Preparation method of dopamine-modified bimetallic organic framework antibacterial composite material

Abstract

The present invention relates to a method for preparing a dopamine-modified bimetallic organic framework antibacterial composite material, which belongs to the fields of biomedicine and materials chemistry. The present invention adopts an in-situ solvent thermal method to successfully dope Co into Fe-MIL-53-NH2 to obtain a Co / Fe-MIL-53-NH2 bimetallic-organic framework material, which greatly improves the Fenton catalytic performance of Fe-MIL-53-NH2; with the help of a dopamine in-situ polymerization method, the surface of Co / Fe-MIL-53-NH2 is successfully modified to obtain Co / Fe-MIL-53-NH2@PDA nanoparticles, which have better photothermal performance. The composite material synthesized by the present invention has both Fenton catalysis and photothermal antibacterial properties, and the synergistic antibacterial rate is as high as 98%. The chemical kinetics and photothermal synergistic antibacterial performance have been verified, and it has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the technical fields of biomedicine and material chemistry, and specifically provides a dopamine-modified bimetallic organic framework composite material with antibacterial properties utilizing an in-situ solvothermal method and a polymerization method, and a preparation method thereof. Background Art

[0002] Food production, processing, and distribution often introduce bacteria that can cause human disease. The widespread use of antibiotics and bacterial infections pose a serious threat to human health. Inappropriate use of antibiotics can lead to the emergence of drug-resistant bacteria. Therefore, the development of new antimicrobial materials is urgently needed to reduce antibiotic usage and mitigate the adverse consequences of inappropriate antibiotic use.

[0003] In recent years, chemodynamic therapy (CDT), photothermal therapy (PTT), and other novel multimodal antimicrobial therapies have garnered widespread attention. Polydopamine (PDA), a photothermal agent, offers advantages such as ease of synthesis and modification, high photothermal conversion efficiency, and good biocompatibility. Under near-infrared laser irradiation, it converts photothermal energy into localized heat to kill bacteria.

[0004] Metal-organic frameworks (MOFs) are one-, two-, or three-dimensional periodic porous materials formed by the self-assembly of organic ligands and metal ions. They possess advantages such as porosity, large specific surface area, adjustable pore size and porosity, ease of functionalization, and catalytic activity. Combining the strengths of both inorganic and organic antimicrobial agents, MOFs further demonstrate their superiority and address the shortcomings of existing antimicrobial agents. These significant advantages give MOFs enormous potential for application in antimicrobial therapy.

[0005] Currently, various photothermal materials have been reported. The heat generated can kill bacteria and prevent biofilm formation, and they exhibit strong near-infrared light absorption. Therefore, photothermal therapy (PTT) has been recognized as a new and effective way to eliminate bacterial infections. Polydopamine (PDA), as a photothermal agent, has the advantages of easy synthesis and modification, high photothermal conversion rate, and good biocompatibility. Under near-infrared laser irradiation, it converts photothermal heat into localized heat to kill bacteria.

[0006] However, the single material used in the photothermal therapy in the prior art has an unsatisfactory chemotherapy effect, is prone to non-specific distribution of organisms, and may also cause toxic side effects. Summary of the Invention

[0007] To address the shortcomings of the existing technology, the present invention provides a multimodal antibacterial bimetallic organic framework composite material and its preparation method. The method has a simple preparation process, is easy to operate, and has a stable and reliable process. The Co / Fe-MIL-53-NH2 is doped with Co and modified with PDA functionalization, which can achieve both Fenton catalysis and photothermal therapy, resulting in a synergistic antibacterial effect.

[0008] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0009] A dopamine-modified bimetallic organic framework composite material with antibacterial properties utilizing in-situ solvothermal and polymerization methods is disclosed. Cobalt is doped by a solvothermal method to synthesize a Co / Fe-MIL-53-NH2 metal-organic framework material. Dopamine is in situ polymerized to uniformly distribute it on the surface of Co / Fe-MIL-53-NH2. The framework composite material is Co / Fe-MIL-53-NH2@PDA.

[0010] Furthermore, in the above-mentioned metal-organic framework composite material, the measured photothermal conversion efficiency of the Co / Fe-MIL-53-NH2@PDA nanoparticles is 24.50%, and the antibacterial rate is 98%; the Co / Fe-MIL-53-NH2 is a nano-sized metal-organic framework material synthesized by a solvent thermal method with 2-aminoterephthalic acid as a ligand and iron ions and cobalt ions as metal ions.

[0011] The above-mentioned method for modifying a bimetallic organic framework (BOF) composite material with dopamine using in situ polymerization includes the following steps: mixing FeCl3·6H2O, CoCl2·6H2O, and NH2-BDC in DMF, reacting in a sealed polytetrafluoroethylene autoclave, cooling, centrifuging, collecting, and drying the resulting mixture. This yields a cobalt-doped BOF composite material, also referred to as Co / Fe-MIL-53-NH2. The Co / Fe-MIL-53-NH2 is then added to ultrapure water and ultrasonically homogenized. Dopamine hydrochloride in Tris buffer is then added, and the mixture is stirred in a blender. The mixture is centrifuged to obtain a black precipitate, ultimately yielding a PDA-encapsulated bimetallic organic framework composite material, also referred to as Co / Fe-MIL-53-NH2@PDA.

[0012] Furthermore, the Fe-MIL-53-NH2 is a metal organic framework (MOF) material, and Fe-MIL-53-NH2 can be prepared by methods disclosed in the prior art.

[0013] Furthermore, when preparing Co / Fe-MIL-53-NH2, the reaction was carried out in a closed polytetrafluoroethylene high-pressure reactor at 150°C for 24 hours.

[0014] Furthermore, when preparing Co / Fe-MIL-53-NH2, the molar ratio of FeCl3·6H2O and CoCl2·6H2O was 8:1.

