Zirconium / molybdenum-porphyrin metal organic framework nano-enzyme as well as preparation method and application thereof

Abstract

The invention relates to a zirconium / molybdenum-porphyrin metal organic framework nano-enzyme as well as a preparation method and application thereof, and belongs to the technical field of biological medicine.In the preparation process of the zirconium / molybdenum-porphyrin metal organic framework nano-enzyme, two different topological linkers are introduced, so that the zirconium / molybdenum-porphyrin metal organic framework nano-enzyme is prepared; the Mo / PCN has near-infrared enhanced peroxidase activity and excellent photo-thermal performance and can have good sterilization and biofilm formation destroying performance on gram-positive bacteria, gram-negative bacteria and gram variation bacteria, and the minimum sterilization concentration is 10 micrograms / mL to 15 micrograms / mL. The zirconium / molybdenum-porphyrin metal organic framework nano-enzyme has excellent POD-like activity, and generated active oxygen and NIR promote an electron transfer mechanism in a bacterial membrane to trigger an electron transfer process, so that electrons enter a bacterial kernel to finally cause bacterial death. In-vitro experiments show that Mo / PCN has excellent biocompatibility and relatively strong antibacterial and anti-biofilm activity, and the effect of Mo / PCN is superior to that of a positive control drug.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a zirconium / molybdenum-porphyrin metal organic framework nanozyme and its preparation method and application. Background Art

[0002] Metal-organic frameworks (MOFs) are a new class of porous materials composed of metal ions / clusters linked to organic ligands through coordination bonds. Due to their high porosity, controllable pore size / shape, and unique physicochemical properties, MOFs have shown promise in applications such as gas storage, separation, sensors, and catalysis. Compared to single-linker MOFs, hybrid MOFs exhibit the following advantages: (i) the increased number of linkers imparts enhanced stability to hybrid MOFs, and (ii) their tunability allows for systematically regulated pores and diverse functionalities. Nanozymes, a class of nanomaterials with enzyme-mimicking catalytic properties, have emerged as a novel antibacterial therapeutic agent, primarily through their unique ability to produce toxic products that can effectively disrupt bacterial biofilms. MOF-based nanozymes have shown great potential for application in nanozyme-photothermal therapy (NPT) for anti-infective treatment. They can overcome drug resistance, reduce drug dosage, and avoid potential systemic side effects, making them a promising alternative strategy to antibiotics for combating infections.

[0003] Bacterial vaginosis (BV) is the most common vaginal infection, accounting for nearly half of all vaginal infections. Patients may experience symptoms such as vaginal itching and abnormal vaginal discharge. Patients with BV need timely treatment, otherwise the risk of spontaneous abortion, postpartum endometritis, and other conditions increases. Currently, antibacterial drugs such as metronidazole are the preferred treatment for BV, but long-term use can easily lead to the development of drug-resistant strains. Due to antibiotic resistance in pathogens and their biofilms, the greatest challenge in BV treatment is its high recurrence rate. To address this challenge in BV treatment, there is an urgent need to develop new antibacterial alternatives with different bactericidal mechanisms than traditional antibiotics, to overcome bacterial resistance and persistence without causing new resistance or affecting vaginal probiotics such as Lactobacillus. Summary of the Invention

[0004] Based on the problems raised in the background technology, the present invention provides a near-infrared light-enhanced zirconium / molybdenum-porphyrin metal-organic framework nanozyme (Mo / PCN) and its synthesis method. Two different topological connectors are introduced during the synthesis to successfully obtain a metal-organic framework (MOF) nanozyme. The resulting nanozyme exhibits near-infrared (NIR)-enhanced peroxidase-like (POD-like) activity and excellent photothermal performance. It can effectively kill and disrupt biofilm formation against Gram-positive, Gram-negative, and Gram-mutant bacteria, with a minimum bactericidal concentration (MFC) of 10 μg / mL to 15 μg / mL. This is attributed to the reactive oxygen species generated by the excellent POD-like activity of the Zr / Mo-porphyrin metal-organic framework nanozyme and the NIR-promoted electron transfer mechanism within the bacterial membrane, triggering the electron transfer process, causing electrons to enter the bacterial core and ultimately leading to bacterial death. In vitro experiments showed that Mo / PCN has excellent biocompatibility. Combined with its POD-like activity and photothermal properties, Mo / PCN has strong antibacterial and anti-biofilm activity, which is better than the positive control drug. It is expected to be used to develop or enhance antibacterial therapies to prevent drug-resistant bacterial infections.

