A covalent organic framework antibacterial material based on o-phenanthroline and its application

Abstract

The present invention discloses a covalent organic framework antibacterial material based on o-phenanthroline and its application, belonging to the field of biomedicine technology. The covalent organic framework antibacterial material is obtained by the coordination reaction of 5,5'-[(1E,1'E)-({1,10-phenanthroline-5,6-diyl}bis(azeninyl))bis(methylene)]bis(1,2,4-benzenetriol) PRSA with copper to obtain a covalent organic framework antibacterial material PRSA-Cu based on o-phenanthroline. The PRSA-Cu prepared by the present invention can utilize photothermal capacity to combine simulated photothermal (PTT), photodynamic therapy (PDT), peroxidase (PODs) and glutathione peroxidase (GSH-Px) to achieve synergistic antibacterial effects. It can also utilize the inherent characteristics of the infection microenvironment to induce bacterial death and significantly accelerate the recovery of bacterial-infected wounds by regulating the microenvironment of the bacterial infection site.

Description

Technical Field

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

[0002] Pathogens are the primary cause of bacterial infections, which have become a devastating medical problem worldwide and a serious threat to human health. Currently, treatment for bacterial infections relies heavily on antibiotics, leading to a rapid increase in drug-resistant bacteria and even the emergence of superbugs, which in turn reduces treatment efficacy and even contributes to the spread of infectious diseases. In recent years, emerging nanozymes have emerged as a promising new generation of antibiotics due to their broad-spectrum antimicrobial activity, low toxicity, and resistance to 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, which attack bacterial membranes at weakly acidic infection sites, achieving a bactericidal effect. ROS are nonspecific in their bacterial killing and therefore do not require a specific target to exert their effect. Compared to antibiotics, ROS-based bactericidal methods can kill bacteria by destroying their cell membranes, DNA, proteins, and more, thereby avoiding the development of bacterial resistance.

[0004] O-phenanthroline, also known as 1,10-phenanthroline, is a commonly used metal chelator that can form complexes with various transition metals. Complexes formed with copper and their derivatives exhibit certain DNA-cleaving activity, making them useful as non-oxidative nucleases and, consequently, anticancer activities. For example, patent application number CN114106020A discloses a highly anticancer complex based on a pyrimidine Schiff base-copper complex of o-phenanthroline and its preparation method. This patent utilizes o-phenanthroline and copper to form a complex for anticancer purposes. Due to the many excellent properties of copper complexes, there have been reports of o-phenanthroline coordinated with copper for antibacterial purposes. Patent application number CN111808123A discloses the preparation of a solid antibacterial agent based on a copper complex, using a coordination polymer containing copper sulfate. However, this polymer is a crystalline material that only utilizes the antibacterial activity of copper, resulting in a limited antibacterial capacity and low efficacy. 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 can also use the intrinsic properties of the infection microenvironment (IME) to accurately treat and induce bacterial death. It can also significantly accelerate the recovery of bacterial-infected wounds by regulating the microenvironment of the bacterial infection site. Summary of the Invention

[0005] In response to the above-mentioned prior art, the present invention aims 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(azeninyl))bis(methylene)]bis(1,2,4-benzenetriol) (PRSA) coordinated with copper to form a porous polymer PRSA-Cu. This material utilizes photothermal capacity to combine simulated photothermal (PTT), photodynamic therapy (PDT), peroxidases (PODs), and glutathione peroxidase (GSH-Px) to achieve synergistic antibacterial effects. It can also utilize the inherent characteristics of the infection microenvironment to induce bacterial death and significantly accelerate the recovery of bacterial-infected wounds by regulating the microenvironment of the bacterial infection site.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention provides a covalent organic framework antibacterial material based on o-phenanthroline, 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

[0008] .

[0009] The 1,10-phenanthroline involved in the present invention can also be written as 1,10-phenanthroline, also known as o-phenanthroline.

[0010] Preferably, the covalent organic framework antibacterial material is prepared by the following method:

[0011] PRSA and copper salt are added to a mixed solvent and a solvent thermal 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.

