An antibacterial gallium-copper bimetallic MOF nanomaterial and its preparation method and application

Abstract

The present invention relates to the field of biomaterial technology, specifically to an antibacterial gallium-copper bimetallic organic framework (Ga / Cu-MOF) nanomaterial and its preparation method and application. The Ga / Cu-MOF nanomaterial meets the requirements of biosafety and can be loaded with antibiotics to eliminate drug-resistant bacteria. The antibacterial application is that the Ga / Cu-MOF nanomaterial is loaded with antibiotics to eliminate intracellular bacteria and biofilms, and is effectively used in mouse skin defect infection models and rat osteomyelitis infection models.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular to a gallium-copper bimetallic organic framework (Ga / Cu-MOF) nanomaterial, a preparation method thereof, and antibacterial applications thereof. Background Art

[0002] With the widespread abuse and misuse of antimicrobial drugs, human health is facing severe challenges from multi-drug resistant super pathogens. The rapid growth of these resistant strains, especially those with complex drug resistance, poses an unprecedented challenge to clinical treatment. In response to this global crisis, the World Health Organization has developed a list of global priority pathogens, including multi-drug resistant strains such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE), because they can cause serious infections and pose challenges to existing treatments.

[0003] Clinically, the emergence of drug-resistant bacteria such as MRSA, whose resistance mechanisms include target changes, the production of antimicrobial inactivating enzymes, and a reduction in the amount of drugs reaching the target, has made traditional antibiotics such as vancomycin, rifampicin, and daptomycin ineffective. Vancomycin, as a tricyclic glycopeptide antibiotic, works by inhibiting the synthesis of bacterial cell walls, but with long-term use, bacteria may develop tolerance and even resistance. Therefore, it is particularly important to find new solutions to overcome antibiotic resistance.

[0004] Recent studies have shown that metal ions in bimetallic or multimetallic MOFs can synergistically kill bacteria, providing a scientific basis for the development of new antibacterial materials and strategies to overcome the clinical treatment challenges posed by multidrug-resistant pathogens. Summary of the invention

[0005] In view of the problems existing in the prior art, the purpose of the present invention is to provide an antibacterial gallium-copper bimetallic organic framework (Ga / Cu-MOF) nanomaterial, which meets the requirements of biosafety and can improve the sterilization effect of corresponding antibiotics. After loading antibiotics, the Ga / Cu-MOF effectively eliminates drug-resistant bacteria, including biofilms and intracellular bacteria, and is effectively used in mouse infection defect models and osteomyelitis models, providing more antibacterial application scenarios for the treatment of infections.

[0006] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a Ga / Cu-MOF nanomaterial having a three-dimensional porous structure, which comprises two metal ions Ga 3+ and Cu 2+ .

[0008] Furthermore, the size of the nanomaterial is in the range of 20 to 500 nm and the shape is a porous structure.

[0009] In a second aspect of the present invention, a method for preparing the Ga / Cu-MOF nanomaterial is provided, and the specific preparation process comprises:

[0010] S1, gallium nitrate Ga(NO3)3, dimethylimidazole (2-MI), terephthalic acid (H3tbc), polyvinylpyrrolidone (PVP) and trifluoroacetic acid are uniformly dispersed in N,N-dimethylformamide (DMF) to obtain a mixed solution;

[0011] S2, uniformly dispersing copper nitrate Cu(NO3)2 in DMF to obtain a dispersion;

[0012] S3, mixing the solutions obtained in step 1 and step 2, ultrasonically stirring and uniformly stirring until the solution is clear;

[0013] S4, transferring the obtained solution to a stainless steel high-pressure reactor lined with polytetrafluoroethylene, and heating the reaction in an oven;

[0014] S5. Cool the reactor to room temperature, centrifuge, wash, and vacuum dry to obtain Ga / Cu-MOF nanoparticles.

[0015] Furthermore, in step 1, the doping molar concentration of Ga(NO3)3 and dimethylimidazole (2-MI) is 1:1~8.

[0016] Furthermore, in step 1, the doping molar concentration of Ga(NO3)3 and terephthalic acid (H3tbc) is 1:0.5~4.

[0017] Furthermore, in step 1, the molar concentration ratio of Ga(NO3)3 to polyvinylpyrrolidone (PVP) is 1:0.5~4.

[0018] Furthermore, in step 1, the molar concentration ratio of Ga(NO3)3 to trifluoroacetic acid is 1:1~8.

