to perform reflux reaction;

[0025] The reaction solution is added into water and allowed to stand; the solid phase is collected by suction filtration and washed to obtain the product.

[0026] The second aspect of the present application provides a transition metal-based nanozyme with multiple enzyme activities prepared by the above preparation method.

[0027] The third aspect of the present application provides the use of the above-mentioned transition metal-based nanozyme with multiple enzyme activities in antibacterial drugs.

[0028] Compared with the prior art, the beneficial effects of this application are:

[0029] The preparation method of the present application is highly safe and does not require high-temperature and high-pressure equipment such as high-temperature reactors and high-temperature furnaces, which is conducive to mass production; it can prepare transition metal-based nanozymes with synergistic effects of photodynamic therapy and photothermal therapy without the need for photosensitizers, greatly simplifying the preparation process.

[0030] The transition metal-based nanozyme with multiple enzyme activities prepared by the preparation method of the present application can simultaneously have photothermal, photodynamic and multiple enzyme activities, and can be used as a peroxidase-like catalyst, a superoxide dismutase catalyst, a catalase catalyst, a haloperoxidase catalyst and a glutathione peroxidase catalyst, and can be combined with photothermal and photodynamic to efficiently kill bacteria. The medicine using the transition metal-based nanozyme with multiple enzyme activities prepared by the present application has excellent antibacterial properties. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments recorded in the present application, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0032] Figure 1 is the SEM spectrum of transition metal-based nanozymes;

[0033] Figure 2 This is a graph showing the test results of the peroxidase-like (POD) activity of transition metal-based nanozymes;

[0034] Figure 3 This is a graph showing the test results of superoxide dismutase (SOD) activity of transition metal-based nanozymes;

[0035] Figure 4 This is a graph showing the test results of hydrogen peroxide (CAT) activity of transition metal-based nanozymes;

[0036] Figure 5 This is a graph showing the test results of the haloperoxidase (HPO) activity of transition metal-based nanozymes;

[0037] Figure 6 This is a graph showing the results of the glutathione peroxidase-like activity (Gpx) of transition metal-based nanozymes;

[0038] Figure 7 This is a photothermal effect test result of transition metal-based nanozymes;

[0039] Figure 8 This is the antibacterial test result of transition metal-based nanozyme TP-Ru. DETAILED DESCRIPTION

[0040] The technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0041] In the following description of this embodiment, the terms "include", "comprising", "having" and "containing" are all open terms, meaning including but not limited to.

[0042] In the following description of this embodiment, the term "and / or" is used to describe the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can represent: A exists alone, B exists alone, and A and B exist at the same time. A and B can be singular or plural. The character " / " generally indicates that the associated objects before and after are in an "or" relationship.

[0043] In the following description of this embodiment, the term "at least one" refers to one or more, and "plurality" refers to two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b or c", or "at least one of a, b and c", can all represent: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, c can be single or multiple, respectively.

[0044] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "an" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0045] Those skilled in the art should understand that in the following description of the embodiments of the present application, the order of serial numbers does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0046] Those skilled in the art will appreciate that the numerical ranges in the embodiments of the present application are to be construed as specifically disclosing each intermediate value between the upper and lower limits of the scope. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present application. The upper and lower limits of these smaller ranges may be independently included or excluded in the scope.

[0047] Unless otherwise specified, the technical / scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this application belongs. Although this application only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of this application. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0048] In a first aspect, the present application provides a method for preparing a transition metal-based nanozyme having multiple enzyme activities, comprising:

[0049] Prepare organic compound I, whose chemical structure is shown in formula (1):

[0050]

[0051] The organic compound I and the metal salt are reacted by a solvent stirring method, a liquid-liquid interface method or a solvothermal method to obtain a transition metal-based nanozyme with multiple enzyme activities. Preferably, the molar ratio of the organic compound I to the metal salt is 0.1:1.

[0052] In the present application, the organic compound I is prepared by the following method:

[0053] Dissolve 4-(4-formylphenyl)-2,2':6',2"-terpyridine in propionic acid and heat to 140-160°C for reflux; then add pyrrole solution dropwise for reflux reaction; wherein the molar ratio of 4-(4-formylphenyl)-2,2':6',2"-terpyridine to pyrrole is preferably 1:1.