[0012] The PRSA-Cu is an amorphous coordinated covalent organic framework polymer.

[0013] Preferably, the PRSA is prepared by the following method:

[0014] (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;

[0015] (2) 2,4,5-Trihydroxybenzaldehyde and 5,6-diamino-1,10-phenanthroline were dispersed in ethanol and subjected to solvent thermal reaction under protective atmosphere to obtain PRSA.

[0016] 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.

[0017] 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.

[0018] Preferably, the copper salt is copper acetate; and the protective atmosphere is argon.

[0019] Preferably, the molar ratio of PRSA to copper acetate is less than 3:7.

[0020] 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.

[0021] The second aspect of the present invention provides the use of a covalent organic framework antibacterial material in the preparation of an antibacterial drug.

[0022] Preferably, the antibacterial drug has peroxidase activity and glutathione peroxidase activity.

[0023] Beneficial effects of the present invention:

[0024] (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). This material utilizes photothermal capacity to combine and simulate PTT, PDT, PODs and GSH-Px treatments. It can not only induce bacterial death by utilizing the inherent characteristics of the infection microenvironment, but also significantly accelerate the recovery of bacterial-infected wounds by regulating the microenvironment of the bacterial infection site.

[0025] (2) The PRSA-Cu prepared by the present invention produces a good photothermal conversion effect through 638nm wavelength laser irradiation, and can also convert endogenous H2O2 into hydroxyl radicals. The enzyme activity is further increased after photothermal treatment. In addition, PRSA-Cu has high biocompatibility, with a lysis rate of less than 4% for red blood cells, little effect on the cell viability of 3T3 cells, and can promote wound healing, which is beneficial for its application in the biological field. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] 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;

[0027] 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;

[0028] Figure 3 : EDS spectrum of PRSA-Cu;

[0029] 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, and (e) O 1s spectrum;

[0030] Figure 5 : Photothermal properties 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 at different laser powers for 10 min; (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 versus negative natural logarithm of temperature;

[0031] Figure 6: Catalase-like activity of PRSA-Cu, including (a) absorbance of TMB, TMB + H2O2, PRSA-Cu + H2O2, H2O2, PRSA-Cu + TMB, and PRSA-Cu + H2O2 + TMB at 652 nm; (b) absorbance change of PRSA-Cu + H2O2 + TMB at 652 nm before and after 638 nm laser irradiation; (c) absorbance of PRSA-Cu (100 μg / mL) in 0.3% H2O2 at different pH environments; (d) absorbance of PRSA-Cu at different concentrations in 0.3% H2O2 at pH 5.5;

[0032] Figure 7 : Electron spin resonance spectrum (ESR) of PRSA-Cu;

[0033] Figure 8 : Glutathione oxidase detection kit was used to detect the consumption of glutathione at different concentrations of PRSA-Cu;

[0034] Figure 9 : 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 at different concentrations and quantitative diagram of PRSA-Cu + H2O2 + laser plate count method; (d) plate count method photos of Staphylococcus aureus and Escherichia coli treated with different treatment methods; (e) plate count method quantitative diagram of Staphylococcus aureus treated with different treatment methods; (f) plate count method quantitative diagram of Escherichia coli treated with different treatment methods;

[0035] Figure 10 : TEM images of bacteria, including (a) Staphylococcus aureus after different treatments, (b) TEM images of Escherichia coli after different treatments, the red tips in the figures represent the damage locations;

[0036] Figure 11 : Bacterial staining images, including (a) fluorescence images of Staphylococcus aureus after different treatments incubated with SYTO-9 / PI live / dead stain; (b) fluorescence images of Escherichia coli after different treatments incubated with SYTO-9 / PI live / dead stain;

[0037] Figure 12 : Hemolysis rate of different concentrations of PRSA-Cu (n=3, error bars indicate standard deviation);

[0038] Figure 13: Cell viability (%) after co-culture of 3T3 cells with different concentrations of PRSA-Cu (n=3, error bars represent standard deviation);

