Phenanthroline-based covalent organic framework antibacterial material and application thereof

Abstract

The invention discloses a phenanthroline-based covalent organic framework antibacterial material and application thereof, and belongs to the technical field of biological medicines. The covalent organic framework antibacterial material is a covalent organic framework antibacterial material PRSA-Cu based on phenanthroline, and the covalent organic framework antibacterial material PRSA-Cu is obtained by carrying out a coordination reaction on 5, 5 '-[(1E, 1' E)-({1, 10-phenanthroline-5, 6-diyl} bis (azenyl)) bis (methylene)] bis (1, 2, 4-trihydroxybenzene) PRSA and copper. The PRSA-Cu prepared by the invention can be used for synergistically resisting bacteria by combining photo-thermal capability with the effects of simulated photo-thermal (PTT), photodynamic (PDT), peroxidase (PODs) and glutathione peroxidase (GSH-Px), can also be used for inducing death of bacteria by utilizing the intrinsic characteristics of an infected microenvironment, and can also be used for inhibiting bacteria infection by adjusting the microenvironment of a bacterial infected part. The recovery of bacterial infection wounds is obviously accelerated.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a covalent organic framework antibacterial material based on o-phenanthroline and application thereof. Background Art

[0002] Pathogens are the main cause of bacterial infectious diseases. Bacterial infections have become a fatal medical problem worldwide, seriously threatening human health. At present, the treatment of bacterial infectious diseases relies heavily on antibiotics, which has led to a rapid increase in drug-resistant bacteria and even the emergence of super bacteria, resulting in reduced treatment effects and even the spread of infectious diseases. In recent years, emerging nanozymes have become a new generation of antibiotics due to their excellent characteristics such as broad-spectrum antibacterial activity, low toxicity and lack of drug resistance.

[0003] Typically, nanozymes with peroxidase-like activity specifically catalyze the conversion of hydrogen peroxide into highly toxic reactive oxygen species (ROS), such as hydroxyl radicals, singlet oxygen, and superoxide anions, to attack the bacterial membrane at the weakly acidic infection site to achieve a bactericidal effect. ROS are non-specific in killing bacteria, so no specific target is required to exert their effects. Compared with antibiotics, ROS-based bactericidal methods can kill bacteria by destroying bacterial cell membranes, DNA, proteins, etc.; they can avoid the occurrence of bacterial resistance.

[0004] O-phenanthroline, also known as 1,10-phenanthroline, is a commonly used metal chelator that can form complexes with a variety of transition metals. The complexes formed with copper and their derivatives have certain cutting activity on DNA and can be used as non-oxidative nucleic acid cutting enzymes, thereby having certain anti-cancer activity. For example, the patent with application number CN114106020A discloses a high anti-cancer activity complex based on pyrimidine Schiff base combined with o-phenanthroline-copper and its preparation method. This patent uses o-phenanthroline and copper to form a complex for anti-cancer. Since copper complexes have many excellent properties, there have been reports on the coordination of o-phenanthroline with copper for antibacterial purposes. The patent with application number CN111808123A discloses the preparation of a copper complex solid antibacterial agent, and prepares a coordination polymer containing copper sulfate; however, the polymer is a crystalline material that only utilizes the antibacterial effect of copper, has a single antibacterial ability, and has a low antibacterial effect. Therefore, it is necessary to develop a covalent organic framework antibacterial material based on o-phenanthroline and copper, which not only has multiple antibacterial modes, but also can utilize the intrinsic properties of the infection microenvironment (IME) to exert accurate treatment and induce bacterial death. It can also significantly accelerate the recovery of bacterially infected wounds by regulating the microenvironment of the bacterial infection site. Summary of the invention

[0005] In view of the above-mentioned prior art, the purpose of the present invention is to provide a covalent organic framework antibacterial material based on o-phenanthroline and its application. The present invention uses 5,5'-[(1E,1'E)-({1,10-phenanthroline-5,6-diyl}bis(azepine))bis(methylene)]bis(1,2,4-benzenetriol) (PRSA) to coordinate with copper to form a porous polymer PRSA-Cu. The material uses photothermal capacity to combine simulated photothermal (PTT), photodynamic (PDT), peroxidase (PODs) and glutathione peroxidase (GSH-Px) to perform synergistic antibacterial, and can also use the inherent characteristics of the infection microenvironment to induce bacterial death, and can also significantly accelerate the recovery of bacterial infection wounds by regulating the microenvironment of the bacterial infection site.

[0006] To achieve the above object, the present invention adopts the following technical solution: In a first aspect of the present invention, a covalent organic framework antibacterial material based on o-phenanthroline is provided, wherein the covalent organic framework antibacterial material is an amorphous porous polymer formed by coordination of PRSA and copper; the PRSA is 5,5'-[(1E,1'E)-({1,10-phenanthroline-5,6-diyl}bis(nitrile))bis(methylene)]bis(1,2,4-benzenetriol), and its structural formula is .

[0007] The 1,10-phenanthroline involved in the present invention can also be written as 1,10-phenanthroline, also known as o-phenanthroline.

[0008] Preferably, the covalent organic framework antibacterial material is prepared by the following method: PRSA and copper salt are added into a mixed solvent, and a solvothermal reaction is carried out under a protective atmosphere. After the reaction is completed, the covalent organic framework antibacterial material PRSA-Cu based on o-phenanthroline is obtained after washing and drying.

[0009] The PRSA-Cu is an amorphous coordinated covalent organic framework polymer.

[0010] Preferably, the PRSA is prepared by the following method: (1) Dissolve 1,2,4-benzenetriol and triethyl orthoformate in tetrahydrofuran, add anhydrous aluminum chloride in portions under a protective atmosphere, and react at room temperature to obtain 2,4,5-trihydroxybenzaldehyde; (2) 2,4,5-trihydroxybenzaldehyde and 5,6-diamino-1,10-phenanthroline are dispersed in ethanol and subjected to a solvothermal reaction under a protective atmosphere to obtain PRSA.