[0015] Furthermore, when preparing Co / Fe-MIL-53-NH2, the amount of 2-amino-1,4-benzenedicarboxylic acid used was 362 mg and the amount of DMF was 35 mL.

[0016] The cobalt-doped metal-organic framework (Co / Fe-MIL-53-NH2) obtained by the above method can be further subjected to in-situ polymerization of dopamine to uniformly distribute the cobalt on the surface of the Co / Fe-MIL-53-NH2. Specifically, the Co / Fe-MIL-53-NH2 is added to ultrapure water and homogenized by sonication. Dopamine hydrochloride is then added, followed by Tris buffer, and the mixture is placed in a stirrer to obtain the encapsulated bimetallic organic framework (Co / Fe-MIL-53-NH2@PDA).

[0017] Furthermore, the mass ratio of the Co / Fe-MIL-53-NH2 to dopamine hydrochloride is 1:3.

[0018] Furthermore, the Co / Fe-MIL-53-NH2 and dopamine hydrochloride were mixed and stirred, dopamine hydrochloride and Co / Fe-MIL-53-NH2 were weighed according to a mass ratio of 3:1, 10 mg of Co / Fe-MIL-53-NH2 was weighed and added to 30 mL of ultrapure water and ultrasonically uniformly mixed, and then 30 mg of dopamine hydrochloride was added and 30 mL of 10 mM Tris buffer was added and placed in a stirrer. The stirring speed was 300-500 r / min, the stirring time was 1 hour, the reaction temperature was room temperature, and the mixture was centrifuged at 10,000 r / min for 30 minutes. The black precipitate was collected, which was the Co / Fe-MIL-53-NH2@PDA nanoparticles.

[0019] This invention synthesizes a Co / Fe-MIL-53-NH2 metal-organic framework (MOF) by solvothermal doping with cobalt. Dopamine is then uniformly distributed on the surface of the Co / Fe-MIL-53-NH2 via in situ polymerization, yielding Co / Fe-MIL-53-NH2@PDA nanoparticles. These nanoparticles exhibit both Fenton catalytic and photothermal synergistic antibacterial properties. Co doping significantly enhances the Fenton performance of the nanoparticles, and PDA modification demonstrates excellent photothermal performance. The chemical kinetics and photothermal synergistic antibacterial properties were also investigated using the plate diffusion method, and CCK8 colorimetry confirmed their good biocompatibility. Using a mouse wound model, Co / Fe-MIL-53-NH2@PDA demonstrated significant wound healing under near-infrared laser irradiation, confirming the chemical kinetics and photothermal synergistic antibacterial properties. H&E results also demonstrated no significant side effects on normal tissues and organs, significantly advancing the application of multimodal synergistic antibacterial therapies.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) The present invention utilizes a composite material synthesized by a solvothermal method and then uniformly distributed on the surface of a metal-organic framework via in situ polymerization of dopamine. The preparation method is simple and the process is stable and reliable. Not only is the Fenton performance significantly improved, but the PDA modification also exhibits excellent photothermal performance. The photothermal conversion efficiency of Co / Fe-MIL-53-NH2@PDA nanoparticles was verified to be 24.50%, and the antibacterial rate was as high as 98%.

[0022] (2) The present invention utilizes an in-situ polymerization method to realize a dopamine-encapsulated bimetallic organic framework composite material: a nanocomposite material Co / Fe-MIL-53-NH2 was successfully prepared by a solvothermal method, and polydopamine (PDA) encapsulation was achieved. This system not only performs Fenton catalysis but also exhibits photothermal effects, demonstrating both Fenton catalysis and photothermal effects. Cell experiments have shown that the material has good biocompatibility, and animal experiments have demonstrated that the nanosystem has good antibacterial properties, providing a new direction for antibacterial applications and offering potential possibilities for its clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1Figures are morphological and structural characterizations, (a) scanning electron microscopy image of Fe-MIL-53-NH2 nanoparticles (scale bar is 400 nm); (b) scanning electron microscopy image of Co / Fe-MIL-53-NH2 (scale bar is 500 nm); (c) scanning electron microscopy image of Co / Fe-MIL-53-NH2@PDA (scale bar is 500 nm); (d) XRD spectra of Co / Fe-MIL-53-NH2 prepared with different reactant concentrations; (e) X-ray diffraction (PXRD) patterns of the synthesized corresponding materials; (f) Fourier transform infrared spectra of the corresponding materials;

[0024] Figure 2 (a) particle size of the corresponding material, (b) zeta potential of the corresponding material;

[0025] Figure 3 For Fenton performance test, (a) UV-Vis absorption spectra of Co / Fe-MIL-53-NH2 at different ratios in the catalytic TMB (pH 5.0), where H2O2 is 400mM; (b) absorbance change curve of Co / Fe-MIL-53-NH2@PDA within 10 minutes; (c) absorbance change curve of 100μg / mL Co / Fe-MIL-53-NH2@PDA at different pH values ​​(7.4, 6, 5, 4) within 10 minutes (the inset corresponds to the digital image of the data, with pH decreasing from top to bottom); (d) absorbance change curve of 100μg / mL Co / Fe-MIL-53-NH2@PDA at different H2O2 values ​​(40, 100, 200, 400mM) within 10 minutes;