[0039] Figure 14 : Wound images of mice in different groups on days 1, 3, 5, 7, and 9 after treatment;

[0040] Figure 15 :(a) Wound healing rate of mice (%);(b) Changes in mouse body weight during treatment;

[0041] Figure 16 : H&E and Masson staining were used for histological analysis of each group;

[0042] Figure 17 : Staining results of organs treated in each group;

[0043] Figure 18 : Synthesis route of PRSA-Cu. DETAILED DESCRIPTION

[0044] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0045] As introduced in the background technology section, the antibacterial ability of the currently reported 1,2-phenanthroline-copper coordination polymers is single, and the antibacterial effect needs to be improved.

[0046] Based on this, the present invention aims to provide a covalent organic framework antibacterial material based on o-phenanthroline and its application. This invention utilizes a pocket-shaped 5,5'-[(1E,1'E)-({1,10-phenanthroline-5,6-diyl}bis(azeninyl))bis(methylene)]bis(1,2,4-benzenetriol) as the main framework, coordinated with copper to form a porous polymer PRSA-Cu. PRSA is typically prepared from 2,4,5-trihydroxybenzaldehyde and 5,6-diamino-1,10-phenanthroline in a molar ratio of 2:1. However, to avoid an incomplete reaction and the formation of other byproducts, the present invention adds 5,6-diamino-1,10-phenanthroline dropwise to 2,4,5-trihydroxybenzaldehyde in an excess of 2,4,5-trihydroxybenzaldehyde, resulting in a molar ratio of 2,4,5-trihydroxybenzaldehyde to 5,6-diamino-1,10-phenanthroline greater than 2:1. The molar ratio of PRSA to copper acetate should be 1:2.33 or 3:7, but in order to ensure that all copper can be coordinated on PRSA, copper acetate needs to be in excess, so the molar ratio of PRSA to copper acetate is less than 3:7.

[0047] The copper contained in PRSA-Cu can convert endogenous H2O2 into highly toxic •OH, thereby effectively regulating the infection microenvironment. PRSA-Cu can achieve localized temperature increases through photothermal conversion, effectively rupturing bacterial membranes. In summary, the inherent photothermal and photokinetic properties of this material, combined with peroxidase-mimicking (PODs) and GSH-Px therapy, can serve as an intelligent platform. This platform can not only leverage the intrinsic properties of the infection microenvironment (IME) to precisely induce bacterial death, but also significantly accelerate the recovery of bacterially infected wounds by regulating the microenvironment at the site of bacterial infection. Furthermore, at its optimal antibacterial concentration, PRSA-Cu exhibits virtually no hemolytic effect and has little effect on normal cell growth, making it promising for biological applications.

[0048] 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 with reference to specific embodiments.

[0049] 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.

[0050] Example 1: Preparation of PRSA-Cu

[0051] (1) Preparation of 2,4,5-trihydroxybenzaldehyde: 1.0 g of 1,2,4-benzenetriol 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 3 M 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.

[0052] (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, PRSA was washed with methanol and ethanol and dried.

[0053] (3) Preparation of PRSA-Cu: 96 mg of PRSA and 140 mg of copper acetate were added to a mixed solution consisting of 12 mL of 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 under vacuum at 100°C overnight to obtain PRSA-Cu. See the synthetic route for details. Figure 18 .

[0054] Example 2: Characterization of PRSA-Cu

[0055] (1) Determination of infrared spectrum

[0056] The structures of PRSA and PRSA-Cu were determined by infrared spectroscopy. Figure 1 As shown in (a), the OH (3300 cm -1 ) stretching vibration band almost disappears, and a new Cu-O (490 cm -1 ) stretching vibration band, the presence of a broad peak can be attributed to the formation of an amorphous coordination polymer. After the complexation of PRSA and metal ions, a unique broad peak appears 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.

[0057] (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.