[0054] The reaction solution was added into water and allowed to stand for 24 hours; the solid phase was collected by suction filtration and washed with methanol and chloroform in sequence to obtain organic compound I.

[0055] In the present application, the metal salt includes chloride, nitrate or acetate of cobalt, nickel, iron, zinc, manganese, copper or ruthenium. When the metal salt is a ruthenium salt, the preferred ruthenium salt is Ru(DMSO)4Cl2, and the preparation method thereof is as follows: reflux RuCl3·3H2O and DMSO in a reaction bottle at 160°C for 15 minutes, then vacuum concentrate to half volume, add acetone, filter to obtain a precipitate, wash the precipitate with acetone and ether, and then vacuum dry to obtain a yellow powder product, namely Ru(DMSO)4Cl2.

[0056] In the present application, the solvent stirring method for preparing transition metal-based nanozymes with multiple enzyme activities specifically includes:

[0057] Dissolving the organic compound I in a first solvent to obtain an organic solution; wherein the first solvent is at least one of DMF, methanol, chloroform or dichloromethane; preferably DMF, a mixture of methanol and chloroform, or a mixture of methanol and dichloromethane;

[0058] The metal salt is dissolved in a second solvent to obtain a metal salt solution; wherein the second solvent is DMF or methanol.

[0059] The metal salt solution is slowly added dropwise to the organic solution and stirred at 50-120°C for 8-24 hours. After the reaction, the reaction solution is centrifuged to collect the solid phase product, which is washed, dried and activated at 90-110°C to obtain a transition metal-based nanozyme with multiple enzyme activities.

[0060] In the present application, the liquid-liquid interface method is used to prepare transition metal-based nanozymes with multiple enzyme activities, specifically:

[0061] The metal salt aqueous solution is slowly added to the chloroform solution of organic compound I, and the mixture is allowed to stand for 72 to 84 hours. The reaction solution is centrifuged to collect the solid phase product, which is then washed, dried and activated at 90 to 110° C. to obtain a transition metal-based nanozyme with multiple enzyme activities.

[0062] In the present application, the solvothermal method is used to prepare transition metal-based nanozymes with multiple enzyme activities, specifically:

[0063] The organic compound I and the metal salt are dissolved in DMF, ultrasonically dissolved, heated to 110-130°C for reaction for 6-10 hours, the reaction solution is centrifuged to collect the solid phase product, washed, dried and activated at 90-110°C to obtain a transition metal-based nanozyme with multiple enzyme activities.

[0064] The preparation method of the present application is highly safe and does not require high-temperature and high-pressure equipment such as high-temperature reactors and high-temperature furnaces, which is conducive to mass production; it can prepare transition metal-based nanozymes with synergistic effects of photodynamic therapy and photothermal therapy without the need for photosensitizers, greatly simplifying the preparation process.

[0065] The transition metal-based nanozyme with multiple enzyme activities prepared in the present application is denoted as TP-M, and its chemical structure is shown in formula (2):

[0066]

[0067] wherein M is selected from Co, Ni, Fe, Mn, Cu, Zn or Ru.

[0068] The transition metal-based nanozyme with multiple enzyme activities prepared by the preparation method of the present application has multiple enzyme-like activities and can be used as a peroxidase-like catalyst, a superoxide dismutase catalyst, a catalase catalyst, a haloperoxidase catalyst, and a glutathione peroxidase catalyst; it has photothermal, photodynamic and multiple enzyme activities, and can combine photothermal and photodynamic to efficiently kill bacteria. It can be used to prepare drugs for antibacterial treatment and has excellent antibacterial properties.

[0069] The present application is further described below by way of examples.

