Nano-enzyme system based on Ag-polydopamine nano-microsphere (at) MOF-74 structure as well as preparation method and application of nano-enzyme system

Abstract

The invention provides a nano-enzyme system based on an Ag-polydopamine nano-microsphere (at) MOF-74 structure as well as a preparation method and application of the nano-enzyme system, and belongs to the technical field of biological medicines. The nano-enzyme system takes a polydopamine microsphere as a core, and an MOF-74 shell layer grows on the surface of the polydopamine microsphere in situ to form a composite microsphere with a core-shell structure; and by utilizing the reduction property of polydopamine, Ag nanoparticles are reduced in situ and uniformly dispersed in the MOF-74 framework, so that the composite nano material with the enzyme-like activity and the photo-thermal function is constructed. The system shows excellent horse radish peroxidase-like activity, and can effectively promote electron transfer between hydrogen peroxide and a substrate and catalyze a chromogenic reaction. Meanwhile, the composite system has good photo-thermal conversion performance, shows a remarkable photo-thermal sterilization effect on gram-negative bacteria and gram-positive bacteria, and can effectively inhibit and destroy the formation of bacterial biofilms. An in-vitro antibacterial experiment result shows that the MOF-74-based nano enzyme system has relatively high antibacterial activity.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine and nanocatalysis, and particularly relates to a nanoenzyme system based on an Ag-polydopamine nanomicrosphere@MOF-74 structure and a preparation method and application thereof. BACKGROUND

[0002] Metal-organic framework (MOF) materials have broad application prospects in the field of biological medicine due to their high specific surface area, adjustable pore structure and diverse functionality. Among them, MOF-74 (constructed by nickel, magnesium and other metal ions and 2,5-dihydroxy terephthalic acid ligand) becomes one of the ideal platforms for constructing biological nanoenzymes due to its open metal sites, good biocompatibility and relatively high stability in aqueous environment.

[0003] However, the morphology and size of MOF-74 crystals synthesized by traditional methods are difficult to accurately control, and its function is relatively single, which to some extent limits its further biological application. SUMMARY

[0004] In view of the problems of morphology control difficulty and single function of existing MOF materials in biological application, the application provides a nanoenzyme system based on an Ag-polydopamine nanomicrosphere@MOF-74 structure and a preparation method and application thereof.

[0005] The application provides a preparation method of a nanoenzyme system based on an Ag-polydopamine nanomicrosphere@MOF-74 structure, which comprises the following steps: (1) 100-300 mg of hydrochloric acid dopamine is weighed and dissolved in 50-200 mL of Tris-HCl buffer solution with a concentration of 0.25-0.50 mM and a pH of 7.8; under the magnetic stirring of 500-1000 rpm, reaction is carried out at 25-30 DEG C for 12-48 hours; after the reaction is completed, centrifugal separation is carried out at 8000-10000 rpm, polydopamine microspheres are obtained, and the polydopamine microspheres are freeze-dried for standby; (2) The polydopamine microspheres are dispersed in an aqueous solution to prepare a dispersion liquid with a concentration of 1-3 mg / mL, 1-3 mL of the polydopamine microsphere dispersion liquid is taken for standby; in addition, magnesium chloride hexahydrate and 2,5-dihydroxy terephthalic acid are weighed and dissolved in a mixed solvent under ultrasonic assistance to obtain a mixed solution for standby; the mixed solution and the polydopamine microsphere dispersion liquid are combined and transferred to a hydrothermal reaction kettle, and reaction is carried out at 135 DEG C for 22 hours to obtain polydopamine microsphere@MOF-7 material; (3) After the reaction is completed, the above polydopamine microspheres MOF-7 material is washed with N,N-dimethylformamide and anhydrous methanol in turn for three times, and then freeze-drying is carried out to obtain a freeze-dried MOF material; 100 mg of the above freeze-dried MOF material is dispersed in 50 mL of water, 10 mL of a silver nitrate solution with a concentration of 1 mg / mL is added, and after 24 hours of reaction, freeze-drying is carried out again to obtain an Ag-polydopamine microspheres MOF-74 material.

[0006] Further, in step (2), the final concentration of magnesium chloride hexahydrate is 0.5-1.8 mM.

[0007] Further, in step (2), the final concentration of 2,5-dihydroxyterephthalic acid is 0.1-0.5 mM.

