Metal organic framework compound constructed by antibacterial agent as well as preparation method and application of metal organic framework compound

Abstract

The invention discloses a metal organic framework compound constructed by an antibacterial agent as well as a preparation method and application of the metal organic framework compound, and relates to the technical field of biomedical materials. The preparation method comprises the following steps: dissolving a soluble metal salt and 2-nitroimidazole in an organic solvent, carrying out a mixed reaction, centrifuging to obtain a precipitate, and cleaning the precipitate to obtain the metal organic framework compound. An antibacterial drug metal organic framework compound with ultrasonic and acid responsiveness is constructed by utilizing a single antibacterial agent, active oxygen and active nitrogen can be generated under ultrasonic stimulation, meanwhile, controllable release of the antibacterial agent in a biological membrane infected slightly-acidic environment is achieved, and the antibacterial drug metal organic framework compound has a good antibacterial effect on deep tissue bacterial infection. The compound shows a potential application effect on prevention of oral caries.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and particularly to a metal-organic framework compound constructed by an antibacterial agent, its preparation method and application. Background Art

[0002] A variety of diseases caused by bacterial infections are closely related to the formation of biofilms. These biofilms are the result of bacteria adhering to tissue surfaces and multiplying, and then secreting extracellular polymeric matrixes to gradually construct complex and robust structures. As biofilms mature, they not only provide a protective microenvironment for bacteria, but also enhance resistance to drug treatment. Inside the biofilm, the horizontal transfer of drug-resistant genes exacerbates the development of drug resistance, thus greatly reducing the effectiveness of traditional antibiotic therapies. Therefore, developing a new generation of drugs that can effectively remove drug-resistant biofilms is crucial for overcoming this clinical challenge.

[0003] Metal-organic frameworks (MOFs) are a class of compounds self-assembled from metal ions or clusters and polydentate organic ligands. This type of material combines the advantages of inorganic and organic components, and has characteristics such as high specific surface area, adjustable size and porosity, and is widely used in multiple fields such as catalysis, gas adsorption, sensing, and drug delivery. However, in traditional drug delivery systems, MOFs usually carry drug molecules in a physical encapsulation manner, which limits the effective control of drug release behavior. Although there have been studies attempting to involve drug molecules as one of the building units in the synthesis process of MOFs - for example, the research work of Professor Shilin Qi's team at Nankai University demonstrated that voriconazole and 2-methylimidazole together form ZIF-8 (Adv. Funct. Mater., 2020, 2000537), it is still a direction worthy of in-depth exploration to achieve more efficient utilization of a single drug to construct MOFs without additional organic ligands, thereby increasing the drug loading and achieving a more precise drug release mechanism. Summary of the Invention

[0004] The purpose of the present invention is to provide a metal-organic framework compound constructed by an antibacterial agent, its preparation method and application, so as to solve the problems existing in the above-mentioned prior art. The present invention constructs an antibacterial drug metal-organic framework compound with ultrasonic and acid responsiveness using a single antibacterial agent, and can generate reactive oxygen and reactive nitrogen under ultrasonic stimulation, while realizing the controllable release of the antibacterial agent in the slightly acidic environment of biofilm infection, showing potential application effects on deep tissue bacterial infections and the prevention of dental caries.

[0005] To achieve the above purpose, the present invention provides the following solution:

[0006] The present invention provides a preparation method of a metal-organic framework compound constructed by an antibacterial agent, comprising the following steps:

[0007] Dissolve the soluble metal salt and 2-nitroimidazole in an organic solvent. After mixing and reacting, centrifuge to obtain a precipitate. After cleaning the precipitate, the metal-organic framework compound is obtained.

[0008] Further, the soluble metal salt is a zinc salt compound.

[0009] Further, the zinc salt compound is zinc nitrate.

[0010] Further, the molar ratio of the zinc nitrate to the 2-nitroimidazole is 1:1.

[0011] Further, the temperature of the mixing reaction is 90 - 110 °C and the time is 24 - 48 h.

[0012] The present invention also provides a metal-organic framework compound prepared by the above preparation method.

[0013] The present invention also provides the application of the above metal-organic framework compound in the preparation of antibacterial products.

[0014] Further, the antibacterial product is a drug or an oral cleaning product. Oral cleaning products include, but are not limited to, toothpaste and mouthwash.