[0070] Example Preparation of Organic Compounds I

[0071] 5.93 mmol of pyrrole was dissolved in 6 mL of propionic acid to obtain a pyrrole solution; 5.93 mmol of 4-(4-formylphenyl)-2,2':6',2"-terpyridine was dissolved in 60 mL of propionic acid solvent, heated to 150°C for reflux, and then the pyrrole solution was added dropwise for reflux reaction for 1 hour. After the reaction system was cooled to room temperature, the reaction mixture was poured into 200 mL of water and allowed to stand in a refrigerator at 4°C overnight. The solid phase was collected by filtration, and the solid phase was washed with methanol and chloroform in turn to obtain organic compound I. The organic compound I was subjected to nuclear magnetic resonance detection, and its nuclear magnetic hydrogen spectrum was:

[0072] 1H NMR (600MHz, CDCl3): 9.09 (s, 8H), 8.99 (s, 8H), 8.80 (d, J = 4.0Hz, 8H), 8.78 (d, J = 7.8Hz, 8H), 8 .42 (d, J=7.8Hz, 8H), 8.33 (d, J=7.8Hz, 8H), 7.94 (t, J=7.8Hz, 8H), 7.40 (m, 8H), -2.66 (s, 2H).

[0073] Example 1

[0074] This embodiment provides a method for preparing a transition metal-based nanozyme having multiple enzyme activities, comprising:

[0075] Dissolve 1.5 mmol of RuCl3·3H2O in 9 mL of DMSO, heat to 160°C and reflux for 15 min, then concentrate in vacuo to half the solution, add 100 mL of acetone, filter, and wash the precipitate with ether to obtain RuCl2(DMSO)4.

[0076] Under argon atmosphere, 0.035 mmol of compound I was dissolved in a mixed solution of 25 mL CHCl3 and 5 mL MeOH, and stirred to dissolve to obtain an organic solution; 0.35 mmol of RuCl2(DMSO)4 was dissolved in 1 mL MeOH to obtain a salt solution;

[0077] The salt solution was slowly added dropwise to the organic solution, and refluxed and stirred at 60° C. for 24 h. After the reaction was completed, the reaction solution was allowed to stand overnight, centrifuged at 8000 r / min for 5 min, washed with chloroform, acetone, and water, centrifuged 3 times, and then dried at 100° C. for 12 h for activation, thereby obtaining a transition metal-based nanozyme TP-Ru with multiple enzyme activities.

[0078] Example 2

[0079] This embodiment provides a method for preparing a transition metal-based nanozyme having multiple enzyme activities, comprising:

[0080] Under argon atmosphere, 0.035 mmol of compound I was dissolved in 14 mL of DMF and stirred to dissolve to obtain an organic solution; 0.35 mmol of NiCl2·6H2O was dissolved in 3 mL of DMF to obtain a salt solution;

[0081] The salt solution was slowly added dropwise to the organic solution and refluxed at 120°C for 15 hours. After the reaction was completed, the mixture was allowed to stand overnight, centrifuged at 8000 r / min for 5 minutes, washed with chloroform, acetone, and water, centrifuged three times, and then dried at 100°C for 12 hours for activation to obtain transition metal-based nanozyme TP-Ni with multiple enzyme activities.

[0082] Example 3

[0083] This embodiment provides a method for preparing a transition metal-based nanozyme having multiple enzyme activities, comprising:

[0084] Under argon atmosphere, 0.035 mmol of compound I was dissolved in a mixed solution of 25 mL of CH2Cl2 and 5 mL of MeOH, and the mixture was stirred to dissolve to obtain an organic solution; 0.35 mmol of MnCl2·4H2O was dissolved in 1 mL of MeOH to obtain a salt solution;

[0085] The salt solution was slowly added dropwise to the organic solution and refluxed at 60°C for 5 hours. After the reaction was completed, the mixture was allowed to stand overnight, centrifuged at 8000 r / min for 5 minutes, washed with chloroform, acetone, and water, centrifuged three times, and then dried at 100°C for 12 hours for activation to obtain transition metal-based nanozyme TP-Mn with multiple enzyme activities.

[0086] Example 4

[0087] This embodiment provides a method for preparing a transition metal-based nanozyme having multiple enzyme activities, comprising:

[0088] Dissolve 30.5 mg of CuCl2·2H2O in 20 mL of water to obtain a salt solution; dissolve 20 mg of compound I in 20 mL of chloroform to obtain an organic solution; slowly add the salt solution to the organic solution, let it stand in the dark for 144 hours, carefully remove the water layer and the chloroform layer, and slowly wash the product of the middle layer with chloroform and water to obtain a transition metal-based nanozyme TP-Cu with multiple enzyme activities.