[0005] The first object of the present invention is to provide a method for preparing a zirconium / molybdenum-porphyrin metal organic framework nanozyme, specifically:

[0006] 35.0-40.0 mg of zirconium chloride, 6.0-7.0 mg of tetrakis(4-carboxyphenyl)porphyrin, 70.0-90.0 mg of sodium molybdate, and 1.0-2.0 g of benzoic acid were weighed and added to 10-20 mL of N,N-dimethylformamide. After mixing, the mixture was ultrasonically treated for 15-20 minutes. The mixed solution was transferred to an autoclave and heated to 130°C-140°C for reaction. After the reaction was cooled to room temperature, the gray-green product was centrifuged and washed with anhydrous ethanol and deionized water. After vacuum drying, Mo / PCN nanozyme was obtained.

[0007] The second object of the present invention is to provide a zirconium / molybdenum-porphyrin metal organic framework nanozyme obtained by the above preparation method.

[0008] The third object of the present invention is to provide the use of zirconium / molybdenum-porphyrin metal organic framework nanozymes mixed with peroxides as antibacterial drugs under near-infrared light irradiation.

[0009] Furthermore, the peroxide is hydrogen peroxide, sodium persulfate or ammonium persulfate; the near-infrared light has a wavelength of 808 nm and a power of 0.50-1.0 W / cm 2 .

[0010] Beneficial effects of the present invention: 1. The zirconium / molybdenum-porphyrin metal organic framework nanozyme Mo / PCN synthesized by the present invention has an enhanced response to infrared light due to the introduction of TCPP, and the introduction of molybdenum generates oxygen vacancies, which ultimately makes Mo / PCN have excellent peroxidase-like activity and photothermal performance. Under near-infrared irradiation, the peroxidase-like activity of Mo / PCN is greatly improved. The concentration of Mo / PCN is between 10-20 μg / mL and 1.0 W / cm 2 When irradiated with 808 nm NIR for 10 min, the sterilization rate of Gram-positive bacteria, Gram-negative bacteria, and Gram-mutant bacteria was greater than 99%, and the biofilm formation was destroyed. The Mo / PCN has excellent POD-like activity, and the reactive oxygen species produced and NIR promote the electron transfer mechanism in the bacterial membrane to trigger the electron transfer process, thereby causing electrons to enter the bacterial core and ultimately lead to bacterial death.

[0011] 2. In vitro experiments showed that Mo / PCN has excellent biocompatibility and low toxicity. Combined with its POD-like activity and photothermal properties, its antibacterial effect is better than that of the positive control drug metronidazole. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 TEM image of Mo / PCN prepared in Example 1 of the present invention; Figure 2 The O 1s, Mo 3d and Zr 3d high-resolution XPS spectra of Mo / PCN prepared in Example 1 of the present invention; Figure 3 The UV absorption spectra of Mo / PCN at different concentrations at 808 nm in Example 1 of the present invention are shown in FIG. Figure 4 For different concentrations of Mo / PCN in Example 1, 1.0 W / cm 2 , 808 nm irradiation for 10 min, and the obtained temperature-time curve; Figure 5 This is the photothermal conversion curve of 150 μg / mL Mo / PCN in Example 1 of the present invention; Figure 6 The Mo / PCN + H2O2 + TMB in Example 1 of the present invention is 1.0 W / cm 2 , UV absorption spectra under 808 nm NIR irradiation for 10 min and without irradiation; Figure 7 is the Michaelis-Menten equation of Mo / PCN under fixed H2O2 and changing TMB concentration under natural light; Figure 8 is the Michaelis-Menten equation of Mo / PCN under natural light with fixed TMB and changing H2O2 concentration; Figure 9is the Michaelis-Menten equation of Mo / PCN under infrared light with fixed H2O2 and varying TMB concentration; Figure 10 is the Michaelis-Menten equation of Mo / PCN under infrared light with fixed TMB and varying H2O2 concentration; Figure 11 Effects of different concentrations of Mo / PCN on the viability of Hep G2 cells and HUVECs cells at 24 h and 48 h; Figure 12 The antibacterial properties of different concentrations of Mo / PCN against different pathogens under 808 nm NIR irradiation and without irradiation are shown; Figure 13 The anti-biofilm activity of Mo / PCN against different pathogens under 808 nm NIR irradiation and without irradiation was obtained by CV staining method; Figure 14 The antibacterial SEM images of Mo / PCN against different pathogens under 808 nm NIR irradiation and without irradiation; Figure 15 is the outer membrane permeability of pathogenic bacteria under Mo / PCN treatment; Figure 16 Leakage of pathogenic bacterial proteins under Mo / PCN treatment. DETAILED DESCRIPTION

[0013] In order to make the objectives, technical solutions and beneficial effects of the present invention clearer, preferred embodiments of the present invention will be described in detail below to facilitate understanding by technicians.