[0015] Further, the bacteria targeted by the antibacterial product is Streptococcus mutans.

[0016] The present invention also provides an antibacterial product, the active ingredient of which includes the above metal-organic framework compound.

[0017] The present invention discloses the following technical effects:

[0018] The present invention innovatively develops a metal-organic framework compound based on a single antibacterial agent, 2-nitroimidazole. This antibacterial agent can coordinate with Zn 2+ to form a unique MOF structure. This compound can generate reactive oxygen species (ROS) and reactive nitrogen species (RNS) under ultrasonic stimulation. These reactive substances can not only dissociate biofilms but also effectively prevent the generation of bacterial drug resistance. In addition, under the conditions of the slightly acidic environment of biofilm infection, this metal-organic framework compound can release the 2-nitroimidazole antibacterial agent, and cooperate with ROS and RNS to achieve the effect of efficiently clearing bacteria and biofilms. The present invention is the first to use a single antibacterial agent to construct an antibacterial drug metal-organic framework compound with ultrasonic and acid responsiveness, and can generate reactive oxygen species and reactive nitrogen species under ultrasonic stimulation, while realizing the controllable release of the antibacterial agent in the slightly acidic environment of biofilm infection.

[0019] The following are the main advantages of the present invention:

[0020] 1. The present invention uses a one-pot method to prepare a carrier-free single antibacterial metal-organic framework compound, which simplifies the synthesis steps and improves the production efficiency.

[0021] 2. The present invention first realizes the ability of metal-organic framework compounds to generate ROS and RNS under ultrasonic stimulation, making the generation process of active substances more controllable and enhancing the antibacterial effect.

[0022] 3. The metal-organic framework compound prepared by the present invention can intelligently respond and release antibacterial agents in the acidic environment of biofilm infection, and cooperate with ROS and RNS to effectively inhibit and dissociate the biofilm formed by drug-resistant bacteria, showing great potential in preventing oral diseases such as dental caries. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0024] Figure 1 is a flowchart of the preparation method of the metal-organic framework compound;

[0025] Figure 2 is a scanning electron micrograph of the metal-organic framework compound;

[0026] Figure 3 is the FTIR spectrum of the metal-organic framework compound (NOF), zinc nitrate (Zn 2+ ), and 2-nitroimidazole (2-NIM); among them, A is the spectrum in the wavelength range of 2000 - 3250 cm -1 ; B is the spectrum in the wavelength range of 200 - 1750 cm -1 ;

[0027] Figure 4 is the ESR test result of the metal-organic framework compound; among them, a is the ESR test spectrum of the O 2- signal; b is the concentration change diagram of O 2- ; c is the ESR test spectrum of the NO signal; d is the concentration change diagram of NO; US represents ultrasonic treatment;

[0028] Figure 5 is the test result diagram of the hemolysis experiment of different materials; among them, NOF represents the metal-organic framework compound prepared in Example 1, Zn 2+ represents zinc nitrate, and 2-NIM represents 2-nitroimidazole;

[0029] Figure 6 Graph showing the test results of the cytotoxicity experiment of different materials; among them, NOF represents the metal-organic framework compound prepared in Example 1, Zn 2+ represents zinc nitrate, and 2-NIM represents 2-nitroimidazole;

[0030] Figure 7 Graph (a) showing the effect of different materials on the formation of biofilms by Streptococcus mutans and statistical graph (b) of the biofilm thickness; among them, NOF represents the metal-organic framework compound prepared in Example 1, Zn 2+ represents zinc nitrate, 2-NIM represents 2-nitroimidazole, and US represents ultrasonic treatment. Detailed implementation manners

[0031] The various exemplary implementation manners of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0032] It should be understood that the terms used in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0033] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0034] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the description of the present invention, which are obvious to those skilled in the art. Other implementation manners obtained from the description of the present invention are obvious to those skilled in the art. The description and examples of the present invention are only exemplary.