[0089] Example 5

[0090] This embodiment provides a method for preparing a transition metal-based nanozyme having multiple enzyme activities, comprising:

[0091] Dissolve 47.6 mg of CoCl2·6H2O in 20 mL of water to obtain a salt solution; dissolve 20 mg of compound I in 20 mL of chloroform to obtain an organic solution; slowly add the salt solution to the organic solution, let it stand in the dark for 144 hours, carefully remove the water layer and the chloroform layer, and slowly wash the product of the middle layer with chloroform and water to obtain a transition metal-based nanozyme TP-Co with multiple enzyme activities.

[0092] Example 6

[0093] This embodiment provides a method for preparing a transition metal-based nanozyme having multiple enzyme activities, comprising:

[0094] 0.02mmol of compound I and 0.2mmol of FeCl3·6H2O were dissolved in 15mL of DMF, added to the reactor after being fully dissolved by ultrasound, and reacted at 120℃ for 6h. The reaction solution was centrifuged at 8000r / min for 5 minutes, washed with DMF and acetone, centrifuged 3 times, and dried at 100℃ for 12 hours to activate the transition metal-based nanozyme TP-Fe with multiple enzyme activities.

[0095] Example 7

[0096] This embodiment provides a method for preparing a transition metal-based nanozyme having multiple enzyme activities, comprising:

[0097] 0.02mmol of compound I and 0.2mmol of Zn(NO3)2·6H2O were dissolved in 15mL of DMF, added to the reactor after being fully dissolved by ultrasound, and reacted at 120℃ for 6h. The reaction solution was centrifuged at 8000r / min for 5 minutes, washed with DMF and acetone, centrifuged 3 times, and dried at 100℃ for 12 hours to activate the transition metal-based nanozyme TP-Zn with multiple enzyme activities.

[0098] The transition metal-based nanozyme TP-M prepared in Example 1-7 was characterized by morphology test, and its SEM image is as follows: Figure 1 shown. Figure 1 In the figure, from top to bottom are the test images of TP-Co, TP-Ni, TP-Fe, TP-Zn, TP-Mn, TP-Cu and TP-Ru, among which the leftmost image in the test image of each transition metal-based nanozyme is a SEM image, and the rest are EDS images.

[0099] from Figure 1SEM showed that TP-Ni, TP-Mn, TP-Fe and TP-Co were in the form of flakes, TP-Cu was in the form of blocks, TP-Ru was in the form of wrinkled flakes, and TP-Zn was in the form of aggregated particles. EDS elemental analysis showed that each metal element was complexed with the pyridine group, and seven transition metal-based nano TP-Ms (M represents cobalt, nickel, iron, zinc, manganese, copper and ruthenium) were successfully synthesized, and each metal element was evenly distributed.

[0100] The enzyme-like activity test of the transition metal-based nanozyme TP-M prepared in Examples 1-7 was performed as follows:

[0101] 1. Peroxidase-like (POD) activity test

[0102] 20 μL of 1 mg / mL transition metal-based nanozyme TP-M, 0.1 mL of 3.3 mmol / L H2O2, and 0.1 mL of 1.7 mmol / L TMB were added to 1 mL of buffer solution at pH 6.0, respectively. After incubation at 37 °C in the dark for 10 min, the colorless TMB was oxidized to a blue oxidation state by reactive oxygen species for characterization. The UV-visible absorption spectrum was measured using a UV-visible spectrophotometer. The peroxidase-like catalytic activity results are shown in Figure 2. Figure 2 As shown, TP-Ru, TP-Co, and TP-Fe nanozymes have obvious peroxidase-like activity compared with the blank TMB group.

[0103] 2. Superoxide dismutase (SOD) activity test

[0104] Superoxide anion (O 2- ) can react with the cell metabolic activity detection reagent WST-1 to generate water-soluble yellow formazan with an absorption peak at 450nm.