[0011] Preferably, in step (1), the ratio of the added amounts of 1,2,4-benzenetriol, triethyl orthoformate and anhydrous aluminum chloride is 1.0 g:6.54 mL:1.628 g.

[0012] Preferably, in step (2), the molar ratio of 2,4,5-trihydroxybenzaldehyde to 5,6-diamino-1,10-phenanthroline is greater than 2:1; the temperature of the solvent thermal reaction is 80° C. and the time is 3 days.

[0013] Preferably, the copper salt is copper acetate; and the protective atmosphere is argon.

[0014] Preferably, the molar ratio of PRSA to copper acetate is less than 3:7.

[0015] Preferably, the mixed solvent is DMF and distilled water mixed in a volume ratio of 5:1; the temperature of the solvent thermal reaction is 100° C. and the time is 3 days.

[0016] The second aspect of the present invention provides the use of covalent organic framework antibacterial materials in the preparation of antibacterial drugs.

[0017] Preferably, the antibacterial drug has peroxidase activity and glutathione peroxidase activity.

[0018] Beneficial effects of the present invention: (1) The PRSA-Cu of the present invention is a covalent organic framework antibacterial material with a pocket structure of 5,5'-[(1E,1'E)-({1,10-phenanthroline-5,6-diyl}bis(nitrile))bis(methylene)]bis(1,2,4-benzenetriol). The material uses photothermal capacity to combine simulated PTT, PDT, PODs and GSH-Px treatments, which can not only induce bacterial death by utilizing the intrinsic characteristics of the infection microenvironment, but also significantly accelerate the recovery of bacterial infection wounds by regulating the microenvironment of the bacterial infection site.

[0019] (2) The PRSA-Cu prepared by the present invention produces a good photothermal conversion effect through laser irradiation with a wavelength of 638nm, and can convert endogenous H2O2 into hydroxyl radicals, and the enzyme activity is further increased after photothermal treatment. In addition, PRSA-Cu has high biocompatibility, a lysis rate of red blood cells of less than 4%, a small effect on the cell viability of 3T3 cells, and can promote wound healing, which is conducive to its application in the biological field. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1:(a)IR spectra of PRSA and PRSA-Cu;(b)Low-temperature N2 absorption isotherm of PRSA-Cu at 77 K;(c)Pore size distribution curve of PRSA-Cu;(d)Thermogravimetric curve of PRSA-Cu;(e)X-ray diffraction pattern of PRSA-Cu; Figure 2 : Morphological characterization of PRSA-Cu, including (a) SEM of PRSA-Cu at 1 μm scale, (b) SEM of PRSA-Cu at 200 nm scale; (c) TEM of PRSA-Cu at 200 nm scale; (d) TEM of PRSA-Cu at 50 nm scale; (e) HR-TEM of PRSA-Cu at 5 nm scale; (f) HAADF-STEM of PRSA-Cu; (g) N element mapping; (h) C element mapping; (i) O element mapping; (j) Cu element mapping; Figure 3 : EDS spectrum of PRSA-Cu; Figure 4 : X-ray photoelectron spectrum of PRSA-Cu, including (a) XPS analysis spectrum; (b) C 1s spectrum, (c) Cu2p spectrum, (d) N 1s spectrum, (e) O 1s spectrum; Figure 5 : Photothermal performance of PRSA-Cu, including (a) concentration-dependent photothermal effect of PRSA-Cu under laser irradiation; (b) photothermal effect of PRSA-Cu with the same concentration (100 μg / mL) irradiated for 10 min at different laser powers; (c) infrared thermal images corresponding to different concentrations of PRSA-Cu; (d) temperature change diagram of PRSA-Cu (100 μg / mL) after 5 cycles; (e) 638 nm laser (1.2 W / cm 2 ) Heating and cooling temperature change curves of PRSA-Cu (100 μg / mL) under irradiation and the curve of cooling time and negative natural logarithm of temperature; Figure 6 : Catalase-like activity of PRSA-Cu, including (a) the absorption of TMB, TMB + H2O2, PRSA-Cu + H2O2, H2O2, PRSA-Cu + TMB and PRSA-Cu + H2O2 + TMB at 652 nm; (b) the change of absorbance of PRSA-Cu + H2O2 + TMB at 652 nm before and after 638 nm laser irradiation; (c) the absorbance of PRSA-Cu (100 μg / mL) in 0.3% H2O2 at different pH environments; (d) the absorbance of different concentrations of PRSA-Cu under 0.3% H2O2 at pH 5.5; Figure 7 : Electron spin resonance spectrum (ESR) of PRSA-Cu; Figure 8 : PRSA-Cu used glutathione oxidase detection kit to detect the consumption of glutathione at different concentrations; Fig. 9 : The bactericidal effect diagram of PRSA-Cu, including (a) plate count method photos of Staphylococcus aureus treated with PRSA-Cu + H2O2 + laser at different concentrations; (b) plate count method photos of Escherichia coli treated with PRSA-Cu + H2O2 + laser at different concentrations; (c) bactericidal effect diagram of PRSA-Cu + H2O2 + laser plate count method quantification diagram; (d) plate count method photos of Staphylococcus aureus and Escherichia coli treated with different treatment methods; (e) plate count method quantification diagram of Staphylococcus aureus treated with different treatment methods; (f) plate count method quantification diagram of Escherichia coli treated with different treatment methods; Fig.10 : TEM images of bacteria, including (a) Staphylococcus aureus after different treatments, (b) TEM images of Escherichia coli after different treatments, the red tip in the figure represents the damage location; Fig.11 : Bacterial staining images, including (a) fluorescence images of Staphylococcus aureus after different treatments and incubation with SYTO-9 / PI live / dead stain; (b) fluorescence images of Escherichia coli after different treatments and incubation with SYTO-9 / PI live / dead stain; Fig.12 : Hemolysis rate of different concentrations of PRSA-Cu (n=3, error bars indicate standard deviation); Fig.13 : Cell viability (%) after co-culture of 3T3 cells with different concentrations of PRSA-Cu (n=3, error bars represent standard deviation); Fig.14 : Wound images of mice on days 1, 3, 5, 7, and 9 after treatment in different groups; Fig.15 :(a) Wound healing rate of mice (%);(b) Changes in mouse body weight during treatment; Fig.16 : H&E and Masson staining were used for histological analysis in each group; Fig.17 : Organ staining results of each group; Fig.18 : Synthesis route of PRSA-Cu. DETAILED DESCRIPTION