[0026] Figure 4 For the photothermal performance test, (a) 1mg / mL different materials at 2.22W / cm 2 Temperature variation curves after 808 nm laser irradiation for 10 min; (b) Co / Fe-MIL-53-NH2@PDA with different concentrations at 2.22 W / cm 2 Temperature variation curves after 808 nm laser irradiation for 10 min; (c) Co / Fe-MIL-53-NH2@PDA with different concentrations at 2.22 W / cm 2 Figure 2. Temperature variation of 0.2 mg / mL Co / Fe-MIL-53-NH2@PDA after 808 nm laser irradiation for 5 min (error bars represent the standard deviation of three independent measurements); (d) Temperature variation curve of 0.2 mg / mL Co / Fe-MIL-53-NH2@PDA after 808 nm laser irradiation for 10 min at different laser power densities; (e) Temperature variation curve of 0.2 mg / mL Co / Fe-MIL-53-NH2@PDA after 2.22 W / cm 2Temperature change curves after five cycles of 808nm laser irradiation; (f) is a photothermal conversion efficiency image of Co / Fe-MIL-53-NH2@PDA;

[0027] Figure 5 Figures for in vitro antibacterial effect evaluation: (a) Temperature curves of E. coli treated with photothermal treatment in both the Co / Fe-MIL-53-NH2@PDA and PBS groups (the inset corresponds to the actual image of the solid agar plate with the data); (b) Temperature curves of S. aureus treated with photothermal treatment in both the Co / Fe-MIL-53-NH2@PDA and PBS groups (the inset corresponds to the actual image of the solid agar plate with the data);

[0028] Figure 6 (a) Photos of solid agar plates of Escherichia coli and Staphylococcus aureus treated with different materials; (b) The corresponding statistical survival percentages of Escherichia coli; (c) The corresponding statistical survival percentages of Staphylococcus aureus;

[0029] Figure 7 Fluorescence microscopy and scanning electron microscopy images of (a) Escherichia coli and (b) Staphylococcus aureus treated with PBS, Fe-MIL-53-NH2, Co / Fe-MIL-53-NH2, Fe-MIL-53-NH2+H2O2, Co / Fe-MIL-53-NH2+H2O2, Co / Fe-MIL-53-NH2@PDA, and Co / Fe-MIL-53-NH2@PDA+H2O2+NIR;

[0030] Figure 8 Cytotoxicity pictures show the relative viability of 3T3 cells treated with different concentrations of Fe-MIL-53-NH2, Co / Fe-MIL-53-NH2 and Co / Fe-MIL-53-NH2@PDA; A: Fe-MIL-53-NH2, B: Co / Fe-MIL-53-NH2, C: Co / Fe-MIL-53-NH2@PDA;

[0031] Figure 9 Representative photographs of mice with wounds in each group were taken every other day over 9 days. (a) Actual image of wound healing in mice on day 9; (b) Bacteria isolated from wound tissues after different treatments were cultured on agar plates; (c) Histological images of S. aureus-infected damaged tissues after different treatments stained with H&E, scale bar: 100 μm; (d) Weight change curves of mice in each group over 9 days; (e) Changes of wounds in mice in different groups over time;

[0032] Figure 10(a) Thermal images and (b) temperature change curves of mice treated with Co / Fe-MIL-53-NH2@PDA+H2O2 under near-infrared irradiation for 2 minutes;

[0033] Figure 11 H&E staining of the main organs of mice treated in different groups. Scale bar: 100 μm. DETAILED DESCRIPTION

[0034] In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, a detailed description will be given below with reference to specific embodiments and the accompanying drawings.

[0035] Unless otherwise specified, the reagents and materials used in the examples and comparative examples can be obtained through commercial channels.

[0036] The present invention provides a dopamine-modified bimetallic organic framework composite material with antibacterial properties utilizing an in-situ solvothermal method and a polymerization method, and a preparation method thereof. Specific embodiments are as follows.

[0037] Example 1

[0038] A method for preparing an antibacterial dopamine-modified bimetallic organic framework composite material using an in-situ solvothermal method and a polymerization method comprises the following steps:

[0039] (1) Using the solvothermal method, 540 mg of FeCl3·6H2O, 59.5 mg of CoCl2·6H2O, and 362 mg of NH2-BDC were mixed in 35 mL of DMF. The mixture was heated at 150°C in a sealed polytetrafluoroethylene autoclave for 24 h. After cooling, the resulting mixture was centrifuged at 10,000 rpm for 30 min. The precipitate was collected and dried to obtain Co / Fe-MIL-53-NH2.

[0040] (2) First, prepare 10 mM Tris buffer (pH 8.5);

[0041] Dopamine hydrochloride and Co / Fe-MIL-53-NH2 were weighed in a mass ratio of 3:1. 10 mg of Co / Fe-MIL-53-NH2 was added to 30 mL of ultrapure water and ultrasonically homogenized. Then, 30 mg of dopamine hydrochloride and 30 mL of 10 mM Tris buffer were added and placed in a blender. Stirred for 1 h, the mixture was centrifuged to obtain a black precipitate, which was Co / Fe-MIL-53-NH2@PDA nanoparticles.

[0042] Comparative Example 1

[0043] Co / Fe-MIL-53-NH2 was synthesized by a solvothermal method: 540 mg of FeCl3·6H2O, 59.5 mg of CoCl2·6H2O and 362 mg of NH2-BDC were mixed in 35 mL of DMF. The mixture was heated at 150°C in a sealed polytetrafluoroethylene autoclave for 24 h. After cooling, the resulting mixture was centrifuged at 10,000 rpm for 30 min. The precipitate was collected and dried to obtain Co / Fe-MIL-53-NH2.

[0044] Comparative Example 2

[0045] Fe-MIL-53-NH2 was synthesized using a solvothermal method: FeCl3·6H2O (2 mmol) and NH2-BDC (2 mmol) were mixed, 35 mL of DMF was added, and the mixture was placed in a sealed Teflon-lined autoclave for polymerization at 120°C for 24 hours. After cooling, the mixture was centrifuged, collected, and dried to obtain Fe-MIL-53-NH2 nanoparticles.

[0046] The properties of the nanoparticles prepared in the above examples and comparative examples were characterized as follows.

