A bimetallic organic framework multifunctional enzyme and its preparation method and application

Abstract

The present invention discloses a bimetallic organic framework multifunctional enzyme and its preparation method and application, belonging to the technical field of biomedicine. The bimetallic organic framework multifunctional enzyme is obtained by copolymerizing a copper-based cyclic trinuclear complex and a gold-based cyclic trinuclear complex through a Schiff base condensation reaction. The copper-based cyclic trinuclear complex is obtained by a solvothermal reaction of Cu2O and 3,5-dimethyl-1H-pyrazol-4-amine; the gold-based cyclic trinuclear complex is obtained by first preparing 4-(3,5-dimethyl-1H-pyrazol-4-yl)benzaldehyde and then reacting it with AuCl4H. The COF-CTU-AuCu of the present invention exhibits triple enzyme activities: it can simulate CAT, GSHOx, and POD for antibacterial and wound-healing treatment of infected wounds. In addition, COF-CTU-AuCu can also be used as a photothermal agent to generate local heating under red light irradiation, and laser irradiation significantly enhances the therapeutic effect of the combined antibacterial therapy.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a bimetallic organic framework multifunctional enzyme and a preparation method and application thereof. Background Art

[0002] Overuse and misuse of antibiotics have led to the rapid emergence of antibiotic resistance worldwide, and the antibiotic crisis has highlighted the urgent need for innovative antibiotics. The emerging antibiotic crisis urgently requires the development of new antimicrobial agents and antimicrobial strategies. Artificial enzymes have attracted widespread attention in the biomedical field due to their specificity and lesion site specificity.

[0003] To date, a variety of artificial enzymes have been developed that have shown significant effects against both Gram-negative and Gram-positive bacteria in a variety of animal models. The most notable feature of artificial enzymes is their remarkable versatility, which enables them to cleverly integrate multiple therapeutic modalities into a single entity, thereby achieving synergistic therapeutic effects and significantly improving therapeutic efficiency. However, the practical application of artificial enzymes also faces many limitations and challenges. For example, the peroxidase (POD)-like enzymes that are currently most commonly used to combat pathogens can convert endogenous hydrogen peroxide (H2O2) into highly toxic hydroxyl radicals (•OH) at pathological weakly acidic infection sites, thereby achieving in situ bacterial inactivation. However, in neutral or strongly acidic environments, their antibacterial effects are significantly reduced, resulting in a weakened effect in disrupting the redox homeostasis of bacterial cells. The catalytic rates of most simulated PODs are very limited at the infection site (pH 5.5~6.5), which greatly affects their biological activity. At the same time, although the infection-induced H2O2 levels are elevated compared to normal tissues, they are still insufficient to support enzyme therapies that rely on the generation of hydroxyl radicals (•OH) from H2O2. In addition, abnormally high levels of glutathione (GSH) in infected tissues will scavenge the reactive oxygen species (ROS) produced, severely hindering the efficacy of ROS-based treatments. The protective effect of biofilms further reduces the sensitivity of treatment, thereby exacerbating the reduction in treatment efficiency. At the same time, the hypoxic microenvironment of infected wounds also greatly limits the wound healing process. Therefore, the rational design and construction of multifunctional artificial enzymes can not only optimize the simulated POD-like activity in the infected microenvironment, but also simulate CAT-like enzyme activity to release oxygen to improve the hypoxic microenvironment of infected wounds, while consuming glutathione (GSH) produced by oxidative stress and eliminating biofilms, which is crucial to improving treatment efficiency.

[0004] Covalent organic frameworks (COFs) are porous crystalline materials composed of organic monomers connected by covalent bonds. They have high specific surface area, crystallinity, porosity, stability and tunability, making them very attractive in a variety of applications such as drug delivery, phototherapy, radiotherapy and immunotherapy. The antibacterial effect can be further enhanced by introducing metal active centers into the COF structure to form metal covalent organic framework polymers (MCOFs). However, existing metal covalent organic framework polymers rarely have the effects of multiple enzymes, and generally they need to be compounded with other substances to have the effects of multiple enzymes. Therefore, it is very necessary to develop a metal covalent organic framework polymer with multiple enzyme effects. Summary of the invention

[0005] In view of the above-mentioned prior art, the purpose of the present invention is to provide a bimetallic organic framework multifunctional enzyme and its preparation method and application. The bimetallic organic framework multifunctional enzyme (COF-CTU-AuCu) of the present invention is obtained by copolymerization of a copper-based cyclic trinuclear complex and a gold-based cyclic trinuclear complex. COF-CTU-AuCu exhibits triple enzyme activity: it can simulate CAT, GSHOX and POD to perform antibacterial and healing treatment of infected wounds. In addition, COF-CTU-AuCu can also be used as a photothermal agent to produce local heating under red light irradiation, and laser irradiation significantly enhances the therapeutic effect of combined antibacterial therapy.

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

[0007] The first aspect of the present invention provides a method for preparing a bimetallic organic framework multifunctional enzyme, comprising the following steps:

[0008] S1 Synthesis of Cu-CTC-NH2: Dissolve Cu2O and 3,5-dimethyl-1H-pyrazol-4-amine in a mixed solvent, heat to react, collect the yellow solid, wash and dry to obtain Cu-CTC-NH2;

[0009] S2.1 Synthesis of Au-CTC-CHO: Dissolve 3,5-dimethylpyrazole-4-boronic acid pinacol ester in CHCl3, add triethylamine and reflux; then add triphenylmethane dissolved in chloroform, continue to reflux, cool, wash and dry to obtain a yellow solid;

[0010] S2.2 Add the yellow solid, 4-bromobenzaldehyde, cesium carbonate and tetrakis(triphenylphosphine)palladium into a mixed solvent, stir and heat to react to obtain a brown liquid, extract and purify to obtain a light yellow solid;

[0011] S2.3 Dissolve the light yellow solid in a solvent containing hydrochloric acid, stir and heat to react, and sequentially neutralize, extract and purify the reaction solution to obtain a white solid 4-(3,5-dimethyl-1H-pyrazol-4-yl)benzaldehyde;

[0012] S2.4 Add 4-(3,5-dimethyl-1H-pyrazol-4-yl)benzaldehyde to the solvent and mix, then add AuCl4H and stir to react in the dark, filter, wash and dry to obtain a purple-gray solid, which is Au-CTC-CHO;

[0013] S3 Synthesis of bimetallic organic framework multifunctional enzyme: Add acetic acid, Cu-CTC-NH2 and Au-CTC-CHO into a mixed solvent, heat the reaction to obtain a dark brown solid, and wash and dry to obtain a bimetallic organic framework multifunctional enzyme.

[0014] Preferably, in step S1, the molar ratio of Cu2O to 3,5-dimethyl-1H-pyrazole-4-amine is 1:3; and the mixed solvent is obtained by mixing ethanol and pyridine in a volume ratio of 10:1.

[0015] Preferably, in step S1, the temperature of the heating reaction is 120° C., and the heating reaction time is 72 hours.

[0016] Preferably, in step S2.1, the molar ratio of 3,5-dimethylpyrazole-4-boronic acid pinacol ester to triphenylmethane is 1:1; and the reflux temperature is 50°C.

[0017] Preferably, in step S2.2, the molar ratio of the yellow solid, 4-bromobenzaldehyde, cesium carbonate and tetrakis(triphenylphosphine)palladium is 2.15:1.7:6.9:0.75; and the mixed solvent is obtained by mixing 1,4-dioxane and H2O in a volume ratio of 5:1.