[0105] According to this principle, a commercially available SOD activity detection kit (WST-1) was used to perform the test according to the instructions, where the concentration of the nanozyme was 100 μg / mL. After the test data was processed, the SOD enzyme inhibition rate results were obtained as follows: Figure 3 As shown, TP-Co and TP-Fe nanozymes have obvious superoxide dismutase activity.

[0106] 3. Hydrogen peroxide (CAT) activity test

[0107] Catalase can catalyze the decomposition of H2O2 into O2 and H2O, generating bubbles. The transition metal-based nanozymes prepared in Examples 1-7 were reacted with hydrogen peroxide, and the CAT enzyme activity was observed and photographed to analyze the CAT enzyme activity. The generation of bubbles is shown in FIG. Figure 4 As shown, TP-Fe and TP-Ru nanozymes have obvious catalase activity.

[0108] 4. Haloperoxidase (HPO) activity test

[0109] A 0.2 mmol / L azure blue solution was prepared with an acetic acid-sodium acetate buffer solution at pH 5.0, and 20 μL of 1 mg / mL transition metal-based nanozyme TP-M, 20 μL of 5 mmol / L H2O2, 1.92 mL of azure blue solution, and 40 μL of 0.1 mmol / L NaCl were reacted. After each group of reactants was incubated at 37°C for 20 min, the UV-visible absorption spectrum was measured using a UV-visible spectrophotometer. The catalytic activity of haloperoxidase-like Figure 5 As shown, TP-Ru and TP-Fe had obvious haloperoxidase-like activity compared with the blank CP group.

[0110] 5. Glutathione peroxidase (Gpx) activity test

[0111] 50 μL of 50-100 μg / ml transition metal-based nanozyme TP-M and 50 μL of 2 mmol / L GSH were added to 1.9 mL of acetic acid-sodium acetate buffer solution at pH 7.4. After incubation of each group of reactants at 37°C for 10 min, 50 μL of the mixture was added to 30 μL of DTNB, and its UV-visible absorption spectrum was measured using a UV-visible spectrophotometer. The glutathione peroxidase-like catalytic activity was as follows: Figure 6 As shown, TP-Co has obvious glutathione peroxidase-like activity.

[0112] The photothermal effect test of the transition metal-based nanozyme TP-M prepared in Examples 1-7 is as follows:

[0113] Take the transition metal-based nanozyme TP-M prepared in Examples 1-7 (concentration of 150 μg / mL, volume of 500 mL) and add it to a 1.5 mL centrifuge tube. Add water to each centrifuge tube until the volume is 1 mL. Place each centrifuge tube in a water bath with a wavelength of 808 nm and a power density of 1.5 W cm 2 The temperature was displayed on a thermal imaging display. The temperature was read every 30 seconds within 8-10 seconds and plotted. The results are shown in the figure. Figure 7 As shown. Figure 7 It can be seen that the seven transition metal-based nanozymes all have good photothermal activity, and the highest temperature can reach the temperature required for antibacterial properties.

[0114] The activity test results of the transition metal-based nanozyme TP-M (M is selected from Co, Ni, Fe, Mn, Cu, Zn or Ru) with multiple enzyme activities prepared in Examples 1-7 are summarized in Table 1.

[0115] Table 1 Test results of transition metal-based nanozyme TP-M with multiple enzyme activities

[0116]

[0117] Through the above tests, it can be seen that the transition metal-based nanozymes prepared in Examples 1-7 all have multiple enzyme activities, photothermal and photodynamic properties, and can achieve efficient cascade antibacterial. Among them, TP-Ru nanozyme has four enzyme activities of POD enzyme, SOD enzyme, CAT enzyme, HPO enzyme and free radical scavenging ability at the same time, and the photothermal effect can reach 79°C; TP-Co nanozyme has four enzyme activities of POD enzyme, SOD enzyme, CAT enzyme, GPx enzyme and free radical scavenging ability at the same time, and the photothermal effect can reach 50°C; TP-Fe nanozyme has four enzyme activities of POD enzyme, SOD enzyme, CAT enzyme, HPO enzyme at the same time, and the photothermal effect can reach 60°C.

