Phthalocyanine-based MOF nano material and preparation method and antibacterial application thereof

Abstract

The invention relates to the technical field of biological materials, and particularly provides an antibacterial phthalocyanine-based metal organic framework (Pc-MOF) nano material as well as a preparation method and application thereof. The Pc-MOF nano material meets the requirement of biological safety, has good photodynamic and photo-thermal reaction performance, and can load antibiotics to eliminate drug-resistant bacteria. The antibacterial application example is that the Pc-MOF nano material is loaded with antibiotics and is combined with photo-thermal and photodynamic reaction to eliminate a biological membrane, and the Pc-MOF nano material is effectively applied to a mouse skin defect infection model.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular to a phthalocyanine-based metal organic framework (Pc-MOF) nanomaterial and a preparation method and application thereof. Background Art

[0002] In recent years, with the widespread abuse and misuse of antibiotics, multidrug-resistant super pathogens have posed a severe challenge to human health. The rapid growth of these resistant strains, especially those with complex drug resistance, has brought unprecedented challenges to clinical treatment. In response to this global crisis, the World Health Organization has developed a global list of priority pathogens, including multidrug-resistant strains such as methicillin-resistant Staphylococcus aureus (MRSA), which can cause serious infections and challenge existing treatments.

[0003] In clinical treatment, 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 led to the failure of traditional antibiotics such as vancomycin, rifampicin, and daptomycin. In addition, photodynamic and photothermal reactions do not have drug resistance problems and can be combined with antibiotics for synergistic antibacterial effects. Therefore, it is particularly important to find new solutions to overcome antibiotic resistance.

[0004] Recent studies have shown that MOFs can be used as carriers of a variety of drugs to achieve sustained release due to their unique porous structure and high specific surface area. By precisely designing their composition and pore structure, MOFs can be endowed with the properties of generating heat and free radicals under stimulation. Combined with other materials, MOFs can serve as a platform to effectively kill bacteria through the synergistic effect of multiple mechanisms. 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 phthalocyanine-based metal organic framework (Pc-MOF) nanomaterial, which meets the requirements of biosafety, has good photodynamic and photothermal reaction performance, and can improve the sterilization effect of the corresponding antibiotics. After the Pc-MOF is loaded with antibiotics, it effectively eliminates drug-resistant bacteria, including biofilm in vitro models, and is effectively used in mouse skin defect infection models, providing more antibacterial application solutions and options for the clinical treatment of infectious diseases.

[0006] The purpose of the present invention can be specifically achieved through the following technical solutions: In a first aspect, the present invention provides a phthalocyanine-based metal organic framework (Pc-MOF) nanomaterial, wherein the nanomaterial has a three-dimensional porous structure and comprises phthalocyanine molecules and metal ions.

[0007] Furthermore, the metal ions in the nanomaterial include but are not limited to one or a combination of copper ions, zinc ions, iron ions, chromium ions, etc., and more preferably, the metal ions are copper ions.

[0008] Furthermore, the nanomaterial has a particle size range of 30 to 300 nm and a porous structure.

[0009] In a second aspect of the present invention, a method for preparing the Pc-MOF nanomaterial is provided, and the specific preparation process includes: Step 1, uniformly dispersing metal salt ions, phthalocyanine (Pc) and polyvinylpyrrolidone (PVP) in N,N-dimethylformamide (DMF) to obtain a mixed dispersion; Step 2, adding trifluoroacetic acid evenly into DMF to obtain a mixed solution; Step 3, mixing the solutions obtained in step 1 and step 2, and stirring them evenly by ultrasonication until the solution is clear; Step 4, transferring the obtained solution to a stainless steel autoclave lined with polytetrafluoroethylene, and heating the reaction in an oven; Step 5: Cool the reactor to room temperature, centrifuge, wash, and vacuum dry to obtain Pc-MOF nanoparticles.

[0010] Furthermore, in step 1, the doping molar concentration of metal ions and phthalocyanine (Pc) is 1:0.5~4.

[0011] Furthermore, in step 1, the molar concentration ratio of metal ions to polyvinyl pyrrolidone (PVP) is 1:0.5-4.

[0012] Furthermore, the volume of trifluoroacetic acid in step 2 is 10 to 200 μL.