[0018] Preferably, in step S2.3, the hydrochloric acid-containing solvent is obtained by mixing CH2Cl2, methanol and hydrochloric acid solution in a volume ratio of 10:3:3; the concentration of the hydrochloric acid solution is 2M; the heating temperature is 50°C and the heating time is 8h.

[0019] Preferably, in step S2.4, the molar ratio of 4-(3,5-dimethyl-1H-pyrazol-4-yl)benzaldehyde to AuCl4H is 1:1; the solvent is obtained by mixing tetrahydrofuran and triethylamine in a mass ratio of 30:1; and the light-proof stirring reaction time is 12 hours.

[0020] Preferably, in step S3, the molar ratio of Cu-CTC-NH2 to Au-CTC-CHO is 1:1; the mixed solvent is obtained by mixing trimethylbenzene and 1,4-dioxane in a volume ratio of 1:1; and the concentration of acetic acid is 6M.

[0021] The second aspect of the present invention provides a bimetallic organic framework multifunctional enzyme obtained by the above preparation method, wherein the bimetallic is Cu and Au; the bimetallic organic framework multifunctional enzyme is obtained by copolymerization of a copper-based cyclic trinuclear complex and a gold-based cyclic trinuclear complex.

[0022] Preferably, the copper-based cyclic trinuclear complex is Cu-CTC-NH2; and the gold-based cyclic trinuclear complex is Au-CTC-CHO.

[0023] The third aspect of the present invention provides the use of a bimetallic organic framework multifunctional enzyme in the preparation of an antibacterial drug, wherein the antibacterial drug achieves antibacterial and healing-promoting treatment of infected wounds by simulating the effects of catalase, glutathione oxidase and peroxidase.

[0024] Beneficial effects of the present invention:

[0025] (1) The bimetallic organic framework multifunctional enzyme (COF-CTU-AuCu) of the present invention is obtained by copolymerization of a copper-based cyclic trinuclear complex and a gold-based cyclic trinuclear complex; COF-CTU-AuCu exhibits triple enzyme activity: it can simulate CAT, GSHOX and POD to perform antibacterial and healing treatment of infected wounds. In addition, COF-CTU-AuCu can also be used as a photothermal agent to produce local heating under red light irradiation, and laser irradiation significantly enhances the therapeutic effect of combined antibacterial therapy.

[0026] (2) The COF-CTU-AuCu prepared by the present invention seamlessly combines mild photothermal therapy with multifunctional enzyme activity, demonstrating significant multimodal synergistic bactericidal ability at low doses, alleviating hypoxia, ultimately enhancing antibacterial and anti-biofilm capabilities, and significantly promoting the healing of infected wounds while minimizing adverse reactions. The triple catalytic cascade enzyme reaction of the COF-CTU-AuCu prepared by the present invention breaks through the limitations of enzyme therapy, and COF-CTU-AuCu has the potential to be a IME (immune microenvironment) responsive multifunctional wound healing material, thus opening up new avenues for related clinical applications.

[0027] (3) Under 638 nm near-infrared laser irradiation, COF-CTU-AuCu can generate high heat for photothermal therapy, while enhancing its catalytic activity, promoting •OH generation, effectively destroying bacterial cell membranes and inhibiting bacterial growth. In addition, it can effectively alleviate hypoxia in the infection environment and promote angiogenesis. In vivo anti-infection treatment further confirmed its strong wound healing ability, biosafety, effective inflammation reduction and angiogenesis promotion. Therefore, COF-CTU-AuCu, as a multifunctional nanozyme, has strong antibacterial and wound healing properties, meets the therapeutic needs of bacterial clearance and tissue repair, and provides new insights into the application of nanomaterials in medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 :(a) IR spectra of Au-CTC-CHO, Cu-CTC-NH2 and COF-CTU-AuCu;(b) IR spectra of COF-CTU-AuCu 13 C solid-state NMR spectrum; (c) XRD pattern of COF-CTU-AuCu; (d) N2 adsorption / desorption curve of COF-CTU-AuCu; (e) Pore size distribution of COF-CTU-AuCu; (f) Thermogravimetric analysis (TG) diagram of COF-CTU-AuCu; (g) XPS measurement spectrum of C element in COF-CTU-AuCu, (h) XPS measurement spectrum of Au element in COF-CTU-AuCu; (i) XPS measurement spectrum of Cu element in COF-CTU-AuCu

[0029] Figure 2 :(a) SEM of COF-CTU-AuCu, scale bar is 1 μm; (b) SEM of COF-CTU-AuCu, scale bar is 500 nm; (c) SEM of COF-CTU-AuCu, scale bar is 100 nm; (d) TEM of COF-CTU-AuCu, scale bar is 500 nm; (e) TEM of COF-CTU-AuCu, scale bar is 200 nm; (f) TEM of COF-CTU-AuCu, scale bar is 50 nm; (g) HR-TEM of COF-CTU-AuCu, scale bar is 10 nm; (h) HR-TEM of COF-CTU-AuCu, scale bar is 5 nm; (i) HR-TEM of another position of COF-CTU-AuCu, scale bar is 5 nm; (j) dark field element spectrum of COF-CTU-AuCu; (k) Au element spectrum of COF-CTU-AuCu; (i) Cu element spectrum of COF-CTU-AuCu; (m) C element spectrum of COF-CTU-AuCu; (n) N element spectrum of COF-CTU-AuCu; (o) O element spectrum of COF-CTU-AuCu; (p) EDS map of COF-CTU-AuCu;

[0030] Figure 3 :(a) COF-CTU-AuCu with different concentrations under laser irradiation (638 nm, 1.0 W / cm 2 ) under different laser powers (638 nm, 100 μg / mL); (c) infrared thermal imaging of COF-CTU-AuCu (1.0 W / cm 2); (d) Temperature curve of COF-CTU-AuCu under four ON-OFF laser irradiation; (e) Temperature curve of COF-CTU-AuCu (100 μg / mL) under laser irradiation (638 nm, 1.0 W / cm 2 ,) and the negative natural logarithm relationship between cooling time and temperature;

[0031] Figure 4 :(a)UV-visible spectra of TMB, TMB+H2O2, TMB+COF-CTU-AuCu and TMB+H2O2+COF-CTU-AuCu;(b)UV-visible spectra of TMB oxidation catalyzed by COF-CTU-AuCu (50 μg / mL) at different pH values;(c)UV-visible spectra of TMB oxidation catalyzed by COF-CTU-AuCu (pH 5.5) at different concentrations;(d)638 nm laser irradiation (1.0 W / cm 2 ) The effect of 1 minute on the catalytic ability of COF-CTU-AuCu nanozyme (50 μg / mL); (e) ESR spectra of COF-CTU-AuCu in the absence and presence of H2O2;

[0032] Figure 5 :(a) Curve of the absorbance of oxTMB at 652 nm after adding COF-CTU-AuCu nanozyme (50 μg / mL) and different concentrations of H2O2 (0, 10, 20, 30, 40, 50 and 60 mM);(b) Curve of the absorbance of oxTMB at 652 nm after adding COF-CTU-AuCu nanozyme (50 μg / mL) and different concentrations of H2O2 (0, 10, 20, 30, 40, 50 and 60 mM);( -2 ) Michaelis-Menten kinetics of the POD-like activity of COF-CTU-AuCu with or without 638 nm laser irradiation (1 W cm -2 ) (substrate H2O2); (d) with and without 638 nm laser (1 W cm -2 ) Michaelis-Menten kinetics of the POD-like activity of COF-CTU-AuCu with or without 638 nm laser irradiation (1 W cm -2 ) when the Lineweaver-Burk plot of POD-like activity (substrate TMB);