[0047] 1. Morphological and structural characterization

[0048] Experimental steps: The Fe-MIL-53-NH2, Co / Fe-MIL-53-NH2 and Co / Fe-MIL-53-NH2@PDA obtained in Example 1 and the comparative example were characterized and measured using scanning electron microscopy (SEM), X-ray diffractometer and Fourier transform infrared (FTIR) spectroscopy.

[0049] Experimental steps: The Fe-MIL-53-NH2, Co / Fe-MIL-53-NH2 and Co / Fe-MIL-53-NH2@PDA obtained in Example 1 and the comparative example were characterized by particle size and Zeta.

[0050] The experimental results are shown in Figure 1 ,Depend on Figure 1 Scanning electron microscopy (SEM) images show Fe-MIL-53-NH2 nanoparticles with a particle size range of approximately 500 nm. The introduction of Co did not alter the shape of Fe-MIL-53-NH2, which remained approximately 500 nm. Dopamine hydrochloride (HDA) is a PDA shell formed by self-polymerization under alkaline conditions. It can adhere to a variety of materials and contains a large number of catechol and amino groups on its surface. Therefore, the surface of Co / Fe-MIL-53-NH2@PDA has a certain thickness and has changed its shape to an elliptical shape, indicating that PDA has been successfully coated on the Co / Fe-MIL-53-NH2 surface.

[0051] The XRD spectra of Co / Fe-MIL-53-NH2 prepared with different reactant concentrations are shown in Figure 1 (d) We found that the ratio of 8:1 was superior to other ratios, and its crystallinity was also the best compared to other ratios. Therefore, we continued to characterize Fe-MIL-53-NH2 (8:1) and found that the diffraction peaks of Fe-MIL-53-NH2 were consistent with the XRD pattern of the simulated MIL-53 (Fe) (CCDC database), indicating that Fe-MIL-53-NH2 was successfully synthesized. The characteristic diffraction peaks of Co / Fe-MIL-53-NH2 and Co / Fe-MIL-53-NH2@PDA did not change significantly, indicating that the crystalline structure was well preserved after the introduction of Co, maintaining good structural integrity. In addition, the PDA wrapping did not affect the crystal structure.

[0052] Next, the Fourier transform infrared (FTIR) spectra of each material were tested. Fe-MIL-53-NH2 has a wavelength of 1580 cm -1 The NH symmetric deformation mode is shown at 1580 cm -1 It shows tensile vibration at 622cm -1 The Fe-O bond can be clearly observed in the figure at 1260 cm -1 The absorption peak near 1618 cm -1 The vibration peak at 3433 cm is attributed to the symmetric and asymmetric stretching modes of the coordination (-COO-) group. -1 There is a broad peak at 1622 cm, which is attributed to the OH and NH bonds in the aromatic ring. -1 and 1510cm -1 The characteristic bands belong to C=O stretching vibration and NH bending vibration peaks respectively. Co / Fe-MIL-53-NH2@PDA shows characteristic absorption peaks of PDA and Co / Fe-MIL-53-NH2, and no new absorption peaks appear.

[0053] Depend on Figure 2It can be seen that the particle size and Zeta potential were used for characterization, and the particle size is consistent with the material size characterized by scanning electron microscopy. The Zeta potential of each material is Fe-MIL-53-NH2 (34.4mV), Co / Fe-MIL-53-NH2 (27.26mV), and Co / Fe-MIL-53-NH2@PDA (-20.26mV). The Zeta potential of Co / Fe-MIL-53-NH2 is positively charged (27.26mV). After PDA coating, the potential of Co / Fe-MIL-53-NH2@PDA becomes negative. This is due to the deprotonation of the phenolic hydroxyl groups in PDA, and the Zeta potential becomes negative (-20.26mV). This shows that Co / Fe-MIL-53-NH2 is successfully encapsulated.

[0054] 2. Chemical kinetics determination

[0055] Experimental Procedure: Using TMB as a probe, we studied the hydroxyl radical (·OH). Based on the TMB-induced reaction, we analyzed the chemical kinetics of Co / Fe-MIL-53-NH2@PDA nanoparticles. First, the Co / Fe-MIL-53-NH2@PDA nanoparticles were dispersed in a pH 5.0 PBS solution containing 400 mmol / L H2O2 at a concentration of 100 μg / mL. Immediately, 100 μL of TMB test solution was added. After a 10-minute reaction, the nanoparticles were analyzed using a UV spectrophotometer. Then, the Co / Fe-MIL-53-NH2@PDA nanoparticles were dispersed in PBS solutions (pH 5.0) containing different concentrations of H2O2 (40, 100, 200, 400 mM), maintaining the concentration of Co / Fe-MIL-53-NH2@PDA in the reaction system at 100 μg / mL. 100 μL of TMB test solution should be added immediately, and the absorbance of the solution at 652 nm was measured every 60 seconds. The Co / Fe-MIL-53-NH2@PDA nanoparticles were dispersed in PBS solutions (H2O2 400 mmol / L) containing different concentrations of pH (pH 4, 5, 6, 7.4), maintaining the concentration of Co / Fe-MIL-53-NH2@PDA in the reaction system at 100 μg / mL. 100 μL of TMB test solution should be added immediately, and the absorbance of the solution at 652 nm was measured every 60 seconds.

[0056] Depend on Figure 3 It can be seen that ·OH is determined by 3,3',5,5'-tetramethylbenzidine (TMB). When H2O2 and acidic conditions are present, ·OH can be oxidized by Co / Fe-MIL-53-NH2@PDA to produce ·OH from H2O2 substrate and converted to the oxidized state of TMB (oxTMB, signal peak at 652 nm).