[0058] (3) The thermal stability of PRSA-Cu was investigated by thermogravimetric analysis. The thermal stability of the synthesized material under N2 atmosphere was investigated by thermogravimetric analysis (TGA). Figure 1 (d) shows that the first mass loss of the PRSA-Cu material is less than 16.9% (<100°C), while the second mass loss occurs above 200°C. The former is attributed to the evaporation of water absorbed in the highly polar porous framework, while the latter is attributed to the decomposition of the porous network. The weight at 800°C remains at 53.66%, indicating the excellent thermal stability of the prepared material.

[0059] (4) The crystallinity and porosity of PRSA-Cu were estimated by powder X-ray diffraction (PXRD) measurement. Figure 1(e) shows a large broad peak at around 25°, which can be attributed to the formation of amorphous coordination polymer.

[0060] (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) reveals that PRSA-Cu is a typical bulk material composed of irregular particles with a uniformly distributed, interconnected macroporous structure. Its rough surface facilitates bacterial adhesion. Transmission electron microscopy (TEM) further reveals that PRSA-Cu has a continuous hierarchical pore structure composed of loosely packed particles and exhibits clear hierarchical porosity.

[0061] Figure 2 (f) High-angle annular dark field scanning transmission electron microscopy (HAADF-STEM) was used to detect the elemental composition and distribution of PRSA-Cu. 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.

[0062] (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 higher Cu + 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) The O1s spectra of PRSA-Cu show that there are two types of O species: O-Cu (530.82 eV) and OC (532.57 eV).

[0063] (7) Photothermal performance of PRSA-Cu

[0064] 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 fully disperse it in 1 mL of distilled water using an ultrasonic instrument to prepare a 1 mg / mL mother solution. Then, 50, 100, 200, 300, and 400 μL of the mother solution were respectively drawn into 950, 900, 800, 700, and 600 μL of distilled water to finally prepare 50, 100, 200, 300, and 400 μg / mL of PRSA-Cu aqueous dispersion. Using pure water as a control, the 638 nm laser (1.2 W / cm 2 , 10 min) to monitor the temperature changes of PRSA-Cu with different concentrations, and preliminarily study the photophysical properties of the synthesized samples.

[0065] like Figure 5 As shown in (a), unlike pure water where the temperature change 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 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. In order 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.

[0066] Furthermore, the photothermal conversion efficiency of PRSA-Cu is η (%) = 49.7%. This efficiency was calculated using the formula described in CN115845086A, "A Photothermal-Fenton-like Artificial Nanozyme, Its Preparation Method, and Application." Figure 5 (e) shows the τS and θ values ​​in the light-to-thermal conversion efficiency calculation formula.

[0067] (8) Peroxidase activity of PRSA-Cu

[0068] The peroxidase activity of PRSA-Cu was studied at different pH levels. The ROS-generating capacity of PRSA-Cu was evaluated using a dual-substrate system: H₂O₂ and 3,3',5,5'-tetramethylbenzidine (TMB), with TMB serving as the chromogenic reagent. TMB is oxidized by ROS to form chromogenic ox-TMB. The presence of copper endows PRSA-Cu with the ability to efficiently generate •OH in acidic media.

[0069] The TMB solution used in the experiment was prepared as follows: 3.606 mg of TMB (0.015 mmol) was weighed and dissolved in 10 mL of ethanol to prepare a 1.5 mmol / L TMB ethanol solution. The H₂O₂ concentration was 30%. 50 mL of PBS (pH 7.4) was placed in a test tube and phosphoric acid was added 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 an ultrasonicator 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, draw 100 μL of the stock solutions into 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 each of TMB and H2O2, 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 of 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.

[0070] like Figure 6As shown in (a), only in the culture medium containing both PRSA-Cu and H2O2 (PRSA-Cu + H2O2 + TMB) can the colorless TMB be oxidized to blue oxTMB, with a characteristic absorbance peak at 652 nm. This result indicates that PRSA-Cu can act as a POD-like enzyme, catalyzing the generation of highly toxic hydroxyl radicals (•OH) and effectively oxidizing TMB. Figure 6 As shown in (b), the enzyme activity of PRSA-Cu is enhanced under 638 nm 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.