[0013] Furthermore, the solution reaction conditions described in step 4 are to react at 100-180° C. for 6-15 h.

[0014] The third aspect of the present invention provides the application of the Pc-MOF nanomaterial in medical devices, in vivo implants or anti-bacterial infection.

[0015] 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.

[0016] Compared with the prior art, the present invention has the following effects: The present invention provides an antibacterial phthalocyanine-based metal organic framework (Pc-MOF) nanomaterial. A novel functionalized Pc-MOF nanomaterial is prepared by adjusting the doping ratio of phthalocyanine molecules to metal ions, adding a polyvinyl pyrrolidone regulator, and adding trifluoroacetic acid to adjust the pH. The Pc-MOF nanomaterial meets the requirements of biosafety, has good photodynamic and photothermal reaction performance, can improve the sterilization effect of the corresponding antibiotics after loading antibiotics, effectively eliminate drug-resistant bacteria, and provide more antibacterial application options for the treatment of skin defect infections, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 . High-magnification transmission electron microscopy (TEM) image of PcCu-MOF nanoparticles in Example 1 of the present invention.

[0018] Figure 2 . Electron microscope energy spectrum mapping diagram of PcCu-MOF nanoparticles in Example 1 of the present invention.

[0019] Figure 3 . X-ray diffraction (XRD) pattern of PcCu-MOF nanoparticles in Example 1 of the present invention.

[0020] Figure 4 . Experimental diagram of CCK-8 toxicity of PcCu-MOF nanoparticles on L929 cells in Example 1 of the present invention.

[0021] Figure 5 . Graph of the hemolysis experiment of PcCu-MOF nanoparticles in Example 1 of the present invention.

[0022] Figure 6 . Photothermal and photodynamic performance diagram of PcCu-MOF nanoparticles in Example 1 of the present invention.

[0023] Figure 7 . Bacterial coating by the dilution plate method, bacterial SYTO9 / PI staining image and bacterial TEM image in Example 2 of the present invention.

[0024] Figure 8 Crystal violet staining, 2D and 3D confocal images of the biofilm elimination effects of different groups in Example 3 of the present invention.

[0025] 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. DETAILED DESCRIPTION

[0026] 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.

[0027] Example 1 Preparation and characterization of PcCu-MOF nanomaterials Cu(NO3)2·3H2O (3.6 mg), Pc (4 mg), PVP (4 mg) and DMF (20 mL) were mixed to obtain a mixed dispersion. 50 μL of trifluoroacetic acid was added to DMF to obtain a mixed solution. The solution was ultrasonically stirred until it was clear. The solution was placed in a 50 mL polytetrafluoroethylene-lined stainless steel autoclave and heated to 150°C in an oven for 8 hours. After the reactor was cooled to room temperature, the obtained reaction product was centrifuged at 12000 rpm and washed three times with ethanol and water. The precipitate was then transferred to a freeze dryer and freeze-dried for 24 hours to obtain the final product PcCu-MOF. PcCu-MOF was characterized, including the following steps: the vancomycin aqueous solution and the PcCu-MOF mixture were shaken on a shaker for 6 hours, and then the product and the supernatant were separated by centrifugation. The obtained Van@PcCu-MOF was washed twice with methanol and then freeze-dried. Figure 1 , 2 As shown in Figures 3 and 4, the TEM and mapping images were obtained using a transmission electron microscope (TEM) and its energy dispersive spectrometer, and the XRD images were obtained using X-ray diffraction (XRD), which showed that the synthesized PcCu-MOF was a nanoparticle with uniform particle size and uniform distribution of copper elements. Figure 4 and 5 As shown in the figure, the biosafety of PcCu-MOF was evaluated, including the use of CCK-8 method to determine that 200 μg / mL PcCu-MOF had little cytotoxicity to L929 cells; the hemolysis experiment was carried out by co-incubating mouse red blood cells with PcCu-MOF, and the hemolysis rate was less than 5%. Figure 6 As shown in Figure 2, the singlet oxygen content produced by 100 ug / ml PcCu-MOF was the highest, and 1.5 W / cm 2 After 808 nm near-infrared laser irradiation of 100 μg / ml PcCu-MOF for 10 min, its temperature can reach ~62℃.