[0057] The experimental results are shown in Figure 3 The UV-Vis absorption spectra of different Co / Fe-MIL-53-NH2 ratios in the catalytic reaction of TMB show that the Fenton effect is most pronounced at the Co / Fe-MIL-53-NH2 (8:1) ratio (a). Subsequently, the Co / Fe-MIL-53-NH2 (8:1) ratio was selected as a representative for subsequent experiments. In the presence of acidic (pH 5.0) and 400 mM H2O2 in the presence of Co / Fe-MIL-53-NH2@PDA nanoparticles in the TMB test solution, the characteristic absorption value gradually increases with increasing reaction time (b), indicating that the generation of ·OH also increases accordingly, indicating that the Co / Fe-MIL-53-NH2@PDA nanoparticles can continuously catalyze the generation of ·OH from H2O2. As shown in the figure, when Co / Fe-MIL-53-NH2@PDA nanoparticles are added to the TMB test solution at pH 7.4, the absorbance at 652 nm increases only slightly over time. However, when Co / Fe-MIL-53-NH2@PDA nanoparticles are added to the TMB test solution at pH 4.0, the absorbance at 652 nm increases significantly (c), and this increase is positively correlated with the reaction time. These studies indicate that Co / Fe-MIL-53-NH2@PDA nanoparticles can only catalyze H2O2 through the Fenton reaction in an acidic environment, generating large amounts of cytotoxic ·OH, which is used to kill bacteria. When the same amount of Co / Fe-MIL-53-NH2@PDA nanoparticles was added to TMB test solutions (pH 5.0) containing different concentrations of H2O2, the absorbance at 652 nm showed a distinct time-dependent curve. The reaction time was constant, and the absorbance increased with increasing H2O2 concentration in the TMB test solution (d). This indicates that the catalytic ability of Co / Fe-MIL-53-NH2@PDA nanoparticles to generate ·OH in the reaction of H2O2 under acidic conditions is positively correlated with the H2O2 concentration. In summary, Co / Fe-MIL-53-NH2@PDA has the potential for effective chemokinetic antibacterial therapy and is promising as a nano-antibacterial agent.

[0058] 3. Photothermal performance test

[0059] Experimental Procedure: The photothermal properties of Fe-MIL-53-NH2, Co / Fe-MIL-53-NH2, and Co / Fe-MIL-53-NH2@PDA prepared in the Examples and Comparative Examples were measured using an 808nm NIR laser in a quartz cuvette. The photothermal conversion efficiency of the Co / Fe-MIL-53-NH2@PDA nanoparticles was experimentally calculated. The photothermal conversion efficiency (η) was calculated using the following equation:

[0060]

[0061] Where h is the heat transfer coefficient, s represents the surface area of ​​the quartz cuvette, T max is the equilibrium temperature, T surr is the temperature of the surrounding environment, I is the laser power, and A is the absorbance of Co / Fe-MIL-53-NH2@PDA at 808 nm, which can be determined by UV-visible spectroscopy.

[0062] In addition, Q dis represents the heat absorption of the quartz cuvette, which can be calculated as:

[0063] Q dis =hs(T max-water -T amb-water )

[0064] Among them, T max-water is the equilibrium temperature of water, and T amb-water is the ambient temperature of the water.

[0065] When the input heat of the system is equal to the output heat, Q dis It is 25.03mV (negligible).

[0066]

[0067] Among them, mi represents the mass of the material plus water, which is 1.2g. is the specific heat capacity of water, which is 4.2 J / g, and τs is the time constant of Co / Fe-MIL-53-NH2@PDA.

[0068] According to the cooling time calculation formula:

[0069]

[0070] Therefore, the time constant τs can be calculated by a linear regression curve with respect to t (time) and T (temperature).

[0071] The experimental results are shown in Figure 4 ,Depend on Figure 4 It can be seen that Co / Fe-MIL-53-NH2@PDA can reach 80℃ under 10 minutes of laser irradiation (a), and the dispersion of Co / Fe-MIL-53-NH2@PDA nanoparticles with different concentrations (0-200 μg / mL) is monitored under 808 nm laser (2.22 W / cm 2) irradiated the sample for 10 minutes, and the temperature change of the Co / Fe-MIL-53-NH2@PDA solution was monitored every 60 seconds. The temperature increased with the increase of concentration (b). The inset is the actual image under laser irradiation (c). The results of the experiments were continued with different power densities (0.97, 1.26, 1.58, 1.90 and 2.22 W / cm 2 ) of Co / Fe-MIL-53-NH2@PDA (200 μg / mL) nanoparticles under near-infrared laser irradiation. The temperature increases with the increase of power density, showing a positive correlation (d).

[0072] To investigate the photothermal conversion efficiency and photostability of Co / Fe-MIL-53-NH2@PDA, we employed five cycles of NIR laser on / off. 200 μg / mL of Co / Fe-MIL-53-NH2@PDA was irradiated with NIR laser for 10 minutes and then naturally cooled to room temperature (32°C). The photothermal performance of Co / Fe-MIL-53-NH2@PDA exhibited negligible degradation over the five on / off cycles, demonstrating excellent photothermal reversibility and stability (e). Furthermore, the heat transfer time constant was calculated using the time-temperature linear relationship of Co / Fe-MIL-53-NH2@PDA and the negative logarithmic relationship of the driving force-temperature obtained during the cooling phase. The photothermal conversion efficiency (η) was calculated based on the linear time data of Co / Fe-MIL-53-NH2@PDA during natural cooling, resulting in a calculated photothermal conversion efficiency of 24.50% for Co / Fe-MIL-53-NH2@PDA. The time constant of Co / Fe-MIL-53-NH2@PDA during the natural cooling stage is τs = 246.73s.