[0035] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0036] The present invention has developed a metal-organic framework compound based on a single antibacterial agent, 2-nitroimidazole. The flow chart of its preparation method is shown in Figure 1 , and the preparation method, characterization, and effect verification of this metal-organic framework compound are described in detail as follows:

[0037] Example 1

[0038] Preparation method of the metal-organic framework compound based on a single antibacterial agent, 2-nitroimidazole:

[0039] Mix 297 mg of zinc nitrate hexahydrate (1.00 mmol) and 113 mg of 2-nitroimidazole (1.0 mmol), dissolve them in 10 mL of an organic solvent, N,N-dimethylformamide (DMF), stir in an oil bath at 90 °C for 24 h, and let it stand for 12 h. Separate the crude product by ultracentrifugation at 10,000 rpm, collect the precipitate, wash the crude product 3 times with 10 mL of PBS, and dry to obtain a pale yellow powder, which is the metal-organic framework compound (labeled as NOF).

[0040] Disperse NOF in methanol, drop 10 μL onto a silicon wafer and let it air dry, and perform scanning electron microscopy tests. The results are shown in Figure 2 , and the results show that NOF is a flaky material.

[0041] Directly detect the powder state of NOF by Fourier transform infrared spectroscopy (FTIR). The results are shown in Figure 3 . From Figure 3 it can be seen that the characteristic peaks at 3155 cm -1 , 2358 cm -1 , 1152 cm -1 , 959 cm -1 in NOF come from 2-nitroimidazole, corresponding to the asymmetric vibration of the C-H bond in -CH3, the N=C=O bond in the nitro group, the C=N bond on the imidazole ring, and the C-N bond on the imidazole ring respectively. In addition, the peak at 658 cm -1 may be the characteristic peak of Zn-O in NOF. Thus, it can be determined that the metal-organic framework compound has been successfully prepared.

[0042] To evaluate the effect of sonodynamic on the ability of NOF to generate ROS, it is known through a detection kit test that the ability of the metal-organic framework compound to generate nitric oxide and superoxide anions will increase after sonodynamic treatment. As Figure 4 shown, through electron spin resonance spectroscopy (ESR) test, it can be observed that superoxide anions are generated after ultrasound, while the O 2- signal of the non-ultrasonicated NOF does not change. This indicates that sonodynamic can improve the yield of ROS through cavitation, and Zn in NOF 2+It may be oxidized by cavitation, thus promoting the separation of electrons and holes to generate a large amount of ROS. At the same time, after sonodynamic treatment, the spectral signal of the metal-organic framework compound gradually changes from a quintet to a septet, indicating an increase in its NO content with sonodynamic treatment. Ultrasonic conditions: 0.5 w / cm 2 , duty cycle 50%, 25 min.

[0043] Example 2

[0044] Preparation method of metal-organic framework compound based on a single antibacterial agent 2-nitroimidazole:

[0045] Mix 297 mg of zinc nitrate hexahydrate (1.00 mmol) and 113 mg of 2-nitroimidazole (1.0 mmol), dissolve them in 10 mL of organic solvent DMF, stir in an oil bath at 110 °C for 48 h, and let it stand for 12 h. Separate the crude product by ultracentrifugation at 10000 rpm, collect the precipitate, wash the crude product 3 times with 10 mL of PBS, and dry to obtain a pale yellow powder, which is the metal-organic framework compound.

[0046] Example 3

[0047] Preparation method of metal-organic framework compound based on a single antibacterial agent 2-nitroimidazole:

[0048] Mix 297 mg of zinc nitrate hexahydrate (1.00 mmol) and 113 mg of 2-nitroimidazole (1.0 mmol), dissolve them in 10 mL of organic solvent DMF, stir in an oil bath at 100 °C for 48 h, and let it stand for 12 h. Separate the crude product by ultracentrifugation at 10000 rpm, collect the precipitate, wash the crude product 3 times with 10 mL of PBS, and dry to obtain a pale yellow powder, which is the metal-organic framework compound.

[0049] Example 4

[0050] Hemolysis experiment: Mix different concentrations of 2-nitroimidazole, zinc nitrate, and the NOF sample prepared in Example 1. Among them, the concentrations of 2-nitroimidazole and zinc nitrate in the control group are the same as those in NOF respectively. Mix them with 4% erythrocytes at a ratio of 1:1, incubate in a 37 °C incubator for 1 h, then obtain the supernatant by centrifuging at 5000 rpm for 15 minutes, and measure the absorbance of the supernatant at 545 nm using an enzyme-linked immunosorbent assay instrument; use phosphate buffer as the negative control and 0.1% Triton solution as the positive control.