[0033] Figure 6:(a)UV-visible spectra of H2O2 at different concentrations;(b)Standard curve of UV absorption of H2O2 at 240nm;(c)UV-visible spectra of COF-CTU-AuCu catalyzed H2O2 decomposition;(d)With and without 638nm laser (1Wcm -2 ) Michaelis-Menten kinetics of CAT-like activity of COF-CTU-AuCu irradiated with or without 638 nm laser irradiation (1 W cm -2 ) when the CAT-like activity is at 1.5 % (substrate H2O2);

[0034] Figure 7 :(a) UV-visible spectra of glutathione and benzoic acid solutions treated with COF-CTU-AuCu at different concentrations;(b) UV-visible spectra of glutathione and benzoic acid solutions treated with COF-CTU-AuCu nanozymes (50µg / mL) at different incubation times;

[0035] Figure 8 :(a) Blood compatibility of COF-CTU-AuCu at different concentrations;(b) Cytotoxicity of COF-CTU-AuCu at different concentrations to L929 cells;

[0036] Fig. 9 :(a)COF-CTU-AuCu treated by photothermal effect S. aureus and E. coli Digital photo of bacterial colonies on agar plates; (b) after photothermal treatment S. aureus and E. coli survival rate;

[0037] Fig.10 : (a) S. aureus and E. coli Photographs of bacterial colonies on agar plates co-cultured with different concentrations of COF-CTU-AuCu; (b) S. aureus and E. coli Survival rate after co-culture with COF-CTU-AuCu alone;

[0038] Fig.11 :(a)COF-CTU-AuCu treated with enzyme activity E. coli Digital photo of bacterial colonies on agar plates; (b) COF-CTU-AuCu after enzyme activity treatment E. coli survival rate;

[0039] Fig.12 :(a)COF-CTU-AuCu treated with enzyme activity S. aureusDigital photo of bacterial colonies on agar plates; (b) COF-CTU-AuCu after enzyme activity treatment S. aureus survival rate;

[0040] Fig.13 :(a)After different grouping processing S. aureus and E. coli Photographs of bacterial colonies on agar plates; (b) after different grouping treatments S. aureus and E. coli survival rate;

[0041] Fig.14 :(a)Staining images of biofilms produced by Staphylococcus aureus and Escherichia coli with different treatments using crystal violet;(b)Analysis of the corresponding optical density at a wavelength of 590 nm;

[0042] Fig.15 :(a) SYTO-9 and PI were used to analyze the different S. aureus and E. coli (a) After fluorescence staining; (b) S. aureus Relative fluorescence intensity of (c) after staining E. coli The corresponding relative fluorescence intensity in ;

[0043] Fig.16 : After different treatments S. aureus and E. coli TEM images (scale bar = 2 μm);

[0044] Fig.17 :Different group processing S. aureus Visual observation of the healing process of infected wounds;

[0045] Fig.18 :(a) Quantitative analysis of the changes in wound area over time in each group; (b) Changes in mouse body weight during the experiment

[0046] Fig.19 : Results of hematological examinations of the corresponding groups (day 9): (a) red blood cell count, (b) white blood cell count, (c) mean corpuscular volume, (d) mean corpuscular hemoglobin, (e) hemoglobin, (f) hematocrit, (g) platelets, (h) mean corpuscular hemoglobin concentration, data using mean ± standard error (n = 6);

[0047] Fig. 20 : H&E and Masson's trichrome-stained wound tissue sections of mice in the control and treatment groups on day 9;

[0048] Fig.21 : H&E stained organ tissue sections of mice in the control group and treatment group on the 9th day. DETAILED DESCRIPTION

[0049] 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.

[0050] As introduced in the background technology section, current artificial enzymes of metal covalent organic framework polymers generally have the activity of 1 to 2 enzymes. Improving the activity of enzymes can only be achieved by loading them on metal covalent organic framework polymers.

[0051] Based on this, the purpose of the present invention is to provide a bimetallic organic framework multifunctional enzyme and its preparation method and application. The present invention uses a simple Schiff base condensation reaction to copolymerize the copper-based cyclic trinuclear complex Cu-CTC-NH2 and the gold-based cyclic trinuclear complex Au-CTC-CHO to construct a bimetallic covalent organic framework artificial enzyme, named COF-CTU-AuCu. COF-CTU-AuCu has multiple enzyme active sites: self-grown AuCu alloy, cyclic trinuclear coordinated Au and Cu, Au and CuO. The unique structure of COF-CTU-AuCu has highly dispersed catalytic active centers and hierarchical porous structures, which can maintain the maximum exposure of active sites, thereby enhancing the activity of the simulated enzyme. In addition, unlike single metal covalent organic framework polymers, the interaction between multiple metal active sites in COF-CTU-AuCu accelerates electron transfer, promotes the mutual conversion between valence states, and has a higher electron transfer efficiency. Therefore, the enzyme activity of the bimetallic covalent organic framework polymer is significantly enhanced compared with the enzyme activity of the two single metal covalent organic framework polymers.

[0052] Both Cu-CTC-NH2 and Au-CTC-CHO monomers have triazole ring structures. After copolymerization, the conjugated system of the product is enlarged, and a significant conjugated effect appears, which makes the product have a stronger photothermal effect. The metal particles are well wrapped by the polymer shell to improve their biocompatibility and promote wound repair. By separating various active sites within the framework, COF-CTU-AuCu exhibits triple enzyme activity and realizes a triple catalytic cascade enzyme reaction: it can generate oxygen and consume GSH through its simulated CAT and simulated glutathione oxidase (GSHOx) activities; it can generate hydroxyl radicals (•OH) through simulated peroxidase (POD)-like activity. In addition, COF-CTU-AuCu can also be used as a photothermal agent to produce local heating under red light irradiation, and it can also enhance the catalytic activity of nanozymes, enhance the generation of hydroxyl radicals (•OH) and O2, and laser irradiation significantly enhances the therapeutic effect of combined antibacterial therapy.

[0053] 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.

[0054] Note: 3,5-Dimethylpyrazole-4-boronic acid pinacol ester was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd., CAS: 857530-80-4.

[0055] Unless otherwise specified, the blank in the figures of the specification refers to the control group or PBS control group; C represents the COF-CTU-AuCu group, L represents the laser group, and H represents the H2O2 group; C / L / H or C+L+H both represent the COF-CTU-AuCu+laser+H2O2 group (in no particular order).

[0056] 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.

[0057] Embodiment 1:

[0058] (1) Synthesis of Cu-CTC-NH2: In a hydrothermal autoclave reactor, Cu2O (42 mg, 0.3 mmol) and 3,5-dimethyl-1H-pyrazol-4-amine (100.0 mg, 0.9 mmol) were dissolved in a mixture of 4 mL ethanol and 0.4 mL pyridine and heated in an oven at 120 °C for 72 h. The yellow solid was collected, washed with ethanol and dried under vacuum for 6 h (70.6 mg, 82.4%). The synthetic route is as follows:

[0059] .