[0073] 4. In vitro antibacterial effect evaluation

[0074] Experimental steps: Prepare 200 μg / mL different materials (PBS, Fe-MIL-53-NH2, Co / Fe-MIL-53-NH2, Fe-MIL-53-NH2+H2O2, Co / Fe-MIL-53-NH2+H2O2, Co / Fe-MIL-53-NH2@PDA, Co / Fe-MIL-53-NH2@PDA+H2O2+NIR) to treat E. coli and S. aureus bacterial suspensions, that is, PBS was used as the blank group, and different material groups were prepared by adding 700 mL PBS and 100 μL bacterial suspension (~10 8 CFU / mL) were incubated for 3 h. The illumination group was irradiated with an 808 nm laser NIR irradiator at 2.22 W / cm 2 After irradiation for 10 minutes, incubate for 3 hours. After 3 hours, dilute the bacterial suspension by 10 6The diluted bacterial solution was then spread on an agar plate, and then placed in a constant temperature incubator for 12 hours for observation. Finally, the number of colonies and bacterial survival rate were counted.

[0075] 100 μL of E. coli, S. aureus bacterial suspension (~10 8 CFU / mL) were placed on the slide and cultured for half an hour to allow the bacteria to adhere to the wall. Then, materials (PBS, Fe-MIL-53-NH2, Co / Fe-MIL-53-NH2, Fe-MIL-53-NH2+H2O2, Co / Fe-MIL-53-NH2+H2O2, Co / Fe-MIL-53-NH2@PDA, Co / Fe-MIL-53-NH2@PDA+H2O2+NIR) were added for treatment. The illumination group was treated with an NIR irradiation device with an 808 nm laser at 2.22 W / cm 2 After 10 minutes of irradiation, the cells were incubated for 3 hours and washed three times with sterile PBS to obtain the bacteria. The above bacterial samples were then stained with live bacteria and dead bacteria at 37°C using a live bacteria / dead bacteria staining kit that uses BBcellProbe N01 / PI green-red fluorescent probes for dual bacterial staining. 2.0 μg / mL of the live cell nuclear staining kit-N01 and 2.0 μg / mL of PI (Propidium Iodide) were incubated separately for 15 minutes each, washed three times with sterile PBS, and the slides were placed on a slide for observation under a fluorescence microscope.

[0076] The bacterial suspension (~10 8 CFU / mL) were placed on the slide and cultured for half an hour to allow the bacteria to adhere to the wall. Then, 100 μL E.coli and S.aureus bacterial solution were treated with different materials (PBS, Fe-MIL-53-NH2, Co / Fe-MIL-53-NH2, Fe-MIL-53-NH2+H2O2, Co / Fe-MIL-53-NH2+H2O2, Co / Fe-MIL-53-NH2@PDA, Co / Fe-MIL-53-NH2@PDA+H2O2+NIR) at a concentration of 200 μg / mL and incubated for 3 h. The illumination group was irradiated with an NIR device of 808 nm laser at 2.22 W / cm 2 After 10 minutes of irradiation and 3 hours of incubation, the bacteria were washed three times with sterile PBS to obtain the bacteria. The bacterial suspension was fixed with electron microscopy fixative solution for 4 hours, dehydrated with gradient ethanol solutions (15%, 35%, 55%, 75%, 95%, 100%), and the bacterial morphology was observed under a scanning electron microscope.

[0077] The experimental results are shown in Figure 5 ,Depend on Figure 5As can be seen, the effects of nanoparticles on the growth of Escherichia coli (a) and Staphylococcus aureus (b) were monitored over temperature. The figure shows a significant increase in the temperature of the PDA-modified Co / Fe-MIL-53-NH2@PDA, while the temperature of the PBS group remained unchanged. The corresponding solid agar plate also clearly demonstrates the excellent antibacterial effect of Co / Fe-MIL-53-NH2@PDA.

[0078] Depend on Figure 6 It can be seen that the bacterial solid agar plate treated with different materials is shown in (a). E. coli, 50μL H2O2+Co / Fe-MIL-53-NH2@PDA+NIR at 2.22W / cm 2 After 10 minutes of 808nm laser irradiation, the temperature reached 52°C, demonstrating a 98% bactericidal effect against both bacteria. Co / Fe-MIL-53-NH2+H2O2 also demonstrated excellent bactericidal activity against Escherichia coli (b), with similar results obtained against Staphylococcus aureus (c). This demonstrates that Co / Fe-MIL-53-NH2 nanoparticles catalyze hydrogen peroxide through the Fenton reaction, producing large amounts of cytotoxic ·OH, which kills bacteria.

[0079] Depend on Figure 7 It can be seen that in the PBS-treated group, which served as the control group, most cells showed green fluorescence. After 10 minutes of 808nm laser treatment, the Co / Fe-MIL-53-NH2@PDA+H2O2+NIR group almost completely killed E. coli and S. aureus. Compared with the bacteria incubated with Co / Fe-MIL-53-NH2@PDA, after Co / Fe-MIL-53-NH2@PDA+H2O2 was treated with 808nm laser, more bacterial cells showed red fluorescence, and only a few bacteria survived and emitted green fluorescence. Co / Fe-MIL-53-NH2+H2O2 also showed a good bactericidal effect, proving that Co / Fe-MIL-53-NH2 nanoparticles catalyze hydrogen peroxide through the Fenton reaction, producing a large amount of ·OH to kill bacteria. Co / Fe-MIL-53-NH2@PDA+H2O2+NIR achieved Fenton-photothermal synergistic treatment to effectively eradicate bacteria.