[0008] Further, in step (2), the mixed solvent is composed of 5.5 mL of N,N-dimethylformamide and 5.5 mL of ethanol.

[0009] The application further provides a nano-enzyme system based on the Ag-polydopamine nanomicrosphere MOF-74 structure prepared by any of the above preparation methods.

[0010] The application further provides an application of the nano-enzyme system based on the Ag-polydopamine nanomicrosphere MOF-74 structure in antibiosis.

[0011] Further, the application comprises: after mixing the nano-enzyme system based on the Ag-polydopamine nanomicrosphere MOF-74 structure with a peroxide, under near-infrared light irradiation, the antibiosis is exerted.

[0012] Further, the peroxide is hydrogen peroxide.

[0013] Further, the wavelength of the near-infrared light is 808 nm, and the power is 0.5 W / cm 2 .

[0014] The application has the following advantages: The preparation method of the nano-enzyme system based on the Ag-polydopamine nanomicrosphere MOF-74 structure provided by the application introduces polydopamine (PDA) microspheres as a core template, and the surface of the polydopamine microspheres is rich in active functional groups such as catechol and amino groups, which not only can effectively adsorb metal ions through coordination to provide an advantage site for the heterogeneous nucleation of MOF-74, but also can accurately control the epitaxial growth of the MOF-74 shell on the surface, so as to finally form a core-shell structure (PDA MOF-74) with uniform morphology and good monodispersity. The structure effectively integrates the adhesion, biocompatibility of the PDA microspheres and the ordered pores and open active sites of the MOF-74.

[0015] To further enhance the antibacterial and catalytic performance of the composite system, silver nanoparticles (AgNPs) are introduced on the basis of the present application. On the one hand, silver nanoparticles have excellent light-heat conversion efficiency and can produce local high temperature under near-infrared laser irradiation, producing a synergistic photothermal effect with MOF-74 and PDA microspheres to efficiently kill pathogenic bacteria. On the other hand, and more importantly, silver nanoparticles have been proven to have excellent horseradish peroxidase (HRP) activity, which can efficiently catalyze the decomposition of hydrogen peroxide (H2O2) to produce strong oxidizing substances such as hydroxyl radicals (·OH), thus achieving chemical kinetic sterilization without light and catalyzing the color development substrate reaction. This synergistic mechanism of photothermal therapy and enzyme-catalyzed sterilization endows the nanoenzyme system with efficient and multi-mode antibacterial function, which has great potential in the treatment of drug-resistant bacterial infections and biofilm removal. BRIEF DESCRIPTION OF DRAWINGS

[0016] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed in the embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0017] Figure 1 SEM diagram of polydopamine microspheres in Test Example 1 of the present application.

[0018] Figure 2 SEM diagram of MOF-74 and polydopamine@MOF-74 in Test Example 1 of the present application. Among them, Figure 2 (a) is the SEM diagram of MOF-74 in Test Example 1; Figure 2 (b) is the SEM diagram of polydopamine@MOF-74 in Test Example 1.

[0019] Figure 3 XRD diagram of polydopamine@MOF-74 and Ag-polydopamine@MOF-74 in Test Example 1 of the present application.

[0020] Figure 4 Photothermal effect diagram of Ag-PDA@MOF-74 in Test Example 2 of the present application.

[0021] Figure 5 Photothermal antibacterial effect diagram of the material in Test Example 2 of the present application.

[0022] Figure 6 Effect diagram of the mixed solution in Test Example 3 of the present application, wherein, Figure 6 (a) is the mixed solution without adding materials, Figure 6(b) is the mixed solution after PDA@MOF-74 and Ag-PDA@MOF-74 are added.

[0023] Figure 7 The full wavelength detection of the reaction solution of PDA@MOF-74 and Ag-PDA@MOF-74 in Example 3 of the present application is carried out. DETAILED DESCRIPTION

[0024] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. In the case of no conflict, the embodiments in the present application and the features in the embodiments can be combined with each other.