[0014] The preparation method of the Mo / PCN nanozyme is as follows: 35.0-40.0 mg ZrCl4, 6.0-7.0 mg tetrakis(4-carboxyphenyl)porphyrin TCPP, 70.0-90.0 mg sodium molybdate and 1.0-2.0 g benzoic acid were weighed and added to 10-20 mL DMF and mixed. After mixing, the mixture was ultrasonically treated for 15-20 min. The mixed solution was transferred to a polytetrafluoroethylene autoclave, heated to 130℃-140℃ and reacted for 24 h. After the reaction solution was cooled to room temperature, the gray-green product was centrifuged and washed with anhydrous ethanol and deionized water 3-4 times each. After vacuum drying, Mo / PCN nanozyme was obtained.

[0015] Applications of Mo / PCN nanozymes: The Mo / PCN nanozyme was mixed with peroxide and sonicated for 5-10 min, where the weight ratio of Mo / PCN to peroxide was 10-15:1. Upon application, the nanozyme was irradiated with 808 nm near-infrared light for 10-15 min.

[0016] Wherein, the peroxide is hydrogen peroxide, sodium persulfate or ammonium persulfate.

[0017] The power of the 808nm near-infrared light is 0.50-1.0 W / cm 2 .

[0018] Example 1 Preparation of Mo / PCN nanozymes: 35.0 mg of zirconium chloride (ZrCl4), 6.0 mg of tetrakis(4-carboxyphenyl)porphyrin TCPP, 70.0 mg of sodium molybdate (Na2MoO4) and 1.0 g of benzoic acid (C7H6O2) were weighed and added to 10 mL of N,N-dimethylformamide. After mixing, the mixture was ultrasonically treated for 15 min. The mixed solution was transferred to a polytetrafluoroethylene high-pressure reactor, heated to 130°C and reacted for 24 h. After the reaction was cooled to room temperature, the gray-green product was centrifuged at 8000 r / min for 10 min, washed with anhydrous ethanol and deionized water 3-4 times each, and vacuum dried to obtain Mo / PCN nanozyme.

[0019] Figure 1 This is the TEM image of Mo / PCN. It can be seen from the TEM image that Mo / PCN has irregular particles and stacking structure, which is composed of 2D layers composed of Zr6 clusters and main connector benzoic acid, and supported by auxiliary connector TCPP.

[0020] Figure 2 High-resolution XPS spectra of Mo / PCN and high-resolution O 1s spectra ( Figure 2 a) shows two peaks at 530.28 eV and 532.38 eV, which are oxygen vacancies (Ov) and surface adsorbed water / hydroxyl groups (Os), respectively. Mo 3d shows two peaks at 232.48 eV and 235.58 eV, which correspond to Mo 3d5 / 2 and Mo 3d3 / 2, respectively. Figure 2 b), 182.28 eV and 184.68 eV correspond to Zr 3d5 / 2 and Zr 3d3 / 2 ( Figure 2 c), These results indicate the successful synthesis of Mo / PCN.

[0021] Performance analysis of Mo / PCN nanozyme: (1) Photothermal performance evaluation of Mo / PCN To evaluate the NIR-triggered photothermal performance of Mo / PCN nanozymes, the temperature change of the nanozyme solution under NIR irradiation at 808 nm was measured.

[0022] The UV-visible absorption spectrum of Mo / PCN solution ( Figure 3), it can be seen that: with the increase of concentration (0 ~ 200 μg / mL), the absorbance at 808 nm (0.069 ~ 0.306) increases accordingly. This result shows that the absorption of Mo / PCN to near-infrared light (808 nm) is concentration-dependent, providing a basis for further research on the photothermal properties of Mo / PCN. 2 , 808 nm NIR irradiation for 10 min, and the temperature-time curve was obtained ( Figure 4 ), it can be seen that after 110 minutes of 808 nm NIR light irradiation, the temperature of Mo / PCN (150 μg / mL) increased significantly from 19.3°C to 60.4°C, and the temperature was positively correlated with the concentration, while the temperature of water only increased by 9.1°C. In addition, the photothermal conversion efficiency (η) of Mo / PCN was calculated according to the following formula:

[0023] Where T max is the equilibrium temperature of the sample solution, T surr Corresponding to the ambient temperature of the experiment, Q dis is the heat loss caused by the container absorbing light, where Q dis = (5.4×10 -4 ), I represents the power density of 808 nm laser (1.0 W / cm 2 ), the absorbance of the sample solution at 808 nm is recorded as A λ ; hs is obtained by the following formula:

[0024] Where m represents the mass of the sample solution, C water Corresponding to the heat capacity of water (4.2 J kg -1 K -1 ), is the time constant of the system. The calculated photothermal conversion efficiency of the prepared Mo / PCN is 75.0% ( Figure 5 ab), showing high photothermal conversion efficiency.