[0021] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0022] As introduced in the background technology section, the antibacterial ability of the currently reported o-phenanthroline and copper coordination polymers is single, and the antibacterial effect needs to be improved.

[0023] Based on this, the purpose of the present invention is to provide a covalent organic framework antibacterial material based on o-phenanthroline and its application. The present invention uses 5,5'-[(1E,1'E)-({1,10-phenanthroline-5,6-diyl}bis(nitrile))bis(methylene)]bis(1,2,4-benzenetriol) with a pocket structure as the main skeleton, and coordinates with copper to form a porous polymer PRSA-Cu. PRSA should be prepared from 2,4,5-trihydroxybenzaldehyde and 5,6-diamino-1,10-phenanthroline in a molar ratio of 2:1, but in order to avoid insufficient reaction and generate other by-products, the present invention adds 5,6-diamino-1,10-phenanthroline dropwise to 2,4,5-trihydroxybenzaldehyde, and 2,4,5-trihydroxybenzaldehyde is excessive, so the molar ratio of 2,4,5-trihydroxybenzaldehyde and 5,6-diamino-1,10-phenanthroline should be greater than 2:1. The molar ratio of PRSA to copper acetate should be 1:2.33, or 3:7. However, in order to ensure that copper can be coordinated on PRSA, an excess of copper acetate is required. Therefore, the molar ratio of PRSA to copper acetate is less than 3:7.

[0024] The copper contained in PRSA-Cu can convert endogenous H2O2 into highly toxic •OH, thereby achieving effective regulation of the infection microenvironment. PRSA-Cu can achieve local temperature increase through photothermal conversion to achieve the effect of rupturing the bacterial membrane. In short, the inherent photothermal and photokinetic ability of this material combined with simulated peroxidase (PODs) and GSH-Px treatment can be used as an intelligent platform. It can not only use the intrinsic characteristics of the infection microenvironment (IME) to exert accurate treatment and induce bacterial death, but also regulate the microenvironment of the bacterial infection site to significantly accelerate the recovery of bacterial infection wounds. Moreover, at the optimal antibacterial concentration, PRSA-Cu has almost no hemolytic effect and hardly affects the growth of normal cells, which makes it have good biological application prospects.

[0025] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0026] The test materials used in the examples of the present invention are all conventional test materials in the art and can be purchased through commercial channels.

[0027] Example 1: Preparation of PRSA-Cu (1) Preparation of 2,4,5-trihydroxybenzaldehyde: 1.0 g of 1,2,4-pyrogallol and 6.54 mL of triethyl orthoformate were dissolved in 40 mL of tetrahydrofuran. A total of 1.628 g of anhydrous aluminum chloride was added in three equal batches under an argon atmosphere. The mixture was reacted at room temperature for 12 h. After quenching with 18 mL of 3M hydrochloric acid, the mixture was extracted with 40 mL of dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and purified by column chromatography to obtain 2,4,5-trihydroxybenzaldehyde.

[0028] (2) Preparation of PRSA: 41 mg of 2,4,5-trihydroxybenzaldehyde and 20 mg of 5,6-diamino-1,10-phenanthroline were dispersed in 50 mL of ethanol, purged with argon for 30 min, and refluxed at 80° C. for three days. After the reaction, the mixture was washed with methanol and ethanol and dried to obtain PRSA.

[0029] (3) Preparation of PRSA-Cu: 96 mg PRSA and 140 mg copper acetate were added to a mixed solution of 12 mL DMF and distilled water (10 mL: 2 mL), purged with argon, and reacted at 100 °C for 72 h. After the reaction was completed, the mixture was filtered, washed with tetrahydrofuran, dimethyl sulfoxide, water, methanol, and ethanol in sequence, and dried overnight under vacuum at 100 °C to obtain PRSA-Cu. See the synthetic route for details. Fig.18 .

[0030] Example 2: Characterization of PRSA-Cu (1) Determination of infrared spectrum The structures of PRSA and PRSA-Cu were determined by infrared spectroscopy. Figure 1 As shown in (a), in the OH (3300 cm -1 ) stretching vibration band almost disappeared, and a new Cu-O (490 cm -1 ) stretching vibration band, the presence of broad peaks can be attributed to the formation of amorphous coordination polymers. After the complexation of PRSA and metal ions, a unique broad peak appeared within 450-800 nm, indicating the successful complexation of Cu in the prepared PRSA-Cu, confirming the occurrence of metal coordination. The new characteristic Cu-O (490 cm -1 ) and Cu-N (~ 560 cm -1 ) Telescopic strap.