[0025] In one aspect, the present application provides a preparation method of a nano-enzyme system based on Ag-polydopamine nanomicrosphere@MOF-74 structure, comprising the following steps: (1) 100-300 mg of hydrochloric acid dopamine is weighed and dissolved in 50-200 mL of Tris-HCl buffer solution with a concentration of 0.25-0.50 mM and a pH of 7.8; under the magnetic stirring of 500-1000 rpm, the reaction is carried out at 25-30°C for 12-48 hours; after the reaction is completed, centrifugal separation is carried out at 8000-10000 rpm, and polydopamine microspheres are obtained, which are freeze-dried for standby; (2) The polydopamine microspheres are dispersed in an aqueous solution to prepare a dispersion liquid with a concentration of 1-3 mg / mL, 1-3 mL of the polydopamine microsphere dispersion liquid is taken for standby; another magnesium chloride hexahydrate and 2,5-dihydroxyterephthalic acid are weighed and dissolved in a mixed solvent under ultrasonic assistance to obtain a mixed solution for standby; the mixed solution and the polydopamine microsphere dispersion liquid are combined and transferred to a hydrothermal reaction kettle, and the reaction is carried out at 135°C for 22 hours to obtain polydopamine microsphere@MOF-7 material; (3) After the reaction is completed, the polydopamine microsphere@MOF-7 material is washed with N,N-dimethylformamide and anhydrous methanol respectively for three times, and then freeze-dried to obtain freeze-dried MOF material; 100 mg of the freeze-dried MOF material is dispersed in 50 mL of water, 10 mL of silver nitrate solution with a concentration of 1 mg / mL is added, and the reaction is carried out for 24 hours, and then freeze-dried again to obtain Ag-polydopamine microsphere@MOF-74 material.

[0026] The embodiment of the present application provides a preparation method of a composite nanomaterial taking polydopamine (PDA) microspheres as a core and MOF-74 as a shell. The method utilizes the rich functional groups on the surface of the PDA microspheres to guide the heterogeneous nucleation and epitaxial growth of MOF-74 crystals on the surface of the PDA microspheres, realizes the accurate control of the morphology and size of the MOF-74 shell layer, and forms a core-shell structure with good monodispersity. On this basis, the in-situ reduction and loading of silver nanoparticles (AgNPs) in the MOF channels are realized by using the reducing property of PDA, and a multifunctional nanoscale enzyme system is successfully constructed.

[0027] The material has multiple excellent properties: the AgNPs endow the material with high horseradish peroxidase-like activity, can catalyze hydrogen peroxide to generate hydroxyl radicals, and realize chemical kinetic sterilization; meanwhile, the AgNPs, together with the PDA and the MOF-74, make the composite material exhibit a significant photothermal effect, and can generate a strong hyperthermia effect under near-infrared light irradiation. In addition, the stability of the MOF-74 shell in the aqueous environment provides the composite material with good biocompatibility and carrier function.

[0028] Experiments show that the nanoscale enzyme material exhibits excellent sterilization effect on gram-positive bacteria and gram-negative bacteria, and can effectively destroy bacterial biofilms, and has a wide application prospect in the fields of antibacterial treatment, biofilm removal and infection wound healing.

[0029] In an embodiment of the present application, in step (2), the final concentration of magnesium chloride hexahydrate is 0.5-1.8 mM.

[0030] In an embodiment of the present application, in step (2), the final concentration of 2,5-dihydroxyterephthalic acid is 0.1-0.5 mM.

[0031] In an embodiment of the present application, in step (2), the mixed solvent is composed of 5.5 mL of N,N-dimethylformamide and 5.5 mL of ethanol.

[0032] An embodiment of the present application also proposes a nanoscale enzyme system based on the Ag-polydopamine nanomicrosphere@MOF-74 structure prepared by any of the preparation methods described above. The nanoscale enzyme system constructed in the embodiment of the present application has achieved remarkable results in structure control, function integration and biological activity. Specifically, the polydopamine microspheres act as excellent template agents, effectively guide the ordered growth of MOF-74 on the surface of the polydopamine microspheres, successfully prepare a core-shell structure with uniform morphology and regular size, and significantly improve the dispersity and stability of the MOF material in practical application. In addition, by virtue of the in-situ reduction capacity of polydopamine, silver nanoparticles are uniformly loaded in the composite structure, not only greatly enhance the absorption and heat conversion capacity of the material to near-infrared light, endow the material with excellent photothermal performance, but also significantly improve the horseradish peroxidase-like catalytic activity.

[0033] The application also provides application of the Ag-polydopamine nanosphere@MOF-74 structure-based nanoenzyme system in antibiosis.

[0034] In an embodiment of the application, the application comprises: mixing the Ag-polydopamine nanosphere@MOF-74 structure-based nanoenzyme system with peroxide, and irradiating near-infrared light to exert antibiosis. In an embodiment of the application, the peroxide is hydrogen peroxide; the wavelength of the near-infrared light is 808 nm, and the power is 0.5 W / cm 2 .