[0071] (9) Type I photodynamic validation of PRSA-Cu

[0072] like Figure 7 As shown in Figure 2, PRSA-Cu is more intuitively observed from electron spin resonance (ESR) under laser irradiation. It can be seen that PRSA-Cu exhibits a typical •OH signal peak with a multi-peak distribution of 1:2:2:1, proving that hydroxyl radicals are generated under laser irradiation.

[0073] (10) Verification of GSH-Px activity of PRSA-Cu

[0074] The ability of PRSA-Cu to consume GSH was detected using the source leaf R22075 reduced glutathione (GSH) detection kit, and the detection and graphing were 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.

[0075] Test Example 1: In vitro antibacterial test

[0076] (1) Bacterial culture

[0077] 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 as follows: first, thaw the frozen bacteria at 37°C, take 100 μL of the bacterial solution into a shaking tube containing 5 mL of liquid culture medium, place it on a constant temperature shaker (110 rpm, 37°C) and culture for 12 hours, take 100 μL of the cultured bacterial solution into a 2 mL EP tube containing 900 μL of liquid culture medium, and then dilute it according to the gradient method of 10 -2Dilute 5-10 tubes, take 100 µL of the bacterial solution from each tube, and evenly spread it onto a Petri dish containing solid culture medium using a spreading stick. Incubate at 37°C for 24 hours. Observe the colony morphology and colony count. The dish containing approximately 1000 colonies is considered the first generation. Use a pick stick to pick a colony from the first generation and transfer it to a shake tube containing 5 mL of liquid culture medium. Culture the same way as for the first generation until a dish containing approximately 1000 colonies is considered the second generation. Liquid culture medium is prepared as follows: Disperse 5g of LB broth in 200 mL of distilled water and sterilize by autoclaving to obtain liquid culture medium. Solid culture medium is prepared as follows: Disperse 5g of LB broth and 3g of agar in 200 mL of distilled water and sterilize by autoclaving to obtain solid culture medium.

[0078] (2) Determination of antibacterial activity of PRSA-Cu by plate count method

[0079] The preparation method of the bacterial dispersion solution of 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 ... 8 CFU mL -1 Bacterial solution (S. aureus or E. coli) was added, and then 980, 960, 940, 920, 900, and 880 μL of PBS was added, 10 μL of 30 wt% H2O2 was added to each concentration, and then 0, 20, 40, 60, 80, and 100 μL of 1 mg / mL PRSA-Cu stock solution was added, respectively, to obtain PRSA-Cu with a concentration of 0, 20, 40, 60, 80, and 100 μL. 8 CFU mL -1 The mixed solution of PBS dispersion is the PRSA-Cu solution.

[0080] The concentrations of PRSA-Cu solution were 0, 20, 40, 60, 80, and 100 μg / mL. 10 μL of 30 wt% H2O2 was added to each concentration, and then laser irradiation (λ = 638 nm, 1.2 W / cm 2 , 10min) the above solutions, the above solutions were placed in a constant temperature shaker (110 rpm, 37 ° C) for 12h, and then the cultured bacterial solution was gradient diluted 10 5 Transfer 100 μL of the blown bacterial solution to the solid culture medium and spread it evenly. Incubate at 37°C for 24 hours, count the colonies and compare the bacterial activity of each group. Figure 9 (a) ~ Figure 9As 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.

[0081] (3) In vitro antibacterial activity of PRSA-Cu under different treatments

[0082] The plate count method was used to investigate the antibacterial activity of 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), and PRSA-Cu + H2O2 (laser on). The co-culture solutions for the different groups were prepared as follows: 2 mg of PRSA-Cu powder was thoroughly dispersed in 2 mL of PBS to create a 1 mg / mL PRSA-Cu stock solution.

[0083] PBS (laser off) group: add 10 µL of 10 8 CFU mL -1 Then add 990 μL of PBS to the bacterial solution.

[0084] PBS (laser on): add 10 µL of 10 8 CFU mL -1 Bacterial solution, then 990 μL of PBS was added, and the dispersion was irradiated with laser.