[0080] SEM was used to further investigate the antibacterial behavior and morphological changes of E. coli and S. aureus. For bacteria treated with PBS, no dead bacteria were observed, and the bacteria displayed an intact membrane morphology. For Co / Fe-MIL-53-NH2+H2O2, some dead bacteria could be detected in the SEM images, along with ruptured bacterial membranes. This suggests that the Co / Fe-MIL-53-NH2 nanoparticles catalyze hydrogen peroxide through the Fenton reaction, producing a large amount of cytotoxic ·OH, which kills bacteria and causes damage to the bacterial membrane and the death of some bacteria. For Co / Fe-MIL-53-NH2@PDA+H2O2, after 808nm laser irradiation, almost no live bacteria were observed, and the bacterial membranes were completely destroyed and severely adhered, indicating that the synergistic effect caused by the photothermal effect achieves antibacterial effects, resulting in severe damage.

[0081] 5. Cytotoxicity assay

[0082] Experimental steps: Preparation of experimental solutions for the Fe-MIL-53-NH2 group: Fe-MIL-53-NH2 was dissolved in 1640 culture medium, and Fe-MIL-53-NH2 experimental solutions with concentrations of 0, 25, 50, 100, 200, and 250 μg / mL were prepared using 1640 culture medium;

[0083] Preparation of experimental solutions for the Co / Fe-MIL-53-NH2 group: Co / Fe-MIL-53-NH2 was dissolved in 1640 medium, and 1640 medium was used to prepare Co / Fe-MIL-53-NH2 experimental solutions with concentrations of 0, 25, 50, 100, 200, and 250 μg / mL;

[0084] Preparation of experimental solutions for the Co / Fe-MIL-53-NH2@PDA group: Co / Fe-MIL-53-NH2@PDA was dissolved in 1640 culture medium, and 1640 culture medium was used to prepare Co / Fe-MIL-53-NH2@PDA experimental solutions with concentrations of 0, 25, 50, 100, 200, and 250 μg / mL.

[0085] 3T3 cells in logarithmic growth and in good condition were digested and the original culture medium removed. Each cell was washed twice with 2 mL of PBS solution. 1 mL of trypsin was added and the cells were incubated in an incubator for 4 minutes. After digestion, 4 mL of complete culture medium (containing 10% fetal bovine serum and 1% double-antibody) was added to terminate the digestion. The cells were centrifuged at 1200 rpm for 5 minutes. The supernatant was removed and 9 mL of complete culture medium was added to the cells, which were then pipetted evenly. The cells were counted and a cell suspension was prepared according to experimental requirements. 5,000 cells were plated per well in a 96-well plate and incubated in an incubator for 24 hours. After the cells attached, the original culture medium was removed and 200 μL of 1640 culture medium, Fe-MIL-53-NH2 experimental solution, Co / Fe-MIL-53-NH2 experimental solution, and Co / Fe-MIL-53-NH2@PDA experimental solution were added to each well. After drug addition, the cells were returned to the incubator and incubated for an additional 10 hours. After the incubation period, the drug-containing medium was aspirated, and 100 μL of 10% CCK-8 reagent was added to each well. The cells were then incubated in a 37°C incubator for another 2 h. Finally, the absorbance of each well was measured at a wavelength of 450 nm using a microplate reader. The data was recorded and the corresponding cell viability was calculated using the cell viability calculation formula.

[0086] The cell survival rate formula is as follows:

[0087]

[0088] Among them, OD 实验组 is the OD value of the wells with cells, CCK-8 solution, and drug solution; OD 空白组 is the OD value of the wells with cells and CCK-8 solution but no drug solution; OD 调零组 is the OD value of the well without cells.

[0089] The experimental results are shown in Figure 8 ,Depend on Figure 8 The cytotoxicity of different concentrations of Fe-MIL-53-NH2, Co / Fe-MIL-53-NH2, and Co / Fe-MIL-53-NH2@PDA on 3T3 cells after 24 hours of incubation was determined using a Cell Counting Kit-8 (CCK-8). Cell viability assayed using a standard CCK-8 assay showed that Co / Fe-MIL-53-NH2@PDA had no cytotoxicity towards mouse fibroblasts, even at a high concentration of 250 μg / mL, demonstrating the good biocompatibility of Co / Fe-MIL-53-NH2@PDA.

[0090] 6. In vivo animal experiments and tissue analysis

[0091] Experimental procedures: All animal experimental procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals: 8th Edition, ISBN-10: 0-309-15396-4, and in accordance with international standards for such animal research. Healthy SPF-grade BALB / c female Nude mice were purchased from Beijing Weitonglihua and approved by the Institutional Ethics Committee of Hainan University.

[0092] Mice (4-6 weeks) were divided into 7 groups (6 mice per group). An 8 mm wound was created on the back of each mouse using scissors. Subsequently, the wound was infected with S. aureus suspension (10 8 CFU / mL, 100 μL), and the initial infection time was recorded as day 0. 24 hours later, the wounds of mice in different groups were treated with 100 μL (PBS, Fe-MIL-53-NH2, Co / Fe-MIL-53-NH2, Fe-MIL-53-NH2+H2O2, Co / Fe-MIL-53-NH2+H2O2, Co / Fe-MIL-53-NH2@PDA, Co / Fe-MIL-53-NH2@PDA+H2O2+NIR) suspensions, and then the light group was irradiated with 2.22 W / cm 2 The mice were irradiated with an 808 nm NIR laser for 5 minutes. The treatment day was designated as day 0. The body weight and wound area of ​​each group of mice were recorded daily. After 9 days of observation, the mice were sacrificed, and the wounds, along with the heart, liver, spleen, lungs, and kidneys, were collected for H&E analysis and staining. The wound area of ​​the mice was calculated as follows:

[0093]