[0031] (2) The pore distribution of PRSA-Cu was understood through the N2 adsorption-desorption curve and pore size distribution curve. The BET specific surface area of ​​PRSA-Cu is 51.255 m 2 g -1 .from Figure 1(b) It can be seen that the adsorption curve of PRSA-Cu has typical IV isotherm characteristics, and there is an obvious hysteresis phenomenon at the branch of the adsorption-desorption curve, indicating that its structure is mainly mesoporous. Based on the Barrett-Joyner-Halenda (BJH) method, Figure 1 (c) shows that the average pore size of PRSA-Cu is 9.136 nm. The pore size distribution curve also directly reflects its pore characteristics.

[0032] (3) Thermal stability of PRSA-Cu by thermogravimetric analysis The thermal stability of the synthesized material under N2 atmosphere was studied by thermogravimetric analysis (TGA). Figure 1 (d) shows that the first part of the mass loss of PRSA-Cu materials is less than 16.9% (<100°C), and the second part of the mass loss is above 200°C. The former is due to the evaporation of water absorbed in the highly polar porous material, the latter is attributed to the decomposition of the porous network. The weight at 800°C remains at 53.66%, which shows that the prepared material has excellent thermal stability.

[0033] (4) Powder X-ray diffraction (PXRD) analysis was performed to estimate the crystallinity and porosity of PRSA-Cu. Figure 1 (e) shows a large broad peak at around 25°, the presence of which can be attributed to the formation of amorphous coordination polymers.

[0034] (5) Use Figure 2 (a) ~ Figure 2 (b) SEM and Figure 2 (c) ~ Figure 2 (e) TEM observation of the morphology of PRSA-Cu. Scanning electron microscopy (SEM) shows that PRSA-Cu is a typical bulk material composed of irregular particles and has a uniformly distributed, interconnected macroporous structure, and its rough surface is conducive to bacterial adhesion. Transmission electron microscopy (TEM) further reveals that PRSA-Cu has a continuous multi-level pore structure, which is composed of loosely stacked particles and presents clear multi-level pore characteristics.

[0035] Figure 2 (f) The elemental composition and distribution of PRSA-Cu were detected by high-angle annular dark field scanning transmission electron microscopy (HAADF-STEM). Figure 3 From the energy dispersive X-ray spectroscopy (EDS) spectrum, it can be seen that C (74.99%), N (9.93%), O (12.46%) and Cu (2.59%) elements are evenly distributed on the PRSA-Cu porous framework.

[0036] (6) XPS spectrum analysis of PRSA-Cu. As shown in Figure 4(a), similar to the EDS results, XPS also shows the presence of C, N, O and Cu. Figure 4 (b) shows that there are three types of carbon in the C 1s of PRSA-Cu, namely CC (284.8 eV), CO (285.82 eV), and C=N (288.39 eV). Figure 4 (c) It shows that the two types of Cu in Cu2p of PRSA-Cu are Cu 2+ (934.46 eV) and Cu + (931.48eV), while Cu 2+ and Cu + The corresponding peak area accounts for 56.49% and 43.51% respectively. + The ratio proves the occurrence of electron transfer and indirectly proves the formation of coordination bonds. Figure 4 (d) shows that the two types of N in N1s of PRSA-Cu are C=N (398.33 eV) and C=N-Cu (399.58 eV). Figure 4 (e) It shows that there are two types of O in O1s of PRSA-Cu, namely O-Cu (530.82 eV) and OC (532.57 eV).

[0037] (7) Photothermal performance of PRSA-Cu The PRSA-Cu concentration (50, 100, 200, 300, 400 μg / mL) or the laser power density (0.5, 0.8, 1.0, 1.2, 1.5, and 1.8 W / cm 2 ), the photothermal conversion performance of PRSA-Cu was studied in detail. Among them, the preparation method of PRSA-Cu with different concentrations is as follows: first, weigh 1 mg of PRSA-Cu and use an ultrasonic instrument to fully disperse it in 1 mL of distilled water to prepare a 1 mg / mL mother solution, then draw 50, 100, 200, 300, and 400 μL from the mother solution and add them to 950, 900, 800, 700, and 600 μL of distilled water, and finally prepare 50, 100, 200, 300, and 400 μg / mL of PRSA-Cu aqueous dispersion. Using pure water as a control, a 638 nm laser (1.2W / cm 2 , 10 min) to monitor the temperature changes of PRSA-Cu with different concentrations, and the photophysical properties of the synthesized samples were preliminarily studied.

[0038] like Figure 5As shown in (a), unlike the case where the temperature change of pure water is negligible, PRSA-Cu exhibits a dose-dependent temperature increase behavior, which increases rapidly with increasing concentration. Specifically, the temperature of PRSA-Cu increases with concentrations of 50, 100, 200, 300, and 400 μg mL -1 The temperature of the PRSA-Cu solution rose to 42.9, 51.6, 57.2, 61.2, and 65.8°C, respectively. Figure 5 The laser power intensity in (b) is also found to be positively correlated with the dispersion temperature. Figure 5 The effect image obtained from the thermal imaging camera in (c) demonstrates the excellent photothermal capability of PRSA-Cu. All these results indicate that red light can be effectively converted into local heat by adjusting the PRSA-Cu concentration or the power of the laser. To evaluate the photostability of PRSA-Cu, periodic irradiation experiments were performed, such as Figure 5 (d) The PRSA-Cu shown has a stable laser switching effect, with almost no temperature fluctuation after 5 consecutive laser on / off cycles.

[0039] In addition, the photothermal conversion efficiency of PRSA-Cu is: η (%) = 49.7%. The photothermal conversion efficiency calculation formula is calculated by referring to the photothermal conversion efficiency formula in "A Photothermal-Fenton-like Reaction Artificial Nanozyme and Its Preparation Method and Application" disclosed in application number CN115845086A. Figure 5 (e) shows the τS and θ values ​​in the photothermal conversion efficiency calculation formula.