[0085] H2O2 (laser off) group: add 10 µL of 10 8 CFU mL -1 Bacterial solution, 980 μL of PBS, and then 10 μL of 30wt% H2O2 were added.

[0086] H2O2 (laser on) group: add 10 µL of 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.

[0087] 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 of PBS.

[0088] 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.

[0089] PRSA-Cu+H2O2 (laser off) group: add 10μL10 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.

[0090] PRSA-Cu+H2O2 (laser on) group: add 10μL10 8 CFUmL -1 To 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.

[0091] The laser parameters in the above groups are: λ = 638 nm, 1.2 W / cm 2 , 10 minutes.

[0092] Then, the above groups were placed in a constant temperature shaker (110 rpm, 37°C) and cultured for 12 hours, and then the cultured bacterial solution was gradient diluted 10 times 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 hours, count the colonies and compare the bacterial activity of each group. Figure 9As shown in (d), the H2O2 (laser off) and H2O2 (laser on) groups had only a slight antibacterial effect on bacteria compared to the PBS (laser off) and PBS (laser on) groups. As shown in Groups C and D, laser irradiation enhanced 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 CFU of S. aureus and E. coli to 49.8% and 1.8%, respectively, while the CFU of S. aureus and E. coli in the PRSA-Cu (laser off) group were 93.3% and 92.7%, respectively. Due to a synergistic effect, the CFU of S. aureus and E. coli in the PRSA-Cu + H2O2 (laser off) group were also significantly reduced to 45.5% and 24.9%, respectively, compared to the PRSA-Cu (laser off) group. 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.

[0093] Test Example 2: Bacterial Transmission Electron Microscopy

[0094] 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 according to the method in Experiment 1 (3). Subsequently, 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. The bacteria were then treated with ethanol solutions (30 wt%, 50 wt%, 70 wt%, 90 wt%, 95 wt%, and 100 wt%) for 10 minutes at room temperature to dehydrate. The bacteria were then treated with acetone for 3 hours at room temperature and embedded in a gradient infiltration of embedding medium (epoxy resin) (using a 3:1, 1:1, and 1:3 mass ratio of acetone to epoxy resin for 1 hour, followed by overnight infiltration with pure epoxy resin). Negative staining was performed and sections were mounted on nickel grids. The nickel grids were then placed under a TEM to capture bacterial morphology. TEM was used to examine the integrity of the bacterial membranes from the different treatment groups.

[0095] like Figure 10As shown, Staphylococcus aureus and Escherichia coli treated with PBS (laser off), PBS (laser on), H2O2 (laser off), and H2O2 (laser on) exhibited smooth surfaces with intact cell membranes and flagella. In contrast, bacteria treated with PRSA-Cu under different conditions exhibited distorted and even ruptured cell membranes, demonstrating a significant antibacterial effect of PRSA-Cu. Among these groups, bacteria treated with PRSA-Cu + H2O2 (laser on) exhibited the most pronounced morphological changes, with leakage of cellular contents and complete cell membrane destruction.

[0096] Test Example 3: Bacterial live / dead staining test

[0097] 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 green; PI only penetrates damaged bacterial membranes, marking the bacteria red, while reducing the green color of SYTO-9.

[0098] 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 in each group was mixed with 20 μL of SYTO-9 (1.0×10 -3 M) and 20 µL PI (1.5 × 10 -3 M) and incubated in the dark at 37°C for 15 minutes. After staining, each group was centrifuged in PBS to remove excess SYTO-9 and PI. The bacteria were then resuspended in 50 µL of PBS and placed on the surface of a glass slide. Images of E. coli or S. aureus were then captured using an inverted fluorescence microscope.

[0099] from Figure 11 The significant PTT / CAT / POV synergistic antibacterial effect of PRSA-Cu was also visually observed in the live and dead bacterial staining results. Bacteria treated with PBS (laser off), PBS (laser on), H2O2 (laser off), and H2O2 (laser on) exhibited strong green fluorescence, consistent with the plate count results. In contrast, bacteria treated with PRSA-Cu (laser off), PRSA-Cu (laser on), and PRSA-Cu + H2O2 (laser off) exhibited distinct red fluorescence. Furthermore, the PRSA-Cu + H2O2 (laser on) group demonstrated the highest bactericidal efficiency, showing the death of nearly all bacteria. All bacteria stained red, indicating substantial bacterial death.