[0025] (2) Evaluation of Mo / PCN peroxidase nanozyme activity The peroxidase-like (POD-like) activity of the enzyme was evaluated using TMB as the catalytic substrate. 100 μL of 5 mmol / L TMB, 100 μL of 50 mmol / L H2O2, and 100 μL of 1 mg / mL Mo / PCN were added to 2 mL of 0.1 mol / L HAc-NaAc buffer solution (pH 4.0) in a 5 mL stoppered colorimetric tube. The mixture was thoroughly mixed and the volume was adjusted to 3 mL. The absorbance was measured after 10 min of reaction. 2 The enzyme activity was compared after irradiation at 808 nm for 10 min, and the absorbance was measured at 654 nm. The results showed that ( Figure 6 ), compared with no irradiation under 808 nm near-infrared light, irradiation under near-infrared light significantly enhanced the peroxidase activity of Mo / PCN, and Mo / PCN showed the characteristics of near-infrared light-enhanced peroxidase activity.

[0026] The Michaelis-Menten catalytic kinetic parameters were also determined ( Figures 7 to 10 and Table 1), the Michaelis constant K of Mo / PCN for substrates TMB and H2O2 m were 8.525 mmol / L and 9.696 mmol / L, respectively, and the reaction rate constants were 57.180×10 -8 mol / Ls and 6.667×10 -8 mol / Ls, K after infrared light irradiation m The reaction rates were 3.755 mmol / L and 0.487 mmol / L, respectively, and the reaction rate constants were 173.96×10 -8 mol / Ls and 3.232×10 -8 mol / Ls, indicating that infrared light irradiation increased the affinity and reaction rate of Mo / PCN nanozyme with the substrate.

[0027] Table 1 Michaelis-Menten catalytic kinetic parameters

[0028] (3) Cytotoxicity test The cytotoxicity of nanozymes was detected using a CCK-8 cell viability kit. Specifically, human umbilical vein endothelial cells and hepatocellular carcinoma cells (HUVECs and Hep G2, Beina Chuanglian Biotechnology Co., Ltd.) were seeded in 96-well plates and cultured for 24 h. They were then incubated with different concentrations of Mo / PCN for 24 and 48 h, respectively. The cells were rinsed with PBS in batches, and CCK-8 solution was added to each well to a concentration of 10%. The cells were incubated at 37°C, and the absorbance was measured at 450 nm. CCK-8 analysis ( Figure 11) showed that Mo / PCN had no toxicity to cells.

[0029] (4) Mo / PCN antibacterial test The following bacterial strains were obtained from Beina Chuanglian Biotechnology Co., Ltd.

[0030] Experimental Methods: Staphylococcus aureus (ATCC 43300), methicillin-resistant Staphylococcus aureus (MRSA, ATCC-6538), Escherichia coli (ATCC-8099), and ampicillin-resistant E. coli (SHBCC D25148) were used as representative Gram-positive and -negative strains, as well as Gram-variant bacteria (G. vaginalis). The antibacterial activity of Mo / PCN was determined by counting CFU using the plate count method. First, the above-mentioned strains were incubated in solid Luria-Bertani (LB) medium and solid nutrient broth medium for 24 hours. A small amount of the formed colonies were picked with an inoculation loop and inoculated into the corresponding liquid nutrient broth medium (5 mL). Then, after shaking and incubating for 12 hours at 37°C and 180 rpm in a constant temperature shaker, a bacterial suspension (1×108 CFU / mL) was obtained and diluted to 1×105 CFU / mL with sterile phosphate buffered saline (PBS). The materials were divided into four groups: blank control group, H2O2 group, Mo / PCN group, and Mo / PCN + H2O2 group, in which the Mo / PCN concentration was 10 μg / mL and the H2O2 concentration was 200 μmol / L. The cultured bacteria were added to phosphate buffer as a blank control group and subjected to 1.0 W / cm 2 The bacterial suspension incubated at 37°C for 60 min was diluted (100 μL) and evenly spread on LB solid medium and nutrient broth solid medium, and cultured at 37°C for 24 h. The number of colonies was counted to determine the antibacterial properties, and SEM characterization was performed.