[0019] Furthermore, the molar concentration ratio of Ga(NO3)3 to Cu(NO3)2 in the mixed solution in step 3 is 1:0.1~1.

[0020] Furthermore, the solution reaction conditions in step 4 are to react at 80-150° C. for 4-15 h; further, to react at 100° C. for 6 hours.

[0021] The third aspect of the present invention provides the application of the Ga / Cu-MOF nanomaterial in in vivo implants or anti-bacterial infection.

[0022] Furthermore, the anti-bacterial infection application is for preparing materials for treating infections caused by drug-resistant pathogenic bacteria, wherein the drug-resistant pathogenic bacteria are selected from any one or more drug-resistant bacteria of Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, Escherichia coli, Proteus, Shigella dysenteriae and Salmonella typhi.

[0023] Compared with the prior art, the present invention has the following effects:

[0024] The present invention provides an antibacterial gallium-copper bimetallic organic framework (Ga / Cu-MOF) nanomaterial. By adjusting the ion doping ratio, adding 2-methylimidazole and polyvinyl pyrrolidone regulators, and adding trifluoroacetic acid to adjust the pH, gallium and copper are successfully integrated to prepare a new functionalized bimetallic Ga / Cu-MOF nanomaterial. The MOF nanomaterial meets the requirements of biosafety, and can improve the sterilization effect of the corresponding antibiotics after loading the corresponding antibiotics. After loading the antibiotics, it can effectively eliminate drug-resistant bacteria, providing more antibacterial application scenarios for the treatment of skin defect infections and osteomyelitis infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 . High-magnification transmission electron microscopy (TEM) image of Ga / Cu-MOF nanoparticles in Example 1 of the present invention.

[0026] Figure 2 . Electron microscope energy spectrum mapping diagram of Ga / Cu-MOF nanoparticles in Example 1 of the present invention.

[0027] Figure 3 . X-ray diffraction (XRD) pattern of Ga / Cu-MOF nanoparticles in Example 1 of the present invention.

[0028] Figure 4 . CCK-8 toxicity test diagram of Ga / Cu-MOF nanoparticles on L929, BMSC and Raw264.7 cells in Example 1 of the present invention.

[0029] Figure 5 . Hemolysis experiment and HE (heart, liver, spleen, lung and kidney morphology) diagram of Ga / Cu-MOF nanoparticles in Example 1 of the present invention.

[0030] Figure 6 . Bacterial coating by the dilution plate method, bacterial SYTO9 / PI staining image and bacterial TEM image in Example 2 of the present invention.

[0031] Figure 7 Crystal violet staining, 2D and 3D confocal images of the biofilm elimination effects of different groups in Example 3 of the present invention.

[0032] Figure 8. Effects of different groups in Example 3 of the present invention on eliminating intracellular bacteria: MRSA self-luminescence, confocal image of DAPI and Phalloidin double staining (left); MRSA self-luminescence, confocal image of Raw264.7 bright field (right).

[0033] Fig. 9 . Pictures of back wound healing in the mouse skin defect infection model treated by different groups in Example 4 of the present invention.

[0034] Fig.10 . Pictures of femoral healing in rat osteomyelitis infection models treated by different groups in Example 5 of the present invention. DETAILED DESCRIPTION

[0035] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several variations and improvements may be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0036] Example 1 Preparation and characterization of Ga / Cu-MOF nanomaterials