[0040] (8) Peroxidase activity of PRSA-Cu The peroxidase activity of PRSA-Cu at different pH was studied. The ROS-generating ability of PRSA-Cu was evaluated using a dual substrate system, i.e., a colorimetric system of H2O2 and 3,3',5,5'-tetramethylbenzidine (TMB), in which TMB was used as the colorimetric agent. TMB can be oxidized by ROS to form colorimetric ox-TMB. The presence of copper endows PRSA-Cu with the ability to act as an efficient •OH generator in acidic media.

[0041] The preparation method of TMB used in the experiment is as follows: weigh TMB (3.606 mg, 0.015 mmoL) and dissolve it in 10 mL of ethanol to prepare a 1.5 mmoL / L TMB ethanol solution; the mass concentration of H2O2 is 30%. Take 50 mL of PBS with a pH of 7.4 in a test tube, add phosphoric acid to adjust the pH to 1.5, 2.5, 3.5, 4.5, 5.5 and 6.5 respectively. Preparation of PRSA-Cu with different pH values: Weigh 2 mg of PRSA-Cu and fully disperse it in 2 mL of PBS with different pH values ​​using a sonicator to prepare 1 mg / mL stock solutions with pH values ​​of 1.5, 2.5, 3.5, 4.5, 5.5 and 6.5, respectively. Then, take 100 μL of the stock solutions and add them to 700 μL of PBS with pH values ​​of 1.5, 2.5, 3.5, 4.5, 5.5 and 6.5, respectively. Add 100 μL of TMB and H2O2 each, and finally prepare PBS dispersions of PRSA-Cu with a concentration of 100 μg / mL and pH values ​​of 1.5, 2.5, 3.5, 4.5, 5.5 and 6.5, respectively. Preparation of different concentrations of PRSA-Cu: weigh 1 mg of PRSA-Cu and fully disperse it in 1 mL PBS at pH 5.5 using an ultrasonicator to prepare a 1 mg / mL stock solution. Then, draw 25, 50, 100, 150, 200, and 250 μL from the stock solution and add them to 775, 750, 700, 650, 600, and 550 μL of PBS at pH 5.5, respectively. Then, add 100 μL each of TMB and H2O2 to finally prepare 25, 50, 100, 150, 200, and 250 μg / mL of PRSA-Cu in PBS.

[0042] like Figure 6 As shown in (a), only in the culture medium containing both PRSA-Cu and H2O2 (PRSA-Cu + H2O2 + TMB) can the colorless TMB be oxidized into blue oxTMB, with a characteristic absorbance peak at 652 nm. This result indicates that PRSA-Cu can act as a POD-like enzyme to catalyze the generation of highly toxic hydroxyl radicals (•OH) and effectively oxidize TMB. Figure 6 As shown in (b), the enzyme activity of PRSA-Cu was enhanced under 638nm laser irradiation. Figure 6 (c) and Figure 6 (d) The enzymatic activity of PRSA-Cu was shown to be highly dependent on the pH value of the solution and the concentration of PRSA-Cu, respectively.

[0043] (9) Type I photodynamic validation of PRSA-Cu like Figure 7As shown in Figure 1, PRSA-Cu is more intuitively seen from electron spin resonance (ESR) under laser irradiation. It can be seen that PRSA-Cu presents a typical •OH signal peak with a multi-peak distribution of 1:2:2:1, proving that hydroxyl radicals are generated under laser irradiation.

[0044] (10) Verification of GSH-Px activity of PRSA-Cu The ability of PRSA-Cu to consume GSH was detected using the source leaf R22075 reduced glutathione (GSH) detection kit, and the detection was performed using a UV-visible spectrophotometer. Figure 8 It can be seen that as the concentration of PRSA-Cu increases, the consumption of GSH also increases, and the GSH consumption capacity of PRSA-Cu shows concentration dependence.

[0045] Test Example 1: In vitro antibacterial test (1) Bacterial culture This experiment used two bacteria, Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli), and used second-generation bacteria to complete the following experiments. The specific culture method of the second-generation bacteria is: first revive the frozen bacteria, thaw the frozen bacteria at 37°C, take 100 µL of the bacterial solution in a shaking tube containing 5 mL of liquid culture medium, place it in a constant temperature shaker (110 rpm, 37°C) and culture it for 12 hours, take 100 µL of the cultured bacterial solution and place it in a 2 mL EP tube containing 900 µL of liquid culture medium, and then dilute it according to the gradient method of 10 -2 Dilute 5-10 tubes, take 100 µL of the bacterial solution in each tube, use a coating rod to evenly spread it on a culture dish containing solid culture medium, incubate at 37°C for 24 hours, observe the clone morphology and colony count, and take a culture dish with about 1000 colonies as the first generation of bacteria. Use a bacteria picker to pick out a colony from the first generation of bacteria and add it to a shaking tube containing 5 mL of liquid culture medium. Cultivate it according to the method of culturing first generation bacteria to obtain a culture dish with about 1000 colonies as the second generation of bacteria. The specific configuration method of liquid culture medium is: take 5g of LB broth, disperse it in 200 mL of distilled water, and then sterilize it by high pressure sterilization to obtain bacterial liquid culture medium. The specific configuration method of solid culture medium is: take 5g of LB broth and 3g of agar, disperse them in 200 mL of distilled water, and then sterilize them by high pressure sterilization to obtain solid culture medium.