[0100] Test Example 4: In vitro biocompatibility test

[0101] (1) Hemolysis test

[0102] 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 of PRSA-Cu solution (50, 75, 100, 150, 200 μg / mL) were added (the volume ratio of red blood cell solution: PRSA-Cu solution = 1:9), 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:

[0103] Hemolysis volume (%) = (A-An) / (Ap-An) × 100%;

[0104] Where "A" is the absorbance of the supernatant after adding PRSA-Cu to erythrocytes. "An" is the absorbance of the supernatant after adding PBS to erythrocytes (negative control). "Ap" is the absorbance of the supernatant after adding distilled water to erythrocytes (positive control).

[0105] like Figure 12 As shown, PRSA-Cu exhibited very low hemolytic activity (less than 4%) or no hemolytic activity within the concentration range where it exhibited antimicrobial activity. The hemolytic rate of PRSA-Cu varied with PRSA-Cu concentration, remaining below 4% as the concentration increased from 50 to 200 μg / mL. This suggests that PRSA-Cu has good hemocompatibility and does not damage erythrocyte membranes.

[0106] (2) Cytotoxicity experiment

[0107] 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. 3The cells were seeded at a density of 180 μL per well, and 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. Figure 13 As shown in the figure, 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).

[0108] Experimental Example 5: In vivo wound healing experiment

[0109] Five-week-old BALB / c mice (purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.) were randomly divided into six 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 six mice in each group. Before surgery, the back of each mouse was shaved, a 5 mm diameter wound was created, and then the mice were infected with Staphylococcus aureus (1×10 6 CFU / mL) for 24 hours to establish a wound healing model. Mice were treated according to the requirements of different group settings. The wound surface of each group of mice was recorded on days 1, 3, 5, 7, and 9, and body weight changes were monitored. Changes in wound size were measured using an image analysis program (Image.J).

[0110] like Figure 14 As shown in the figure, the wound area of ​​mice in different groups gradually decreased with time. The degree of healing after 9 days was quite different between different groups. Figure 15 (a) It can be seen that among the six 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 of 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), Figure 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.

[0111] 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. Figure 16 As shown, the PBS-treated group showed significant inflammatory cells and an incomplete epidermis. In contrast, the PRSA-Cu-treated groups demonstrated varying degrees of skin structural regeneration. The PRSA-Cu + H2O2 + laser group had the most significant effect on wound healing, with visible collagen fibers and a naturally mature epidermal layer, indicating complete wound healing.

[0112] 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. Figure 17 As shown, no abnormal lesions or inflammation were observed in major organs, and no histological changes were found. These results confirm that PRSA-Cu has good biosafety in vivo.

[0113] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection 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 PRSA coordinated with copper; the PRSA is 5,5'-[(1E,1'E)-({1,10-phenanthroline-5,6-diyl}bis(azene))bis(methylene)]bis(1,2,4-benzenetriol), and its structural formula is ； PRSA and copper salt are added to a mixed solvent and subjected to a solvothermal reaction 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. The copper salt is copper acetate; the protective atmosphere is argon; and the molar ratio of PRSA to copper acetate is less than 3:

7.

2. The covalent organic framework antibacterial material according to claim 1, characterized in that: The PRSA was 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 were dispersed in ethanol and subjected to solvent thermal reaction under protective atmosphere to obtain PRSA.

3. The covalent organic framework antibacterial material according to claim 2, 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.

4. The covalent organic framework antibacterial material according to claim 2, 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.

5. The covalent organic framework antibacterial material according to claim 1, 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.

6. Use of the covalent organic framework antibacterial material according to any one of claims 1 to 5 in the preparation of antibacterial drugs, characterized in that: The antibacterial drug has photothermal activity, photodynamic activity, peroxidase activity and glutathione peroxidase activity.

Images