[0060] (2) Synthesis of Au-CTC-CHO: 3,5-Dimethylpyrazole-4-boronic acid pinacol ester (2.22 g, 10 mmol) was added to a 250 mL flask and dissolved in 50 mL CHCl3. Then 8 mL of triethylamine was added and refluxed at 50 °C for 30 min. Then triphenylmethane chloride (2.78 g, 10 mmol) dissolved in 50 mL CHCl3 was added to the reaction flask, refluxed at 50 °C for 6 h, and then cooled with ice water to obtain a yellow solid, which was washed with methanol with a yield of 87.3%. 1 H NMR (400 MHz, DMSO-d6, δ) 7.35 (m, 15 H), 2.22 (s, 6 H), 1.23 (s, 12 H).

[0061] The obtained yellow solid (1.00 g, 2.15 mmol) and 4-bromobenzaldehyde (0.32 g, 1.7 mmol), cesium carbonate (2.25 g, 6.9 mmol) and Pd[P(C6H5)3]4 (0.865 g, 0.75 mmol) were added to 1,4-dioxane (125 mL) and H2O (25 mL) in a 500 mL flask. The mixture was degassed by three consecutive freeze-thaw cycles. Then, the reaction mixture was stirred and heated at 100 °C for 12 h. A brown liquid was obtained, which was extracted with CH2Cl2 to obtain a yellow oil, which was purified by column chromatography (eluent: petroleum ether / DCM = 1 / 2, v / v) to obtain a light yellow solid (yield, 65%). 1 H NMR (400MHz, DMSO-d6, δ) 10.03 (s, 1H), 7.91 (d, J = 8.2 Hz, 2H), 7.43 (d, J = 8.2 Hz, 2H), 7.33-7.20 (m, 15 H), 2.28 (s, 3H), 1.46-1.25 (m, 3H).

[0062] The light yellow solid was dissolved in 50 mL CH2Cl2, and then 15 mL methanol and 15 mL 2 M HCl were added, stirred and heated at 50 °C for 8 h. The resulting yellow solution was neutralized with saturated sodium bicarbonate solution to pH = 6, and then extracted with ethyl acetate. Finally, the organic layer was collected and purified by column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 2, v / v). After removing all solvents, a white solid 4-(3,5-dimethyl-1H-pyrazol-4-yl)benzaldehyde was obtained with a yield of 82.3%. 1 H NMR (400 MHz, DMSO-d6, δ) 12.48 (s, 1H), 10.00 (s, 1H), 7.93 (d, J = 8.0 Hz, 2H), 7.53 (d, J = 8.0 Hz, 2H), 2.25 (s, 6H).

[0063] In a 25 mL flask, add 10 mg (0.05 mmol) of 4-(3,5-dimethyl-1H-pyrazol-4-yl)benzaldehyde, 1.5 mL of tetrahydrofuran, and 50 μL of triethylamine. After stirring for 5 minutes, add 16.99 mg (0.05 mmol) of AuCl4H. After stirring for 12 hours in the dark, filter and wash with ethanol to obtain 7.4 mg of purple-gray solid with a yield of 37.40%. 1H NMR (400 MHz, DMSO-d6, δ) 9.99 (s, 1H), 7.91 (d, J = 8.3 Hz, 2H), 7.52 (d, J = 8.1 Hz, 2H), 2.33 (s, 6H). Synthesis route:

[0064] .

[0065] (3) Synthesis of COF-CTU-AuCu: Trimethylbenzene (0.75 mL) and 1,4-dioxane (0.75 mL) were mixed into a reactor (3 mL), and then 6 M acetic acid (0.15 mL), Cu-CTC-NH2 (26.1 mg, 0.05 mmol) and Au-CTC-CHO (59.43 mg, 0.05 mmol) were added and sealed with argon bubbling. Heating at 120 °C for 72 h gave a dark brown solid. The solid was separated by filtration, washed with ether, DMF and acetone, and dried in vacuum at 100 °C for 12 hours. The yield was 78.2%. The synthetic route is:

[0066] .

[0067] Example 2: Characterization

[0068] (1) The chemical structure and bonding of COF-CTU-AuCu were preliminarily revealed by Fourier transform infrared spectroscopy (FT-IR). Figure 1 As shown in (a), the FT-IR spectrum of COF-CTU-AuCu has Au-CTC-CHO (1600 cm -1 C=C at 1350 cm -1 Meanwhile, the aldehydes (∼1700 cm -1 ) and amine groups (3300~3415 cm -1 ) disappeared, and an unbent C=N stretching band (∼1627 cm -1 ), indicating that an aldehyde-amine condensation reaction occurred.

[0069] (2) If Figure 1 As shown in (b), 13C CP / MAS NMR shows a broad absorption including saturated carbon (100~175 ppm) and unsaturated carbon (5~50 ppm) signals. The signals located in the saturated region are attributed to -CH3 in Au-CTC-CHO and Cu-CTC-NH2 units. The unique resonance peak at 145 ppm is attributed to the unopened imine carbon, which further proves the occurrence of Schiff base reaction.

[0070] (3) If Figure 1 (c) The crystal structure of COF-CTU-AuCu was determined by powder X-ray diffraction (PXRD) analysis. The diffraction pattern of COF-CTU-AuCu shows strong crystalline peaks at 2θ of 38.52°, 40.20°, 41.12° and 45.02°, corresponding to Au (1 1 1), CuO (1 1 1), AuCu (1 1 1) and AuCu (2 0 0) diffractions, respectively, indicating that Au, CuO and AuCu alloys were formed simultaneously during the polymerization process.

[0071] (4) If Figure 1 As shown in (d), the gas adsorption of COF-CTU-AuCu conforms to the characteristics of type IV reversible isotherm, with a rapid increase in adsorption at low pressure and an obvious hysteresis loop at the branch of the isotherm. In addition, the relatively rapid growth at high pressure indicates the presence of macropores in COF-CTU-AuCu. The Brunauer-Emmett-Teller (BET) surface area calculated by nonlocal density functional theory (NLDFT) is 424.6 m 2 g -1 , the cumulative pore volume is 0.41 cm 3 g -1 .from Figure 1 The pore size distribution curve (PSD) of (e) also intuitively shows the hierarchical porous structure, showing a wide range of pore distribution from micropores to macropores, with the main peak centered at 2.69 nm.

[0072] (5) If Figure 1 As shown in f, thermogravimetric analysis (TGA) shows that COF-CTU-AuCu remains stable below 380 °C, has good thermal stability, and can be used as a potential photothermal agent.

[0073] (6) Figure 1As shown in (g), the elemental composition of COF-CTU-AuCu was determined by X-ray photoelectron spectroscopy (XPS). The C1s spectrum is decomposed into several peaks of C = C (284.8 eV), C - N (285.5 eV), C = N (288.8 eV) and π - π * (291.6 eV), indicating that COF-CTU-AuCu has a highly conjugated structure. The Au 4f spectrum shows that the COF-CTU-AuCu signal peaks at 84.2 and 87.9 eV belong to the Au 4f of Au0 (0 valence). 5 / 2 and Au 4f 7 / 2 The Cu 2p spectra of Cu(I) were observed. 3 / 2 (933.2 eV) and Cu 2p 1 / 2 (952.9 eV) peak. The measured spectrum shows five peaks related to the elements C, O, N, Cu and Au, among which the atomic percentage of C is calculated to be 63.31%, O is 6.17%, N is 20.60%, Cu is 6.69% and Au is 1.56%.