[0046] (2) Determination of antibacterial activity of PRSA-Cu by plate count method The preparation method of the dispersion of bacteria in different concentrations of PRSA-Cu groups was as follows: 2 mg of PRSA-Cu powder was fully dispersed in 2 mL PBS to prepare a 1 mg / mL PRSA-Cu stock solution, and 10 µL of 10 8 CFU mL -1 Bacterial solution (S. aureus or E. coli), then add 980, 960, 940, 920, 900, 880 μL of PBS, add 10 μL of 30wt% H2O2 to each concentration, then add 0, 20, 40, 60, 80, 100 μL of 1 mg / mL PRSA-Cu stock solution, to obtain 0, 20, 40, 60, 80, 100 μg / mL PRSA-Cu and 10 μL of 1 mg / mL PRSA-Cu. 8 CFU mL -1 The mixed solution of PBS dispersion is the PRSA-Cu solution.

[0047] The concentrations of PRSA-Cu solutions were 0, 20, 40, 60, 80, and 100 μg / mL. 10 μL of 30 wt% H2O2 was added to each concentration and then laser irradiated (λ = 638 nm, 1.2 W / cm 2 The above solutions were placed in a constant temperature shaker (110 rpm, 37°C) for 12 h, and then the cultured bacterial solution was gradiently diluted 10 according to the bacterial culture method. 5 Transfer 100 μL of the blown bacterial solution to the solid culture medium and spread it evenly. Incubate at 37°C for 24 h, count the colonies and compare the bacterial activity of each group. Fig. 9 (a) ~ Fig. 9 As can be seen in (c), PRSA-Cu can achieve significant antibacterial effects at a concentration of 100 μg / mL, with antibacterial rates of 98.3% and 99.4% against Staphylococcus aureus and Escherichia coli, respectively.

[0048] (3) In vitro antibacterial activity of PRSA-Cu under different treatments The plate count method was used to study the antibacterial ability under different treatments. The experiment was divided into eight groups: (A) PBS (laser off), PBS (laser on), (B) H2O2 (laser off), H2O2 (laser on), (C) PRSA-Cu (laser off), PRSA-Cu (laser on), (D) PRSA-Cu+H2O2 (laser off), PRSA-Cu+H2O2 (laser on). The preparation method of co-culture solution in different groups was as follows: first, 2 mg of PRSA-Cu powder was taken and fully dispersed in 2 mL PBS to prepare a 1 mg / mL PRSA-Cu mother solution.

[0049] PBS (laser off) group: add 10 µL of 10 8 CFU mL -1 Then add 990 μL of PBS to the bacterial solution.

[0050] PBS (laser on): add 10 µL of 10 8 CFU mL -1 Bacterial solution, then add 990 μL of PBS, and then irradiate the dispersion with laser.

[0051] H2O2 (laser off) group: add 10 µL 10 8 CFU mL -1 Bacterial solution, add 980 μL of PBS, and then add 10 μL of 30wt% H2O2.

[0052] H2O2 (laser on) group: add 10 µL 10 8 CFU mL -1 The bacterial solution was then added with 980 μL of PBS, followed by 10 μL of 30 wt% H2O2, and the dispersion was irradiated with laser.

[0053] PRSA-Cu (laser off) group: add 10 µL of 10 8 CFU mL -1 Bacterial solution, then add 100 μL of 1 mg / mL PRSA-Cu stock solution, and finally add 890 μL PBS.

[0054] PRSA-Cu (laser on) group: add 10 μL of 10 8 CFUmL -1 To the bacterial solution, add 100 μL of 1 mg / mL PRSA-Cu stock solution, then add 890 μL of PBS, and finally irradiate the dispersion with a laser.

[0055] PRSA-Cu+ H2O2 (laser off) group: add 10 μL 10 8 CFUmL -1 To the bacterial solution, add 100 μL of 1 mg / mL PRSA-Cu stock solution, 10 μL of 30 wt % H2O2, and then 880 μL of PBS.

[0056] PRSA-Cu+ H2O2 (laser on) group: add 10 μL 10 8 CFUmL -1To the bacterial solution, add 100 μL of 1 mg / mL PRSA-Cu stock solution, add 10 μL of 30 wt% H2O2, and then add 880 μL of PBS, and finally irradiate the dispersion with a laser.

[0057] The parameters of the lasers in the above groups are: λ=638 nm, 1.2 W / cm 2 , 10min.

[0058] Then, the above groups were placed in a constant temperature shaker (110 rpm, 37°C) for 12 h, and then the cultured bacterial solution was gradiently diluted 10 according to the bacterial culture method. 5 Transfer 100 μL of the blown bacterial solution to the solid culture medium and spread it evenly. Incubate at 37°C for 24 h, count the colonies and compare the bacterial activity of each group. Fig. 9 As shown in (d), the bacteria in the H2O2 (laser off) group and the H2O2 (laser on) group had only a slight antibacterial effect compared with the PBS (laser off) group and the PBS (laser on) group. It can be seen from groups C and D that laser irradiation can enhance the bactericidal effect of PRSA-Cu. Specifically, comparing the colony counts (CFU) of Staphylococcus aureus and Escherichia coli, the PRSA-Cu (laser on) group significantly reduced the colony counts of Staphylococcus aureus and Escherichia coli to 49.8% and 1.8%, while the colony counts of Staphylococcus aureus and Escherichia coli in the PRSA-Cu (laser off) group were 93.3% and 92.7%, respectively. Compared with the PRSA-Cu (laser off) group, the colony counts of Staphylococcus aureus and Escherichia coli in the PRSA-Cu+H2O2 (laser off) group were also significantly reduced to 45.5% and 24.9% due to the synergistic effect. The sterilization efficiency of the PRSA-Cu + H2O2 (laser on) group was further improved, almost eliminating all bacteria, and the colony counts of Staphylococcus aureus and Escherichia coli were reduced to 3.9% and 0.2% respectively.