[0118] Nanozyme TP-Ru was selected for antibacterial experiments to test its antibacterial properties, as follows:

[0119] Take E. coli to prepare 8 mixed solutions, the total volume of each solution is 1mL, and the concentration of E. coli in the mixed solution is 10 6 CFU / mL, the concentration of H2O2 was 1mM; the concentration of nanozyme TP-Ru was 200μg / mL.

[0120] The above 8 groups of bacterial liquid were used as I: blank group; II: laser group (NIR); III: H2O2 group; IV: H2O2+laser group (NIR); V: TP-Ru group; VI: TP-Ru+laser group (NIR); VII: TP-Ru+H2O2 group; VIII: TP-Ru+H2O2+laser group (NIR). The laser was irradiated with 808nm near-infrared laser with a power density of 1.5W cm 2 The irradiation time is 10 minutes.

[0121] Each mixed solution was placed in a shaker and incubated at 37°C for 2 hours. 100 μL of the mixed solution was transferred to solid culture medium from each group and incubated at 37°C for 12 hours. The antibacterial effect was evaluated by observing and photographing. Figure 8 As shown. Figure 8 It can be seen that TP-Ru has a good bactericidal effect and can effectively inhibit the growth of Escherichia coli.

[0122] Although the present application has been described in detail in general terms and in specific embodiments in this specification, it is obvious to those skilled in the art that some modifications or improvements may be made to the present application. Therefore, these modifications or improvements made without departing from the spirit of the present application are within the scope of protection claimed in the present application.

Claims

1. A method for preparing a transition metal-based nanozyme having multiple enzyme activities, characterized in that: include: Prepare organic compound I, whose chemical structure is shown in formula (1): The organic compound I and the metal salt are reacted by a solvent stirring method, a liquid-liquid interface method or a solvent thermal method to obtain a transition metal-based nanozyme with multiple enzyme activities.

2. The preparation method according to claim 1, characterized in that: The metal salts include chlorides, nitrates or acetates of cobalt, nickel, iron, zinc, manganese, copper or ruthenium.

3. The preparation method according to claim 2, characterized in that: The metal salt is Ru(DMSO)4Cl2.

4. The preparation method according to claim 1, characterized in that: The molar ratio of the organic compound I to the metal salt is 0.1:

1.

5. The preparation method according to claim 1, characterized in that: The solvent stirring method specifically comprises: dissolving an organic compound I in a first solvent to obtain an organic solution; dissolving a metal salt in a second solvent to obtain a metal salt solution; The metal salt solution is slowly added dropwise to the organic solution, stirred at 50-120°C for 8-24 hours, and after the reaction, the reaction solution is centrifuged to collect the solid phase product, washed, dried and activated to obtain a transition metal-based nanozyme with multiple enzyme activities; Wherein, the first solvent is at least one of DMF, methanol, chloroform or dichloromethane; and the second solvent is DMF or methanol.

6. The preparation method according to claim 1, characterized in that: The liquid-liquid interface method is specifically: The metal salt aqueous solution is slowly added to the chloroform solution of the organic compound I, and the mixture is allowed to stand for 72 to 84 hours. The reaction solution is centrifuged to collect the solid phase product, which is then washed, dried and activated to obtain a transition metal-based nanozyme with multiple enzyme activities.

7. The preparation method according to claim 1, characterized in that: The solvothermal method is specifically: The organic compound I and the metal salt are dissolved in DMF, ultrasonically dissolved, heated to 110-130°C for reaction for 6-10 hours, the reaction solution is centrifuged to collect the solid phase product, washed, dried and activated to obtain a transition metal-based nanozyme with multiple enzyme activities.

8. The preparation method according to claim 1, characterized in that: The organic compound I is prepared by the following method: Dissolve 4-(4-formylphenyl)-2,2':6',2"-terpyridine in propionic acid, heat to 140-160°C and reflux; then add pyrrole solution dropwise to perform reflux reaction; The reaction solution is added into water and allowed to stand; the solid phase is collected by suction filtration and washed to obtain the product.

9. A transition metal-based nanozyme with multiple enzyme activities prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the transition metal-based nanozyme with multiple enzyme activities as claimed in claim 9 in antibacterial drugs.

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