[0037] Ga (NO3)3·xH2O (0.87 mmol, 0.11 g), H3btc (0.53mmol, 0.11 g), 2-Methylimidazole (0.87 mmol, 0.22 g), Polyvinyl pyrrolidone (0.99 mmol, 0.11 g), trifluoroacetic acid (400 μL) and DMF (10 mL) were mixed and stirred for 10 min. The mixed solution of Cu (NO3)2·3H2O (0.87mmol, 0.11 g) and DMF (10 mL) was shaken for 10 min, and the two mixed solutions were placed in a 50 mL polytetrafluoroethylene-lined reactor, heated to 100 °C, and the reaction was continued for 6 hours. The obtained reaction product was centrifuged and washed three times with ethanol and water, and then the precipitate was transferred to a freeze dryer for 24 hours to obtain the final product Ga / Cu-MOF. The Ga / Cu-MOF was characterized by the following steps: the vancomycin aqueous solution and the Ga / Cu-MOF mixture were shaken on a shaker for 6 hours, and then the product and the supernatant were separated by centrifugation. The obtained Van@Ga / Cu-MOF was washed twice with methanol and then freeze-dried. The concentration of Van in the supernatant was measured using a high performance liquid chromatography (HPLC) and compared with a standard Van solution. Figure 1 , 2As shown in Figures 3 and 4, the TEM and mapping images were obtained using a transmission electron microscope (TEM) and its energy spectrometer, and the XRD image was obtained using an X-ray diffraction (XRD), which showed that the synthesized Ga / Cu-MOF was a nanoparticle with uniform particle size and uniform distribution of gallium and copper elements. Figure 4 and 5 As shown, the biosafety of Ga / Cu-MOF was evaluated, including the use of CCK-8 method to determine that 200 μg / mLGa / Cu-MOF had basically no cytotoxicity to L929; a hemolysis experiment was carried out using mouse red blood cells co-incubated with Ga / Cu-MOF, and the hemolysis rate was less than 5%; HE staining of the heart, liver, spleen, lung and kidney morphology of mice treated with Ga / Cu-MOF was evaluated to be good.

[0038] Example 2 Antibacterial experiment of Ga / Cu-MOF nanomaterials

[0039] Four experimental groups were set up, including a blank group, a Van (1 μg / mL) group, a Ga / Cu-MOF (200 μg / mL) group, and a Van@Ga / Cu-MOF (200 μg / mL) group. The above experimental groups were co-cultured with MRSA (OD value was 0.02) and placed in a shaker at 37°C for 24 hours to investigate their antibacterial effects. Figure 6 As shown, after the culture was completed, the culture was coated by the dilution plate method. The coating conditions of each experimental group were observed, and it was found that the Ga / Cu-MOF nanomaterial loaded with vancomycin had a better antibacterial effect than the control group.

[0040] Example 3 Experiment on Eliminating Biofilm and Intracellular Bacteria with Ga / Cu-MOF Nanomaterials

[0041] This example aims to evaluate the elimination effect of Ga / Cu-MOF nanomaterials on MRSA biofilm and intracellular bacteria. The specific steps are as follows:

[0042] 1. In order to determine the effect of the material on biofilm, four groups of blank, Van (1 ug / mL), Ga / Cu-MOF (200 ug / mL), and Van@Ga / Cu-MOF (200 ug / mL) were co-cultured with MRSA with an OD value of 0.02 and placed in a 37°C oven for 24 h. Figure 7As shown, it was combined with 30% crystal violet staining solution, incubated for 30 min, then the staining solution was poured out and washed three times, and photographed with an ELISA instrument. It was found that most of the crystal violet staining solution combined with the biofilm in the experimental group was clear, indicating that the biofilm was significantly eliminated. Then it was dissolved with methanol and the OD590 value was observed. After MRSA with the GFP gene was cultured for 24 hours using the above steps, the slide was placed under a confocal microscope to observe its plane and 3D fluorescence intensity. The experimental results show that the Van@Ga / Cu-MOF group can effectively eliminate biofilms.

[0043] 2. In order to determine the effect of the material on intracellular bacteria, Murine Raw 264.7 cells (number of 2×10 4 ), which were seeded on coverslips and cultured overnight. MRSA labeled with green fluorescent protein (GFP) was added to each well to infect 30 times the number of cells. To ensure sufficient contact between bacteria and cells, the cells were incubated with bacteria for 30 minutes. Afterwards, the supernatant was discarded, and the infected cells were washed twice with 1× PBS solution to remove free bacteria that were not bound to the cells. Next, the original DMEM medium was replaced with medium containing 5 µg / mL vancomycin to kill the remaining extracellular bacteria. To further study the effects of different treatments on infected cells, infected Raw 264.7 cells were exposed to Van (1 μg / mL), Ga / Cu-MOF (200 μg / mL), Van@Ga / Cu-MOF (200 μg / mL) and fresh culture medium alone as controls. Figure 8 As shown, after 24 hours of culture, the activity of intracellular bacteria was observed by laser confocal microscopy, and the number of intracellular bacteria in the Van@Ga / Cu-MOF group was significantly less than that in other groups.