[0059] Test Example 2: Bacterial Transmission Electron Microscopy According to the method in Experimental Example 1 (3), bacterial solutions of PBS (laser off), PBS (laser on), H2O2 (laser off), H2O2 (laser on), PRSA-Cu (laser off), PRSA-Cu (laser on), PRSA-Cu + H2O2 (laser off), and PRSA-Cu + H2O2 (laser on) were prepared. Then, 100 μL of the bacterial solution was fixed in 2.5 wt% glutaraldehyde solution (4°C, 2 h), washed three times with PBS, embedded in agar and blocked. Then, the bacteria were treated with ethanol solution (30 wt %, 50 wt %, 70 wt %, 90 wt %, 95 wt % and 100 wt %) at room temperature for 10 min to dehydrate them, and then treated with acetone at room temperature for 3 h, embedded with embedding medium (epoxy resin) in gradient infiltration (immersed for 1 hour with acetone and epoxy resin in a mass ratio of 3:1, 1:1, and 1:3, and finally immersed in pure epoxy resin overnight), negatively stained, and sliced ​​on a nickel grid. The nickel grid was placed under TEM for observation to capture the bacterial morphology. TEM was used to observe the integrity of the bacterial membranes treated in different groups.

[0060] like Fig.10 As shown, the surface of Staphylococcus aureus and Escherichia coli treated with PBS (laser off), PBS (laser on), H2O2 (laser off) and H2O2 (laser on) was smooth, with complete cell membrane and flagella structure. However, the bacteria treated under different conditions of PRSA-Cu showed twisted or even broken cell membranes, indicating that PRSA-Cu has a significant antibacterial effect. Among these groups, the bacteria treated in the PRSA-Cu+H2O2 (laser on) group showed the most obvious morphological changes, with leakage of cell contents and complete destruction of cell membranes.

[0061] Test Example 3: Bacterial live / dead staining test SYTO-9 and PI are used to distinguish between live and dead microbial cells. SYTO-9 can penetrate all bacterial membranes (intact and damaged), thus marking the bacteria in green; PI only penetrates the injured bacterial membrane, marking the bacteria in red, while reducing the green color of SYTO-9.

[0062] According to the method in Experiment 1 (3), PBS (laser off), PBS (laser on), H2O2 (laser off), H2O2 (laser on), PRSA-Cu (laser off), PRSA-Cu (laser on), PRSA-Cu + H2O2 (laser off), and PRSA-Cu + H2O2 (laser on) were prepared. Then, 100 µL of the bacterial suspension of each group was mixed with 20 µL of SYTO-9 (1.0 × 10 -3 M) and 20 µL PI (1.5 × 10 -3M) and co-incubation was performed for 15 min at 37°C in the dark. After staining, each group was centrifuged in PBS to remove excess SYTO-9 and PI. The bacteria were then resuspended in 50 µL PBS and placed on the surface of the slide. Images of E. coli or S. aureus were then captured using a fluorescent inverted microscope.

[0063] from Fig.11 The significant PTT / CAT / POV synergistic antibacterial effect of PRSA-Cu can also be intuitively observed in the detection results of bacterial live and dead staining. The bacteria treated with PBS (laser off), PBS (laser on), H2O2 (laser off), and H2O2 (laser on) groups had strong green fluorescence, which was consistent with the results of the plate count method. In contrast, bacteria treated with PRSA-Cu (laser off), PRSA-Cu (laser on), and PRSA-Cu+H2O2 (laser off) groups showed obvious red fluorescence. At the same time, the PRSA-Cu+ H2O2 (laser on) group showed that almost all bacteria died, with the highest bactericidal efficiency, and all bacteria were stained with red fluorescence, indicating that a large number of bacteria died.

[0064] Test Example 4: In vitro biocompatibility test (1) Hemolysis test Fresh blood was collected from 5-week-old BALB / c female mice (purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.). After centrifugation at 10,000 rpm for 10 minutes, red blood cells were collected and washed with the same amount of PBS until colorless, and the supernatant was discarded. After diluting the red blood cells with PBS at a volume ratio of 3:11, different concentration gradients (50, 75, 100, 150, 200 μg / mL) of PRSA-Cu solution (volume ratio of red blood cell solution: PRSA-Cu solution = 1: 9) were added, incubated at 37°C for 3 hours, and centrifuged at 10,000 rpm for 10 minutes. Then 100 μL of supernatant from each group was placed in a 96-well plate, and the absorbance of each group was measured at 570 nm using an enzyme marker. Distilled water was used as the positive control, and PBS was used as the negative control. The formula for calculating the amount of hemolysis is as follows: Hemolysis volume (%) = (A-An) / (Ap-An) × 100%; Where "A" is the absorbance obtained by taking the supernatant after adding PRSA-Cu to erythrocytes. "An" is the absorbance obtained by taking the supernatant after adding PBS to erythrocytes (negative control). "Ap" is the absorbance obtained by taking the supernatant after adding distilled water to erythrocytes (positive control).

[0065] like Fig.12As shown, PRSA-Cu showed only very low hemolytic activity (less than 4%) or no hemolytic activity within the concentration range showing antibacterial activity. The hemolytic rate of PRSA-Cu varied with the concentration of PRSA-Cu, and as the concentration increased from 50 to 200 μg / mL, the hemolytic rate was less than 4%. This indicates that PRSA-Cu has good blood compatibility and does not damage the red blood cell membrane.