[0031] The results show that (Appendix Figure 12 ) As shown in the figure, the blank control group had almost no antibacterial properties. Mo / PCN + H2O2 showed excellent antibacterial effects against all bacteria under 10 minutes of NIR irradiation, with a sterilization rate of nearly 100% for all bacteria. SEM characterization showed that the control group retained complete cell morphology. However, in the presence of NIR or H2O2, the cell membranes of AREC, MRSA, and Gardnerella vaginalis (G. vaginalis) showed wrinkles and shrinkage, while Mo / PCN + H2O2+ NIR treatment, the cells were completely destroyed.

[0032] To further evaluate the anti-biofilm performance of Mo / PCN, we investigated its anti-biofilm ability by crystal violet (CV) staining and measured the absorbance at 590 nm to quantify the biofilm. + H2O2+ NIR significantly inhibited the formation of bacterial biofilm after incubation for 48 h ( Figure 13 ), showing the lowest biofilm survival rate of 21.79%. In order to further determine the antibacterial ability of Mo / PCN, the Mo / PCN SEM observation of AREC, MRSA, E. coli and S. aureus treated with Mo / PCN + H2O2 + NIR and without Mo / PCN + H2O2 + NIR incubation (e.g. Figure 14 The control group retained intact cell morphology. However, the cell membranes of AREC, MRSA, E. coli, and S. aureus became wrinkled, shrunken, completely ruptured, and even lost their original morphology.

[0033] The hydrophobic fluorescent probe N-phenyl-1-naphthylamine (NPN) was used to measure the penetration of the outer membrane of various pathogenic bacteria. It exhibits weak fluorescence in aqueous solution and strong fluorescence intensity in hydrophobic environment. The results of the fluorescence intensity of NPN by pathogenic bacteria treated with +H2O2+NIR showed that the treatment directly accelerated the permeabilization of the outer membrane, and the fluorescence intensity increased to varying degrees ( Figure 15 ).

[0034] Another way to verify the destruction of pathogenic bacterial membranes is the leakage of intracellular substances, including some key proteins. The BCA assay (BCA) method was used to detect the concentration of pathogenic bacterial protein leakage after Mo / PCN+H2O2+NIR treatment. Figure 16 As shown in the figure, after Mo / PCN treatment and with or without NIR irradiation, bacterial proteins leaked. After adding NIR, protein leakage increased, indicating that the cytoplasmic membrane was damaged to a certain extent, and under Mo / PCN When the concentrations of H2O2 were 100 μg / mL and 200 μmol / L, respectively, the leakage of pathogenic bacterial proteins was observed.

[0035] The above results indicate that the Mo / PCN nanozyme prepared by the present invention has peroxidase-like activity and infrared photothermal properties. Based on the peroxidase-like activity and photothermal properties of the nanozyme, it has a joint antibacterial effect under near-infrared light (NIR) irradiation, and has an effective antibacterial and anti-biofilm effect on pathogens involved in bacterial vaginosis.

[0036] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A method for preparing a zirconium / molybdenum-porphyrin metal-organic framework nanozyme, characterized by: 35.0-40.0 mg of zirconium chloride, 6.0-7.0 mg of tetrakis(4-carboxyphenyl)porphyrin, 70.0-90.0 mg of sodium molybdate, and 1.0-2.0 g of benzoic acid were weighed and added to 10-20 mL of N,N-dimethylformamide. After mixing, the mixture was ultrasonically treated for 15-20 min. The mixed solution was transferred to an autoclave and heated to 130°C-140°C for reaction. After the reaction was completed, it was cooled to room temperature. The gray-green product was centrifuged and washed with anhydrous ethanol and deionized water. After vacuum drying, Mo / PCN nanozyme was obtained.

2. The zirconium / molybdenum-porphyrin metal organic framework nanozyme obtained by the preparation method according to claim 1.

3. Use of the zirconium / molybdenum-porphyrin metal organic framework nanozyme as claimed in claim 1 mixed with peroxide as an antibacterial drug under near-infrared light irradiation.

4. The use according to claim 3, characterized in that: The peroxide is hydrogen peroxide, sodium persulfate or ammonium persulfate; the near-infrared light has a wavelength of 808 nm and a power of 0.50-1.0 W / cm 2 .

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