[0044] Example 4 Experiment on the treatment of skin defect infection model in mice using Ga / Cu-MOF nanomaterials

[0045] To evaluate the effects of Ga / Cu-MOF on bacterial clearance and wound healing on the skin, a skin infection model was established using BALB / c mice. The specific steps are as follows:

[0046] BALB / c mice (6 weeks old, male) were randomly divided into four groups: 1) PBS-treated infected mice as control group; 2) Van-treated infected mice; 3) Ga / Cu-MOF-treated infected mice; 4) Van@Ga / Cu-MOF-treated infected mice. First, mice were anesthetized by intraperitoneal injection of sodium pentobarbital. The dorsal skin was trimmed and disinfected, and a 10 mm diameter defect was created using a circular skin sampler. The defect was inoculated with bioluminescent Staphylococcus aureus Luc-MRSA (1×10 6CFU / mL) in PBS (100 μL). On days 0, 1, and 2, 40 μL of 0.1 mg / mL Van, 100 μL of 2 mg / mL Ga / Cu-MOF, and Van@Ga / Cu-MOF were given subcutaneously, respectively. Fig. 9 As shown, wound photos were taken during the treatment period to observe the survival of the mice. It was found that after 14 days, the Van@Ga / Cu-MOF group had better wound healing effect than the other three groups.

[0047] Example 5 Experiment on the treatment of rat osteomyelitis infection model with Ga / Cu-MOF nanomaterials

[0048] To evaluate the effect of Ga / Cu-MOF in the treatment of bone infection, an osteomyelitis infection model was established using 250g SD rats (8 weeks old, male), and the specific steps were as follows: the rats were randomly divided into four groups: 1) PBS-treated infected rats as the control group; 2) Van-treated infected rats; 3) Ga / Cu-MOF-treated infected rats; 4) Van@Ga / Cu-MOF-treated infected rats. First, the rats were anesthetized by intraperitoneal injection of sodium pentobarbital. The tibia was then prepared and disinfected. The skin was incised to expose the tibia. A cortical bone defect with a diameter of 1.8 mm was formed on the tibia using a dental drill, and the bone marrow cavity was opened. 100 μL of MRSA bacterial suspension (10 7 CFU / mL) to establish an osteomyelitis model. The bone defect was sealed with bone wax and the skin was sutured. Fig.10 As shown, four weeks later, the bone healing of the surviving rats was verified by micro-CT imaging technology, and it was observed that the bone healing of the Van@GaMOF group was significantly better than that of the other groups.

Claims

1. An application of Ga / Cu-MOF nanomaterials in in vivo implants or anti-bacterial infection, characterized in that: The nanomaterial has a three-dimensional porous structure, which contains two metal ions Ga 3+ and Cu 2+ The specific preparation process of the Ga / Cu-MOF nanomaterial includes: S1, uniformly dispersing gallium nitrate Ga(NO3)3, dimethylimidazole, terephthalic acid, polyvinyl pyrrolidone and trifluoroacetic acid in N,N-dimethylamide to obtain a mixed solution; S2, uniformly dispersing copper nitrate Cu(NO3)2 in DMF to obtain a dispersion; S3, mixing the solutions obtained in step S1 and step S2, and stirring them by ultrasonication until the solution is clear; S4, transferring the obtained solution to a stainless steel high-pressure reactor lined with polytetrafluoroethylene, and heating the reaction in an oven; S5. Cool the reactor to room temperature, centrifuge, wash, and vacuum dry to obtain Ga / Cu-MOF nanoparticles.

2. The use according to claim 1, characterized in that: The size of the nanomaterial is in the range of 30 to 300 nm.

3. The use according to claim 1, characterized in that: In step S1, the doping molar concentration ratio of Ga(NO3)3 to dimethylimidazole is 1:1~8.

4. The use according to claim 1, characterized in that: In step S1, the doping molar concentration ratio of Ga(NO3)3 to trimericacid is 1:0.5~4.

5. The use according to claim 1, characterized in that: In step S1, the molar concentration ratio of Ga(NO3)3 to polyvinyl pyrrolidone is 1:0.5~4.

6. The use according to claim 1, characterized in that: The molar concentration ratio of Ga(NO3)3 to trifluoroacetic acid in step S1 is 1:1~8.

7. The use according to claim 1, characterized in that: The molar concentration ratio of Ga(NO3)3 to Cu(NO3)2 in the mixed solution in step S3 is 1:0.1~1.

8. The use according to claim 1, characterized in that: The solution reaction conditions described in step S4 are to react at 80-150° C. for 4-15 h.

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