[0066] (2) Cytotoxicity assay Mouse 3T3 fibroblasts (from the Cell Bank of the Chinese Academy of Sciences) were cultured at 5 × 10 cells per well in a 96-well plate. 3 The cells were seeded at a density of 100 μL cells per well, with 180 μL cells in each well. 200 μL PBS was added to the surrounding duplicate wells for liquid sealing to prevent excessive evaporation. After incubation for 24 hours, 20 μL of PRSA-Cu solution with different concentration gradients was added to prepare a solution with a final concentration of 0, 50, 100, 150, 200, and 250 μg / mL, and incubated for another 24 hours. Then 20 μL of MTT (4 mg / mL) solution was added to each well. After incubation in the incubator for 4 hours, the supernatant was aspirated and 150 μL of dimethyl sulfoxide was added to dissolve MTT (tetramethyl blue azolate). After dissolving on a shaker for 10 minutes, the absorbance of the 96-well plate was measured at 570 nm using a microplate reader. Each group of experiments was repeated three times. Fig.13 As shown, after 24 h of culture, the survival rate of 3T3 cells treated with PRSA-Cu was above 80% even at a high concentration (200 μg / mL).

[0067] Experimental Example 5: In vivo wound healing experiment Five-week-old BALB / c mice (purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.) were randomly divided into 6 groups: PBS group (A), PRSA-Cu group (B), PRSA-Cu +H2O2 group (C), PRSA-Cu + laser group (D), and PRSA-Cu + H2O2 + laser group (E), with 6 mice in each group. Before surgery, the back hair of each mouse was shaved to form a wound with a diameter of 5 mm, and then infected with Staphylococcus aureus (1×10 6 CFU / mL) for 24 hours to establish the wound healing model. The mice were treated according to the requirements of different group settings, and the wound surface of each group of mice was recorded on the 1st, 3rd, 5th, 7th, and 9th days, and the changes in body weight were monitored. The changes in wound size were measured using an image analysis program (Image.J).

[0068] like Fig.14 As shown in the figure, the wound area of ​​mice in different groups gradually decreased with the extension of time. The healing degree of different groups was quite different after 9 days. Fig.15(a) It can be seen that among the 6 groups, the mice in the PRSA-Cu+H2O2+laser group showed the most significant advantages in wound recovery and skin regeneration, with a wound healing rate of over 95%, much higher than that in the PBS group (76.0%), PRSA-Cu group (86.6%), PRSA-Cu + laser group (87.7%), and PRSA-Cu +H2O2 group (86.7%). At the same time, compared with the body weight of the blank group (normal mice without any treatment), Fig.15 (b) It can be seen that no obvious weight change was detected during the entire treatment process, indicating that PRSA-Cu has good biosafety.

[0069] Histological analysis using hematoxylin and eosin (H&E) and Masson staining was performed to directly evaluate the healing of skin tissue after 9 days of treatment. Fig.16 As shown, the PBS-treated group showed obvious inflammatory cells and incomplete skin epidermis. In contrast, the PRSA-Cu-treated group showed varying degrees of skin structure regeneration. The PRSA-Cu+H2O2+laser group had the most significant effect in promoting wound healing, with visible collagen fibers and naturally mature epidermis, indicating that the wound was completely healed.

[0070] In addition, histological sections were collected and H&E staining was performed to evaluate the damage to internal organs (heart, liver, spleen, lung, and kidney) in different groups. Fig.17 As shown, no abnormal lesions or inflammatory manifestations were found in major organs, and no histological changes were observed. These results confirm that PRSA-Cu has good biosafety in vivo.

[0071] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A covalent organic framework antibacterial material based on o-phenanthroline, characterized in that: The covalent organic framework antibacterial material is an amorphous porous polymer formed by the coordination of PRSA and copper; the PRSA is 5,5'-[(1E,1'E)-({1,10-phenanthroline-5,6-diyl}bis(nitrile))bis(methylene)]bis(1,2,4-benzenetriol), and its structural formula is 。 2. The covalent organic framework antibacterial material according to claim 1, characterized in that: The covalent organic framework antibacterial material is prepared by the following method: PRSA and copper salt are added into a mixed solvent, and a solvothermal reaction is carried out under a protective atmosphere. After the reaction is completed, the covalent organic framework antibacterial material PRSA-Cu based on o-phenanthroline is obtained after washing and drying.

3. The covalent organic framework antibacterial material according to claim 2, characterized in that: The PRSA is prepared by the following method: (1) Dissolve 1,2,4-benzenetriol and triethyl orthoformate in tetrahydrofuran, add anhydrous aluminum chloride in portions under a protective atmosphere, and react at room temperature to obtain 2,4,5-trihydroxybenzaldehyde; (2) 2,4,5-trihydroxybenzaldehyde and 5,6-diamino-1,10-phenanthroline are dispersed in ethanol and subjected to a solvothermal reaction under a protective atmosphere to obtain PRSA.

4. The covalent organic framework antibacterial material according to claim 3, characterized in that: In step (1), the ratio of the added amounts of 1,2,4-benzenetriol, triethyl orthoformate and anhydrous aluminum chloride is 1.0 g:6.54 mL:1.628 g.

5. The covalent organic framework antibacterial material according to claim 3, characterized in that: In step (2), the molar ratio of 2,4,5-trihydroxybenzaldehyde to 5,6-diamino-1,10-phenanthroline is greater than 2:1; the temperature of the solvent thermal reaction is 80° C. and the time is 3 days.

6. The covalent organic framework antibacterial material according to claim 2, characterized in that: The copper salt is copper acetate; the protective atmosphere is argon.

7. The covalent organic framework antibacterial material according to claim 6, characterized in that: The molar ratio of PRSA to copper acetate is less than 3:

7.

8. The covalent organic framework antibacterial material according to claim 2, characterized in that: The mixed solvent is obtained by mixing DMF and distilled water in a volume ratio of 5:1; the temperature of the solvent thermal reaction is 100° C. and the time is 3 days.

9. Use of the covalent organic framework antibacterial material according to any one of claims 1 to 8 in the preparation of antibacterial drugs.

10. The use according to claim 9, characterized in that: The antibacterial drug has peroxidase activity and glutathione peroxidase activity.

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