[0035] The application will be described in detail below with reference to the embodiments.

[0036] Embodiment 1 provides a preparation method of an Ag-polydopamine nanosphere@MOF-74 structure-based nanoenzyme system, comprising the following steps: (1) 200 mg of dopamine hydrochloride is weighed and dissolved in 100 mL of Tris-HCl buffer solution with a concentration of 0.50 mM and a pH of 7.8. Under the condition of 1000 rpm magnetic stirring, the reaction is carried out at 30°C for 24 hours. After the reaction is completed, centrifugal separation is carried out at 10000 rpm to obtain uniformly dispersed polydopamine microspheres, which are subjected to freeze-drying treatment.

[0037] (2) The polydopamine microspheres are dispersed in an aqueous solution to prepare a dispersion liquid with a concentration of 2 mg / mL, and 2 mL of the polydopamine microsphere dispersion liquid is taken for standby. In addition, magnesium chloride hexahydrate (final concentration 1 mM) and 2,5-dihydroxyterephthalic acid (final concentration 0.1 mM) are weighed and dissolved in a mixed solvent (composition: 5.5 mL of N,N-dimethylformamide and 5.5 mL of ethanol) under ultrasonic assistance to obtain a mixed solution. The mixed solution and the polydopamine microsphere dispersion liquid are combined and transferred to an autoclave for reaction at 135°C for 22 hours to obtain polydopamine microsphere@MOF-7 material.

[0038] (3) After the reaction is completed, the obtained yellow product is washed with N,N-dimethylformamide and anhydrous methanol, respectively, for three times, and then freeze-dried to obtain a freeze-dried MOF material. 100 mg of the freeze-dried MOF material is dispersed in 50 mL of water, 10 mL of a silver nitrate solution with a concentration of 1 mg / mL is added, and after 24 hours of reaction, freeze-drying is performed again to obtain an Ag-polydopamine microsphere@MOF-74 material. Comparative Example 1 The same as in Example 1, except that step (3) is not performed, and a polydopamine microsphere@MOF-7 material is directly prepared.

[0039] Test Example 1 The SEM images of the products obtained in steps (1), (2), and (3) in the preparation process of the nanoscale enzyme system based on the Ag-polydopamine nanomicrosphere@MOF-74 structure are tested by a scanning electron microscope, and the results are shown in Figure 1 , Figure 2 (a), Figure 2 (b).

[0040] The XRD images of the products obtained in steps (2) and (3) are tested by an X-ray diffraction technique, and the results are shown in Figure 3 . It can be seen from Figure 3 that the XRD schematic diagram shows that Ag nanoparticles are generated in Ag-PDA@MOF-74 compared with PDA@MOF-74.

[0041] Test Example 2 Photothermal performance determination and antibacterial effect of Ag-polydopamine@MOF-74 1 mg of the silver-based catalyst based on Ag-polydopamine@MOF-74 finally obtained in Example 1 is dispersed in 5 mL of deionized water, and 1.5 mL of the dispersion is taken into a liquid vial; a GCSLS-05-7W type semiconductor laser is used to irradiate the liquid in the vial with near-infrared (NIR) light (wavelength 808 nm) at a power density of 2.0 W / cm 2 for 5 min, and a TES-1315 K type thermocouple thermometer is used to record the temperature every 2 s during the irradiation. The results are shown in Figure 4 .

[0042] Single colonies are picked from the recovered bacterial (Escherichia coli and Bacillus subtilis) plates, inoculated into test tubes containing 4 mL of LB liquid medium, and then the test tubes are placed in a constant temperature incubator and cultured at 37°C and 180 rpm on a shaker overnight to obtain a bacterial suspension.

[0043] Specific experimental procedures: After the LB culture medium in the test tubes became turbid, its OD600 value was measured, and the test strains were prepared into 5×10⁶ test tubes using LB liquid medium. 5 CFU / mL bacterial suspension (for agar plate colony counting). Take 50 μL of 0.2 mg / mL Ag-polydopamine@MOF-74 and mix it with 50 μL of the diluted bacterial suspension. After mixing, perform photothermal treatment for 10 min in 96-well plates (using an 808 nm laser with a power density of 2 W / cm²). 2 The treated mixture was then evenly spread onto an agar plate and incubated overnight at 37°C in a shaker. The control group was not exposed to light. Results are as follows: Figure 5 As shown. Among them, Figure 5 In the following categories: A. Pure Escherichia coli; B. Pure Dried hay; C. Material mixed with Escherichia coli; D. Dried hay mixed with the material; E. Photothermal effect after mixing Escherichia coli with the material; F. Photothermal effect after mixing Dried hay with the material.