[0028] Example 2 Antibacterial experiment of PcCu-MOF nanomaterials Four experimental groups were set up, including blank group, Van (1 μg / mL) group, Van@PcCu-MOF (200 μg / mL) group and Van@PcCu-MOF+808 nm (200 μg / mL) group. Each of the above experimental groups was co-cultured with MRSA (OD value was 0.02) for 6 h. The Van@PcCu-MOF+808 nm group was heated with 1.5 W / cm 2 The antibacterial effect was investigated by 808 nm laser irradiation for 10 min. Figure 7 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 PcCu-MOF nanomaterial loaded with vancomycin had a better antibacterial effect than the control group.

[0029] Example 3 Experiment on Eliminating Biofilm and Intracellular Bacteria with PcCu-MOF Nanomaterials This example aims to evaluate the elimination effect of PcCu-MOF nanomaterials on MRSA biofilms, and the specific steps are as follows: In order to determine the effect of the material on biofilm, four groups were set up: blank, Van (1 μg / mL), Van@PcCu-MOF (200 μg / mL) and Van@PcCu-MOF+808 nm (200 μg / mL) and co-cultured with MRSA with an OD value of 0.02, placed in a 37°C oven for 6 h, and the experimental group was heated with 1.5 W / cm 2 808 nm laser irradiation for 10 min. Figure 8 As 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 a microplate reader. 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@PcCu-MOF+808 nm group can effectively eliminate biofilms.

[0030] Example 4 Experiment on the treatment of mouse skin defect infection model using PcCu-MOF nanomaterials To evaluate the effects of PcCu-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: 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) Van@PcCu-MOF-treated infected mice; 4) Van@PcCu-MOF+808 nm near-infrared laser-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 methicillin-resistant Staphylococcus aureus Luc-MRSA (1×10 6 CFU / mL) of PBS (100 μL). On days 0, 1, and 2, 40 μL 0.1 mg / mL Van, 100 μL 2 mg / mL, and Van@PcCu-MOF were subcutaneously injected, respectively. The fourth group was treated with 1.5 W / cm 2 808 nm laser irradiation for 10 min. 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@PcCu-MOF group had better wound healing effect than the other three groups.

Claims

1. A phthalocyanine-based metal-organic framework (Pc-MOF) nanomaterial, characterized in that: The Pc-MOF has a three-dimensional porous structure containing phthalocyanine molecules and metal ions.

2. The Pc-MOF nanomaterial according to claim 1, characterized in that: The nano material has a particle size range of 30 to 300 nm and a porous structure.

3. The Pc-MOF nanomaterial according to claim 1, characterized in that: The metal ions in the nanomaterial are selected from one of copper ions, zinc ions, iron ions and chromium ions or a combination thereof.

4. The Pc-MOF nanomaterial according to claim 1, characterized in that: The metal ions are copper ions.

5. A method for preparing the Pc-MOF nanomaterial according to claim 1, characterized in that: The specific preparation process includes: Step 1, uniformly dispersing metal ions, phthalocyanine (Pc) and polyvinylpyrrolidone (PVP) in N,N-dimethylformamide (DMF) to obtain a mixed dispersion; Step 2, adding trifluoroacetic acid evenly into DMF to obtain a mixed solution; Step 3, mixing the solutions obtained in step 1 and step 2, and stirring them evenly by ultrasonication until the solution is clear; Step 4, transferring the obtained solution to a stainless steel autoclave lined with polytetrafluoroethylene, and heating the reaction in an oven; Step 5: Cool the reactor to room temperature, centrifuge, wash, and vacuum dry to obtain Pc-MOF nanoparticles.

6. The preparation method according to claim 5, characterized in that: In step 1, the doping molar concentration of metal ions and phthalocyanine (Pc) is 1:0.5~4.

7. The preparation method according to claim 5, characterized in that: In step 1, the molar concentration ratio of metal ions to polyvinyl pyrrolidone (PVP) is 1:0.5-4.

8. The preparation method according to claim 5, characterized in that: The volume of trifluoroacetic acid in step 2 is 10~200 μL.

9. The preparation method according to claim 5, characterized in that: The solution reaction conditions described in step 4 are to react at 100-180° C. for 6-15 h.

10. Use of the Pc-MOF nanomaterial according to claim 1 to claim 4 in medical devices, in vivo implants or anti-bacterial infection.

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