[0074] (7) Figure 2 As shown, Figure 2 (a) Using scanning electron microscopy (SEM) and Figure 2 (b) Transmission electron microscopy (TEM) was used to examine the morphology of COF-CTU-AuCu in detail. Scanning electron microscopy images clearly show that COF-CTU-AuCu is a typical ribbon-like material with interconnected open macropores. TEM images further reveal the hierarchical pore structure of COF-CTU-AuCu. Notably, a typical core-shell structure was observed, characterized by ultrafine black nanoparticles corresponding to Au, AuCu, and CuO cores encapsulated in a porous polymer sheath. Consistent with the XRD results, Figure 2 The high-resolution TEM (HR-TEM) of (c) shows clear lattice edges with spacings of 0.236, 0.223, and 0.199 nm, corresponding to the (1 1 1) plane of Au and the (1 1 1) and (2 0 0) planes of AuCu, respectively. Figure 2 (e) Energy dispersive spectroscopy (EDS) confirms the balanced ratio of the two metal elements gold (41.89 wt%) and copper (48.75 wt%).

[0075] Example 3: Photothermal performance test

[0076] Changing the COF-CTU-AuCu concentration (0 to 100 μg / mL) or the laser power (0.5 to 1.0 W / cm 2), the photothermal effect of COF-CTU-AuCu under 638nm laser irradiation was investigated. As shown in Figure 3 (a) and Figure 3 As shown in (b), the temperature increase of COF-CTU-AuCu is positively correlated with the sample concentration and laser power. 2 ), the solution temperature increased from 25°C to 34.2°C (20 μg / mL), 37.4°C (40 μg / mL), 41.0°C (60 μg / mL), 47.8°C (80 μg / mL), and finally to 52.0°C (100 μg / mL). Similarly, when the laser powers were 0.5, 0.75, and 1.0 W / cm 2 When the temperature of COF-CTU-AuCu suspension (100 μg / mL) was 2.37 °C, the peak temperature of COF-CTU-AuCu suspension (100 μg / mL) increased to 38.9, 46.0, and 52.2 °C within 10 min, respectively. The temperature fluctuation under laser irradiation was monitored by a thermal imager. Figure 3 As shown in (c), the irradiation time (638 nm, 1.0 W / cm 2 ), COF-CTU-AuCu gradually emits brighter thermal signals. In addition, the photothermal stability of COF-CTU-AuCu was evaluated by photothermal cycling tests. Figure 3 As shown in (d), the temperature curve of monitoring the heating and cooling cycles shows that the deviation of the peak temperature is extremely small in four consecutive on / off cycles, confirming that COF-CTU-AuCu has excellent photostability and can be used as an ideal candidate material for photothermal antibacterial applications. In order to quantify the photothermal conversion efficiency, according to the photothermal conversion efficiency formula in "A Photothermal-Fenton Reaction Artificial Nanozyme and Its Preparation Method and Application" disclosed in application number CN202310175497.X, the η value of COF-CTU-AuCu was calculated by analyzing a single heating-cooling cycle in Figure 3 (e). The time constant (τ) of COF-CTU-AuCu was determined to be 238.6 seconds, and the η value was 40.52%.

[0077] Example 4: Enzyme-like catalytic performance test

[0078] The peroxidase-like activity of COF-CTU-AuCu was measured by using TMB as a probe in a total reaction volume of 1 mL. All reactions were completed at room temperature with a reaction time of 5 min, and the reaction system was monitored by a UV-Vis-NIR spectrophotometer. The reaction system was made up to 1 mL with PBS, except for the material solution, hydrogen peroxide, and TMB. In this part, the concentration of TMB was 1.5 mmol / L and the concentration of H2O2 was 1 mmol / L. The activity of COF-CTU-AuCu (100 μg / mL) was first determined. Then, the peroxidase-like catalytic effect was investigated under different pH environments and different concentrations. Next, the effect of 638 nm laser irradiation for 1 min on the catalytic effect of COF-CTU-AuCu was studied. Using H2O2 as the substrate, the catalytic activity of COF-CTU-AuCu was significantly increased by adding phosphate buffer (0.85 mL, pH= 5.5), COF-CTU-AuCu (2 mg mL -1 The enzyme kinetics of COF-CTU-AuCu were analyzed by adding 50 μL of TMB (20 mM, 50 μL), 1.0 mM of TMB (20 mM, 50 μL), and different amounts of H2O2 (final working concentration: 0, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 mM). In addition, a 638 nm laser was added during the experiment to verify the light-enhanced POD-like activity and kinetics. Finally, the glutathione oxidase activity of COF-CTU-AuCu was tested using a glutathione peroxidase (GSH-Px) detection kit (colorimetric method).

[0079] (1) If Figure 4 As shown in (a), only in the dual matrix system containing both H2O2 and COF-CTU-AuCu, can a significant absorption peak at 652 nm be detected, which involves the catalytic oxidation of TMB to ox-TMB. On this basis, the effects of solution pH (1.5~7.4) and COF-CTU-AuCu concentration (20~100 μg / mL) on the enzyme-like activity were further explored. Figure 4 As shown in (b), the POD-like activity of COF-CTU-AuCu exhibits pH dependence. It is noteworthy that as the pH value decreases, the simulated POD activity of COF-CTU-AuCu begins to increase and reaches a peak at pH 4.5. Subsequently, as the pH value further decreases, the enzyme activity also decreases. The enzyme activity at pH 6.5 is comparable to that at pH 3.5. Figure 4 (c) shows that the •OH generation capacity is also positively correlated with the sample dosage. Figure 4As shown in (d), the POD-like activity of COF-CTU-AuCu can be further enhanced by laser irradiation. Specifically, under laser irradiation, the UV-visible light adsorption intensity of ox-TMB is greatly enhanced. This enhancement is attributed to the inherent photothermal effect of COF-CTU-AuCu, where the increase in temperature promotes the generation of ROS. At the same time, Figure 4 (e) Electron spin resonance (ESR) spectroscopy shows that the characteristic signal of •OH radicals can only be seen in the presence of COF-CTU-AuCu and H2O2 at a ratio of 1:2:2:1. In contrast, when COF-CTU-AuCu is used alone, no ESR signal of DMPO / •OH adduct is detected.

[0080] In summary, in the dual matrix system containing H2O2 and COF-CTU-AuCu, TMB was catalytically oxidized to ox-TMB, indicating that COF-CTU-AuCu can effectively catalyze the generation of a large number of hydroxyl radicals (•OH) in the presence of H2O2. Further studies on the effects of solution pH and COF-CTU-AuCu concentration on enzyme-like activity showed that the POD-like activity of COF-CTU-AuCu reached a peak at pH 5.5. Laser irradiation further enhanced the POD-like activity of COF-CTU-AuCu.