[0051] The results are as Figure 5As shown, the 2-nitroimidazole group showed more than 5% hemolysis at 625.0 μg / mL, not meeting the clinical drug use standard. However, in the NOF group, even at the highest concentration (1250 μg / mL), the hemolysis was still no more than 5%. This indicates that under normal physiological conditions, the metal-organic framework compound nanoparticles are very stable and will not disassemble, so they will not cause hemolysis.

[0052] Example 5

[0053] Cytotoxicity experiment: Add 100 μL of the suspension of mouse fibroblasts (L929) (10 5 cell / mL) to a 96-well plate, incubate in a 37 °C incubator for 24 h to allow all cells to adhere, and add 10 μL of 2-nitroimidazole, zinc nitrate, and the NOF sample solution prepared in Example 1 with different concentrations to each well, so that the final concentration of the sample reaches 3.13 - 12500 μg / mL (the NOF group is based on the concentration of 2-nitroimidazole, and the concentration of zinc nitrate in the zinc nitrate group and NOF is kept the same). After incubating in a 37 °C incubator for 24 h, add 10 μL of CCK-8 reagent to each well, incubate in a 37 °C incubator for 1 h, and measure the absorbance value at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader; use the PBS treatment group as the positive control and the group without cells and only adding the culture medium as the negative control.

[0054] Cell viability (%) = [OD (sample) - OD (negative control)] / [OD (positive control) - OD (negative control)].

[0055] The results are as Figure 6 shown. Even at the highest concentration, the cell viability of the NOF group can still be maintained above 80%, indicating that the metal-organic framework compound prepared in the present invention has no toxic side effects on normal cells.

[0056] Example 6

[0057] Add 800 μL of different material solutions (2-nitroimidazole, zinc nitrate, NOF prepared in Example 1; each material is equivalent to the concentration of 2-nitroimidazole, prepared with PBS, and the concentration is 6.25 μg / mL) to a confocal dish. After standing for 0.5 h, add 800 μL of Streptococcus mutans solution (2 × 10 8 CFU / mL), and continue to culture in a 37 °C bacterial incubator for 1.0 h. Then suck out the supernatant and wash the precipitate 2 times with PBS. Next, add 800 μL of the above same material solution and 800 μL of 2 × Brain Heart Infusion (BHI) culture medium to each group again. After incubating in a 37 °C incubator for 16 h, suck out the supernatant, gently wash each well 3 times with 200 μL of PBS, and add SYTO TMIncubate with 9 and PI stains for 15 minutes, and observe the thickness and growth of the biofilm using laser confocal microscopy. Use PBS instead of the material solution as a control; in addition, perform ultrasonic treatment on the basis of the NOF group, denoted as the NOF+US group, with ultrasonic parameters of 0.5 w / cm 2 , duty cycle 50%, 15 min. The results are as Figure 7 shown. Figure 7 In Figure 7 , a is the laser confocal microscopy photo of the biofilm viability staining, where the green fluorescence represents live bacteria and the red fluorescence represents dead bacteria.

[0058] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A preparation method of a metal-organic framework compound constructed by an antibacterial agent, characterized in that, It includes the following steps: Dissolve the soluble metal salt and 2-nitroimidazole in an organic solvent. After mixing and reacting, centrifuge to obtain a precipitate. After cleaning the precipitate, the metal-organic framework compound is obtained.

2. The preparation method according to claim 1, wherein The soluble metal salt is a zinc salt compound.

3. The preparation method according to claim 2, characterized in that, The zinc salt compound is zinc nitrate.

4. The preparation method according to claim 3, characterized in that, The molar ratio of the zinc nitrate to the 2-nitroimidazole is 1:

1.

5. The preparation method according to claim 1, wherein, The temperature of the mixing reaction is 90 - 110 °C, and the time is 24 - 48 h.

6. A metal-organic framework compound prepared by the preparation method according to any one of claims 1 - 5.

7. Use of the metal-organic framework compound as claimed in claim 6 in the preparation of an antibacterial product.

8. The application according to claim 7, wherein The antibacterial product is a drug or an oral care product.

9. The application according to claim 8, characterized in that The bacteria targeted by the antibacterial product is Streptococcus mutans.

10. An antibacterial product, characterized in that, The active ingredient includes the metal-organic framework compound as claimed in claim 6.

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