[0094] The experimental results are shown in Figure 9 ,Depend on Figure 9As shown in Figure 1 (a), mice were divided into seven different groups (PBS, Fe-MIL-53-NH2, Co / Fe-MIL-53-NH2, Co / Fe-MIL-53-NH2@PDA+H2O2+NIR, Co / Fe-MIL-53-NH2+H2O2, Co / Fe-MIL-53-NH2@PDA, and Fe-MIL-53-NH2+H2O2). The results (a) show that the wounds of mice in the PBS group had not healed on the ninth day of treatment. Although the wounds of mice in the Fe-MIL-53-NH2+H2O2, Co / Fe-MIL-53-NH2+H2O2, and Co / Fe-MIL-53-NH2@PDA groups were less severe, they did not completely heal either. In contrast, the wounds of mice in the Co / Fe-MIL-53-NH2@PDA+H2O2+NIR group showed no inflammation or edema and completely healed. Therefore, these results indicate that Co / Fe-MIL-53-NH2@PDA+H2O2+NIR treatment can effectively promote the wound healing process, thereby reducing the burden of bacterial infection. The irradiation-induced synergistic antibacterial effect of Co / Fe-MIL-53-NH2@PDA+H2O2+NIR gives it a highly effective anti-infection ability within the wound.

[0095] To quantitatively evaluate the antibacterial effect of each treatment, the wound skin of each group was harvested after treatment and bacterial counts were assessed using the diffusion plate method. The results showed that the number of bacteria in the Co / Fe-MIL-53-NH2@PDA+H2O2+NIR group was far less than that in the other groups, indicating that Co / Fe-MIL-53-NH2@PDA+H2O2+NIR had the strongest bactericidal effect (b). H&E-stained histological images of S. aureus-infected lesions after different treatments (c). Curves of changes in mouse body weight over time in the different groups (d). The weight of mice treated with Co / Fe-MIL-53-NH2@PDA+H2O2+NIR increased over time, with no significant difference compared to the PBS control group. Curves of changes in the wounds of mice in the different groups over time (e). Co / Fe-MIL-53-NH2@PDA+H2O2+NIR was significantly superior to the other treatment groups. After 9 days of treatment, the wound in the Co / Fe-MIL-53-NH2@PDA+H2O2+NIR group was basically healed, indicating that the rapid and effective sterilization of the synergistic effect of Fenton reaction and PTT played an important role in wound healing.

[0096] Depend on Figure 10 It can be seen that the mice were treated with photothermal therapy and irradiated with near-infrared radiation for 2 minutes (808 nm, 1.26 W / cm 2), the temperature of the wound area of ​​mice in the Co / Fe-MIL-53-NH2@PDA+H2O2 treatment group increased from 36.85℃ to 50.65℃, which intuitively proved that Co / Fe-MIL-53-NH2@PDA can be used as a photothermal therapy agent.

[0097] Depend on Figure 11 As can be seen, there were no significant differences between the treatment and control groups, indicating that Co / Fe-MIL-53-NH2@PDA+H2O2+NIR did not cause tissue damage or inflammation. The negligible accumulation and organ toxicity of Co / Fe-MIL-53-NH2@PDA+H2O2+NIR in major organs demonstrates its excellent biocompatibility. All of these results confirm that Co / Fe-MIL-53-NH2@PDA+H2O2+NIR exhibits no significant toxicity and is safe for in vivo use. Highly biosafe nanomaterials are particularly important in practical applications. In this study, H&E-stained sections of major organs (heart, liver, spleen, lung, and kidney) of mice in each group were collected, and no significant physiological morphological changes or cell apoptosis were observed. Thus, after nine days of treatment, no systemic organ damage was caused in mice, demonstrating the good biocompatibility of the nanomaterial. In summary, Co / Fe-MIL-53-NH2@PDA+H2O2+NIR exhibits excellent in vivo therapeutic efficacy with minimal toxic side effects.

[0098] In summary, the present invention successfully prepared the Co / Fe-MIL-53-NH2 nanocomposite using a solvothermal method and achieved polydopamine (PDA) encapsulation. The resulting composite material not only performs Fenton catalysis but also exhibits photothermal properties, demonstrating both Fenton catalysis and photothermal effects. Cell experiments demonstrated good biocompatibility, and animal experiments confirmed the nanocomposite's excellent antibacterial properties, suggesting potential for clinical application.

[0099] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing a bimetallic organic framework composite material, characterized in that: Use the following steps: (1) FeCl3·6H2O, CoCl2·6H2O and NH2-BDC were mixed in DMF, and the mixture was reacted at high temperature in a closed polytetrafluoroethylene autoclave. After cooling, the resulting mixture was centrifuged, collected, and dried to obtain Co / Fe-MIL-53-NH2; the molar ratio of FeCl3·6H2O to CoCl2·6H2O was 8:1; (2) The Co / Fe-MIL-53-NH2 aqueous solution is mixed into a Tris buffer solution of dopamine hydrochloride and stirred, and dopamine is uniformly modified on the surface of Co / Fe-MIL-53-NH2 and in situ polymerized to obtain Co / Fe-MIL-53-NH2@PDA nanoparticles; the mass ratio of the Co / Fe-MIL-53-NH2 to dopamine hydrochloride is 1:

3.

2. The preparation method according to claim 1, characterized in that The high temperature reaction temperature is 150° C. and the reaction time is 24 h.

3. The preparation method according to claim 1, characterized in that The amount of NH2-BDC used was 362 mg, and the amount of DMF used was 35 mL.

4. The preparation method according to claim 1, characterized in that The concentration of dopamine hydrochloride in the Tris buffer of dopamine hydrochloride is 1 mg / mL, and the concentration of Co / Fe-MIL-53-NH2 in the Co / Fe-MIL-53-NH2 aqueous solution is 0.3 mg / mL.

5. The preparation method according to claim 1, characterized in that The Co / Fe-MIL-53-NH2 aqueous solution was mixed into the Tris buffer solution of dopamine hydrochloride and stirred at a stirring speed of 300-500 r / min for 1 hour. The reaction temperature was room temperature.

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