[0081] (2) The peroxidase (POD) activity of COF-CTU-AuCu was evaluated by steady-state kinetics experiments. The kinetic parameters (K m and V max ) is obtained according to the Michaelis-Menten curve as follows: ,in, ν is the initial velocity, V max represents the maximum reaction rate, [S] represents the substrate concentration, K m is the Michaelis constant. The equation can also be converted into a Lineweaver-Burk plot as shown below: By changing the substrate concentration, the Lineweaver-Burk equation was used to generate the Lineweaver-Burk double reverse plot and the Michaelis-Menten curve. Figure 5 As shown in (a), the simulated POD catalytic reaction rate of COF-CTU-AuCu increases linearly when the substrate concentration is low and tends to be stable when the concentration is high. When H2O2 is used as the substrate, COF-CTU-AuCu shows a high affinity for •OH radicals. Figure 5 (b) and Figure 5 As shown in (c), the Michaelis-Menten kinetics was used to analyze the generation rate of •OH and the Michaelis-Menten constant (Km ) is 0.5147 mM, the maximum speed (V max ) is 7.625×10 -8 Ms -1 The absorbance of the laser irradiation group was higher than that of the non-irradiation group, and its K m Value and V max The values ​​were 0.3671 mM and 8.319×10 -8 Ms -1 When TMB is used as substrate, Figure 5 (d) and Figure 5 (e) shows that its K m Value and V max The values ​​were 0.3154 mM and 13.05 × 10 −8 Ms -1 The K of the group without laser irradiation m and V max The values ​​were 0.2648 mM and 14.59×10 -8 Ms -1 , indicating that the photothermal effect can effectively enhance the ability of COF-CTU-AuCu to produce •OH.

[0082] (3) The CAT-like activity of COF-CTU-AuCu was evaluated by the change of H2O2 concentration. 0.1 mL H2O2 (final working concentration was 2 mM) was added to phosphate buffer (0.8 mL, pH = 7.4), followed by 0.1 mL COF-CTU-AuCu (1 mg mL -1 ) solution. The decrease in absorbance at 240 nm was monitored at different time points, such as Figure 6 (a) and Figure 6 As shown in (b), according to the standard curve of H2O2 concentration, the consumption rate of H2O2 (30mM) by COF-CTU-AuCu was 11.15% after 15min of reaction. Similarly, the kinetic parameters of COF-CTU-AuCu CAT activity were evaluated by changing the H2O2 concentration. Figure 6 (c) and Figure 6 (d) shows the calculated K of COF-CTU-AuCu nanozyme. m and V max The values ​​were 4.962 mM and 3.283×10 -6 M / s, compared with K in the laser irradiation group m Value and V max The values ​​were 4.709 mM and 3.925×10 -6M / s, indicating that laser irradiation can also promote the activity of CAT-type enzymes, indicating that COF-CTU-AuCu has the potential to improve the hypoxic conditions in IME.

[0083] (4) The consumption capacity of COF-CTU-AuCu on GSH was determined by detecting the changes in GSH levels. Figure 7 (a) and Figure 7 As shown in (b), after incubation with glutathione, the oxidation behavior of COF-CTU-AuCu was positively correlated with concentration and time, which proved that COF-CTU-AuCu could effectively remove GSH produced by oxidative stress in infected wounds and weaken the scavenging effect of GSH on oxidative free radicals produced by treatment.

[0084] In summary, the application of COF-CTU-AuCu nanozymes to catalyze redox reactions can promote wound healing of bacterial infections, generate a large amount of reactive oxygen species, consume overexpressed endogenous GSH, and disrupt the balance of IME.

[0085] Example 7: Biocompatibility test

[0086] (1) Fresh blood was obtained from BALB / c mice (3-4 weeks old, purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.), and red blood cells (RBCs) were collected by centrifugation at 1500 rpm for 20 minutes, and then washed three times with PBS. RBCs (4% w / w) were incubated with different concentrations of COF-CTU-AuCu at a ratio of 1:9 (v / v) at 37°C for 3 hours, and then centrifuged at 12000 rpm for 20 minutes. Subsequently, it was measured by UV-visible spectroscopy at 540 nm. Distilled water was set as the positive control, and PBS was set as the negative control.

[0087] Hemolysis rate formula:

[0088] Hemolysis rate (%) = (As - An) / (Ap - An) × 100%;

[0089] Wherein ‘As’ is the absorbance of the red blood cell suspension after adding GSF-POP, ‘An’ is the absorbance of the red blood cell suspension after adding PBS (negative control), and ‘Ap’ is the absorbance of the red blood cell suspension after adding distilled water (positive control).

[0090] like Figure 8As shown in (a), except for the dark red supernatant of the control group (distilled water), the supernatants of the other groups (including the PBS group and the COF-CTU-AuCu treatment group) were almost transparent. As the concentration of the COF-CTU-AuCu dispersion increased from 20 μg / mL to 150 μg / mL, the hemolysis rate of COF-CTU-AuCu gradually increased from 0.08% to 2.55%, but it was still significantly lower than the international standard of blood compatibility (<5.0%), indicating that COF-CTU-AuCu has good blood compatibility.

[0091] (2) Mouse fibroblasts (L929) were used to study the cytotoxicity of COF-CTU-AuCu. L929 cells were cultured at 8 × 10 3 The cells were seeded in a 96-well plate at a density of 10 cells and incubated overnight. Then L929 cells were incubated with COF-CTU-AuCu solutions of different concentrations (20 μg / mL~150 μg / mL) for 24 hours. After removing the supernatant, the same volume of PBS (pH=7.4) containing 10 μL MTT (5 mg / mL) was added to each well. After 4 hours, the supernatant was aspirated and 100 μL DMSO was added to dissolve the formamide crystals formed by MTT. After 5 minutes, the absorbance of each well at 490 nm was measured using a full-wavelength microplate reader. Each group was repeated three times in parallel, and the cell survival rate was calculated using the following formula: Cell survival rate (%) = (mean value of the treatment group / mean value of the control group) × 100%.

[0092] like Figure 8 As shown in (b), the survival rate of L929 cells gradually decreased with the increase of sample dose. However, at the optimal therapeutic concentration (50 μg / mL), the cell survival rate remained above 80% (85.1%). This indicates that COF-CTU-AuCu has good biocompatibility and is expected to be used as a therapeutic agent for in vivo treatment.

[0093] Test Example 1: In vitro antibacterial performance

[0094] (1) Antibacterial effect of single treatment mode

[0095] Select Gram-negative Escherichia coli ( E. coli ) and Gram-positive Staphylococcus aureus ( S. aureus ) was used to evaluate the antibacterial activity of COF-CTU-AuCu nanosheets. Bacteria were cultured in Luria-Bertani (LB) medium at 37 °C until the logarithmic growth phase. Subsequently, the bacterial suspension was diluted to 1 × 10 8The COF-CTU-AuCu suspension was then mixed with the bacterial culture, and the final concentration of COF-CTU-AuCu was set to 0, 50, 100, 150, and 200 mg / mL. A 638 nm laser was used to irradiate for 10 min at a power density of 1 W / cm². Appropriate dilutions (50 μL) of the treated bacteria were evenly spread on LB agar plates. After 24 h of incubation, colony forming units (CFUs) were counted to determine E. coli and S. aureus of survival rate.

[0096] Fig. 9 (a) and Fig. 9 (b) shows the COF-CTU-AuCu under different concentrations and laser irradiation (638 nm, 1.0 W cm -2 ) under the condition of COF-CTU-AuCu concentration of 100 μg / mL, the antibacterial rates against Staphylococcus aureus and Escherichia coli were 64.40±3.15% and 60.33±4.18%, respectively. When the COF-CTU-AuCu concentration was increased to 200 μg / mL, almost 100% of the bacteria were killed, and the survival rates of Staphylococcus aureus and Escherichia coli were less than 0.5%.

[0097] In contrast, when COF-CTU-AuCu was co-cultured with bacteria for 24 h without additional stimulation (laser and H2O2), Fig.10 As shown in (a) and (b), COF-CTU-AuCu has effective antibacterial activity at a concentration of 160 μg / mL (antibacterial rate greater than 99.5%).