[0044] Test Example 3: Peroxidase Activity Determination of Materials Obtained in Example 1 and Comparative Example 1 Prepare 70 mM H2O2 solution, 15 mM 3,3',5,5'-tetramethylbenzidine (TMB) ethanol solution, 1 mg / mL Ag-polydopamine@MOF-74 solution obtained in Example 1, and 1 mg / mL polydopamine@MOF-74 solution obtained in Comparative Example 1 for subsequent use. Figure 6 To compare the mixtures before and after adding PDA@MOF-74 and Ag-PDA@MOF-74 respectively, 1 mL of 70 mM H2O2 solution and 500 μL of 15 mM TMB ethanol solution were dissolved in 50 mM sodium acetate buffer at pH 4. 75 μL of 1 mg / mL Ag-polydopamine@MOF-74 was added, and the volume was brought up to 3 mL with sodium acetate buffer. The mixture was shaken at 45 ℃ and 800 rpm for 15 minutes, centrifuged at 8000 rpm for 2 minutes, and 100 μL of the supernatant was transferred to a 96-well plate. The results were measured at all wavelengths using a UV spectrophotometer. (See attached table for details.) Figure 7 .

[0045] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a nanozyme system based on Ag-polydopamine nanospheres@MOF-74 structure, characterized in that, Includes the following steps: (1) Weigh 100-300 mg of dopamine hydrochloride and dissolve it in 50-200 mL of Tris-HCl buffer solution with a concentration of 0.25-0.50 mM and pH 7.8; react at 25-30℃ for 12-48 hours under magnetic stirring at 500-1000 rpm; after the reaction is completed, centrifuge at 8000-10000 rpm to obtain polydopamine microspheres, freeze-dry them for later use; (2) Disperse the above polydopamine microspheres in an aqueous solution to prepare a dispersion with a concentration of 1-3 mg / mL, and take 1-3 mL of the polydopamine microsphere dispersion for later use; separately weigh magnesium chloride hexahydrate and 2,5-dihydroxyterephthalic acid, dissolve them in a mixed solvent under ultrasonic assistance to obtain a mixed solution for later use; combine the above mixed solution with the above polydopamine microsphere dispersion, transfer them to a hydrothermal reactor, and react at 135℃ for 22 hours to obtain polydopamine microspheres@MOF-7 material; (3) After the reaction was completed, the above polydopamine microspheres@MOF-7 material was washed three times with N,N-dimethylformamide and anhydrous methanol respectively, and then freeze-dried to obtain freeze-dried MOF material; 100 mg of the above freeze-dried MOF material was dispersed in 50 mL of water, 10 mL of silver nitrate solution with a concentration of 1 mg / mL was added, and after reacting for 24 hours, it was freeze-dried again to obtain Ag-polydopamine microspheres@MOF-74 material.

2. The preparation method according to claim 1, characterized in that, In step (2), the final concentration of magnesium chloride hexahydrate is 0.5-1.8 mM.

3. The preparation method according to claim 1, characterized in that, In step (2), the final concentration of 2,5-dihydroxyterephthalic acid is 0.1-0.5 mM.

4. The preparation method according to claim 1, characterized in that, In step (2), the mixed solvent consists of 5.5 mL N,N-dimethylformamide and 5.5 mL ethanol.

5. The nanozyme system based on the Ag-polydopamine nanospheres@MOF-74 structure prepared by the preparation method according to any one of claims 1-4.

6. The application of the nanozyme system based on the Ag-polydopamine nanospheres@MOF-74 structure as described in claim 5 in antibacterial applications.

7. The application according to claim 6, characterized in that, The application includes: mixing a nanoenzyme system based on Ag-polydopamine nanospheres@MOF-74 structure with peroxide, and then exerting an antibacterial effect under near-infrared light irradiation.

8. The application according to claim 7, characterized in that, The peroxide is hydrogen peroxide.

9. The application according to claim 7, characterized in that, The near-infrared light has a wavelength of 808 nm and a power of 0.5 W / cm². 2 .

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