[0098] To further investigate the antibacterial properties of COF-CTU-AuCu, the antibacterial activity was evaluated by adding 100 μM exogenous hydrogen peroxide (H2O2) while varying the concentration of COF-CTU-AuCu to 0, 50, 75, 100, 125, and 150 mg / mL. E. coli and S. aureus In addition, Fig.11 (a) and Fig.11 (b) shows the enzymatic antibacterial activity of COF-CTU-AuCu against Escherichia coli. As the culture time was extended from 1 h to 4 h, the antibacterial activity of COF-CTU-AuCu (100 μg / mL) increased from 57.96±2.97% to 91.76±1.99%. Fig.12 (a) and Fig.12The situation of Staphylococcus aureus in (b) is similar, and the inhibition rate increases from 52.43±7.08% to 87.26±1.69%. This indicates that COF-CTU-AuCu has a strong enzymatic antibacterial effect.

[0099] (2) Antibacterial effect of synergistic treatment strategy

[0100] Six treatment groups were established to compare the antibacterial results: PBS control group, H2O2 group, COF-CTU-AuCu group, COF-CTU-AuCu + laser group, H2O2 + COF-CTU-AuCu group, and H2O2 + COF-CTU-AuCu + laser group. The concentrations of H2O2 and COF-CTU-AuCu were set to 100 μM and 100 mg / mL, respectively. Light treatment was performed using a 638 nm laser at a power density of 1 W / cm² for 10 min. Appropriate dilution solutions (50 μL) of the treated bacteria were evenly spread on LB agar plates. After 24 h of incubation, colony forming units (CFU) were counted to determine E. coli and S. aureus The survival rate of Fig.13 (a) and Fig.13 As shown in (b), compared with the control group, after applying laser and H2O2 respectively, the bacterial activity of Staphylococcus aureus and Escherichia coli decreased to 48.50±4.06% and 51.47±3.76%, and 36.70±4.78% and 32.70±3.99%, respectively. When COF-CTU-AuCu was irradiated with laser and H2O2 simultaneously, its bactericidal efficiency was close to 100%, indicating that COF-CTU-AuCu has excellent multi-mode antibacterial ability against these two strains.

[0101] The anti-biofilm ability of COF-CTU-AuCu was tested according to the above grouping. E. coli and S. aureusThey were introduced into 96-well plates with glass bottoms and cultured at 37°C for 24 h to form mature biofilms. The biofilms were treated with H2O2, COF-CTU-AuCu, COF-CTU-AuCu + laser, H2O2 + COF-CTU-AuCu and H2O2 + COF-CTU-AuCu + laser at 37°C and incubated for 24 h. The concentrations of H2O2 and COF-CTU-AuCu were 100 μM and 50 mg / mL, respectively. The laser was irradiated at 638 nm with a power density of 1 W / cm² for 10 min. Then the supernatant was aspirated, and 250 μL PBS was added to rinse 4 times. Each time, the plate was vibrated for 1 min to remove the unabsorbed bacteria and materials, and then dried naturally. Then 200 μL of 99% methanol was added to fix for 15 min, and then the plate was aspirated and dried naturally. 200 μL of 1% crystal violet was added to stain for 5 min, and then the crystal violet was aspirated, and the unabsorbed crystal violet was rinsed with sterile water, and then dried naturally. 160 μL of 33% acetic acid was used to dissolve the crystal violet adsorbed in the 96-well cell culture plate, and the mixture was allowed to stand for 15 min. Finally, the OD was measured with an enzyme marker. 570nm The absorbance value under Fig.14 As shown in (a), COF-CTU-AuCu nanozyme itself has bactericidal activity and has a certain effect on bacterial biofilm. However, this effect is not enough to destroy the overall structural integrity of the biofilm. When laser irradiation is used simultaneously, the photothermal effect greatly destroys the integrity of the biofilm, allowing COF-CTU-AuCu to effectively penetrate the biofilm and exert a stronger antibacterial effect. Similar antibacterial effects can also be achieved by introducing exogenous H2O2 to exert the multi-species enzyme activity of COF-CTU-AuCu nanozyme. In particular, the simultaneous action of laser irradiation and H2O2 on COF-CTU-AuCu can completely destroy the biofilms of these two bacteria, thereby Fig.14 This can also be seen intuitively in the quantitative analysis of the biofilm micrographs in (b), further indicating that COF-CTU-AuCu has excellent multi-modal inhibitory ability against these two strains.

[0102] The multi-mode antibacterial ability of COF-CTU-AuCu against these two strains was further verified. The bacteria after co-culture were stained with SYTO-9 and propidium iodide (PI), and the fluorescence was observed to determine the bacterial death. Fig.15As shown in (a), compared with the COF-CTU-AuCu group, the COF-CTU-AuCu + laser group, the COF-CTU-AuCu + H2O2 group, and the COF-CTU-AuCu + H2O2+ laser group all showed stronger red fluorescence. All bacteria in the COF-CTU-AuCu + H2O2+ laser group were labeled with red fluorescence. Quantitative analysis further confirmed that the COF-CTU-AuCu + H2O2+ laser group had the most outstanding bactericidal ability, and the bactericidal activity of Staphylococcus aureus and Escherichia coli was less than 0.5%, as shown in Figure 2. Fig.15 (b) and Fig.15 (c).

[0103] The killing ability of COF-CTU-AuCu under different treatments on bacteria was intuitively observed from the obvious morphological changes of bacterial strains under different treatments using TEM technology. Fig.16 As shown, the surfaces of both bacteria in the control group maintained an intact and smooth appearance. Similarly, the morphology of Staphylococcus aureus and Escherichia coli treated with laser irradiation, H2O2, and COF-CTU-AuCu alone was comparable to that of the control group. In contrast, in the COF-CTU-AuCu + H2O2 + laser group, bacterial cells showed obvious fractures and wrinkles, with severe damage to structural integrity, surface collapse, and leakage of intracellular matrix. These microscopic observations were consistent with the quantitative data obtained from plate counts and live / dead bacterial staining analysis, further verifying the synergistic antibacterial effect of the combined therapy.

[0104] Test Example 2: In vivo antibacterial test

[0105] The experimental animals were BALB / c female mice (3-4 weeks old, weighing 13-16 g, purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.). The rats were given one week to adapt to the new environment before the test. The mice were in a 12-h light / dark cycle, with a temperature range of 24 ± 2°C and a humidity range of 50 ± 10%. A 5 mm diameter wound was created on the back skin of the mice using a needle biopsy technique (six mice per group), followed by inoculation of logarithmic-growth Staphylococcus aureus (10 μL, 1 × 10 8CFU / mL). Two days after infection, mice were divided into six different treatment groups: PBS control group, H2O2 group, COF-CTU-AuCu group, COF-CTU-AuCu + laser group, H2O2 + COF-CTU-AuCu group, and H2O2 + COF-CTU-AuCu + laser group. Each solution (50 μL) was evenly applied on the wound. The concentration of H2O2 was 100 μM, and the concentration of COF-CTU-AuCu was 100 mg / mL. For the combined treatment group, H2O2 was applied first, followed by COF-CTU-AuCu, and finally light irradiation. Mice were irradiated with 638 nm laser (1 W / cm²) for 10 min to ensure complete coverage of the infected wound area. Antimicrobial treatment started on day 1 after infection, which was recorded as day 0. Treatment was performed on days 1, 3, and 5. The wound area and body weight were measured and photographed every day. On day 9, the mice were killed, and the back wound skin tissues, heart, liver, spleen, lung, and kidney were obtained and fixed in 10% paraformaldehyde, embedded, sectioned, and observed by H&E and Masson's trichrome staining.

[0106] like Fig.17 As shown, the wound area of ​​different groups gradually decreased with time, but the degree of healing varied among the different treatment groups. The wound area of ​​the COF-CTU-AuCu + H2O2+ laser group was the smallest compared with the other groups. Fig.18 (a) Quantitative analysis of wound healing rates at different time points also showed that on the third day of treatment, the wound areas of the COF-CTU-AuCu + Laser, COF-CTU-AuCu + H2O2, and COF-CTU-AuCu + H2O2 + Laser groups were significantly reduced by 33.00 ± 1.95%, 36.87 ± 4.78%, and 70.1 ± 1.51%, respectively. The wound areas of the two control groups remained essentially unchanged. A more obvious trend was observed on day 5, with a 14.23 ± 5.15% decrease in the COF-CTU-AuCu group, a 49.37 ± 5.94% decrease in the COF-CTU-AuCu + Laser group, and a 63.33 ± 4.28% decrease in the COF-CTU-AuCu + H2O2 group. The COF-CTU-AuCu +H2O2 + Laser group decreased by 71.83 ± 2.02%. At the end of treatment, the COF-CTU-AuCu + H2O2+ laser group had the smallest residual wound area, approximately 5.47±0.74%, which was significantly smaller than the control group. The enhanced catalytic activity and photothermal properties of COF-CTU-AuCu contributed to these effective treatment results, even at a dose of only 50 μg / mL. To further determine the biocompatibility of COF-CTU-AuCu, the body weight of mice was monitored during the 9-day treatment period. Fig.18As shown in (b), similar to the control group, the weight gain rate of all treatment groups remained basically unchanged. After the experiment, the mice were anesthetized and blood was collected from the eyes for routine blood tests, such as Fig.19 As shown, the indexes such as red blood cells, white blood cells, and mean corpuscular volume were not much different from those in the blood of healthy mice (blank group), and were within the normal fluctuation range, indicating that the above-mentioned treatments had no obvious toxicity to mice.

[0107] On the ninth day of the in vivo wound healing experiment, histological analysis was performed by hematoxylin and eosin (H&E) staining and Masson's trichrome staining to evaluate the wound healing effect. Fig. 20 It can be seen that, unlike the incomplete epidermis in the PBS-treated group, the wound epidermis in the COF-CTU-AuCu + H2O2 + laser-treated group was intact, the skin structure (including capillaries) was regenerated, and the collagen fibers were continuous and blue. In addition, from the H&E staining results of each group (Figure 21), the important organs such as the heart, liver, spleen, lungs and kidneys of the mice in each group were almost undamaged. These experiments further confirmed the powerful bactericidal effect of COF-CTU-AuCu. At the same time, due to the photothermal amplification of its peroxidase-like activity, it can achieve effective antibacterial effects at extremely low concentrations and has excellent biosafety. It can reduce inflammation by reducing the bacterial load at the site of infection and significantly accelerate wound healing.

[0108] 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 bimetallic organic framework multifunctional enzyme, characterized in that: The bimetallic organic framework multifunctional enzyme has a core-shell structure and is an ultrafine black nanoparticle corresponding to Au, AuCu and CuO cores, encapsulated in a porous polymer sheath; The bimetallic organic framework multifunctional enzyme is obtained by copolymerization of a copper-based cyclic trinuclear complex and a gold-based cyclic trinuclear complex; the structural formula of the copper-based cyclic trinuclear complex is ； The structural formula of the gold-based cyclic trinuclear complex is ； The bimetallic organic framework multifunctional enzyme is prepared by the following method: S1 Synthesis of Cu-CTC-NH2: Dissolve Cu2O and 3,5-dimethyl-1H-pyrazol-4-amine in a mixed solvent, heat to react, collect the yellow solid, wash and dry to obtain Cu-CTC-NH2; S2.1 Synthesis of Au-CTC-CHO: Dissolve 3,5-dimethylpyrazole-4-boronic acid pinacol ester in CHCl3, add triethylamine and reflux; then add triphenylmethane dissolved in chloroform, continue to reflux, cool, wash and dry to obtain a yellow solid; S2.2 Add the yellow solid, 4-bromobenzaldehyde, cesium carbonate and tetrakis(triphenylphosphine)palladium into a mixed solvent, stir and heat to react to obtain a brown liquid, extract and purify to obtain a light yellow solid; S2.3 Dissolve the light yellow solid in a solvent containing hydrochloric acid, stir and heat to react, and sequentially neutralize, extract and purify the reaction solution to obtain a white solid 4-(3,5-dimethyl-1H-pyrazol-4-yl)benzaldehyde; S2.4 Add 4-(3,5-dimethyl-1H-pyrazol-4-yl)benzaldehyde to the solvent and mix, then add AuCl4H and stir to react in the dark, filter, wash and dry to obtain a purple-gray solid, which is Au-CTC-CHO; S3 Synthesis of bimetallic organic framework multifunctional enzyme: Add acetic acid, Cu-CTC-NH2 and Au-CTC-CHO into a mixed solvent, wherein the mixed solvent is obtained by mixing trimethylbenzene and 1,4-dioxane in a volume ratio of 1:1; heat the reaction to obtain a dark brown solid, which is washed and dried to obtain a bimetallic organic framework multifunctional enzyme.

2. The bimetallic organic framework multifunctional enzyme according to claim 1, characterized in that In step S1, the molar ratio of Cu2O to 3,5-dimethyl-1H-pyrazole-4-amine is 1:3; and the mixed solvent is obtained by mixing ethanol and pyridine in a volume ratio of 10:

1.

3. The bimetallic organic framework multifunctional enzyme according to claim 1, characterized in that In step S1, the temperature of the heating reaction is 120° C., and the heating reaction time is 72 hours.

4. The bimetallic organic framework multifunctional enzyme according to claim 1, characterized in that In step S2.1, the molar ratio of 3,5-dimethylpyrazole-4-boronic acid pinacol ester to triphenylmethane is 1:1; and the reflux temperature is 50°C.

5. The bimetallic organic framework multifunctional enzyme according to claim 1, characterized in that In step S2.2, the molar ratio of the yellow solid, 4-bromobenzaldehyde, cesium carbonate and tetrakis(triphenylphosphine)palladium is 2.15:1.7:6.9:0.75; and the mixed solvent is obtained by mixing 1,4-dioxane and H2O in a volume ratio of 5:

1.

6. The bimetallic organic framework multifunctional enzyme according to claim 1, characterized in that In step S2.3, the hydrochloric acid-containing solvent is obtained by mixing CH2Cl2, methanol and hydrochloric acid solution in a volume ratio of 10:3:3; the concentration of the hydrochloric acid solution is 2M; the heating temperature is 50°C and the heating time is 8h.

7. The bimetallic organic framework multifunctional enzyme according to claim 1, characterized in that In step S2.4, the molar ratio of 4-(3,5-dimethyl-1H-pyrazol-4-yl)benzaldehyde to AuCl4H is 1:1; the solvent is obtained by mixing tetrahydrofuran and triethylamine in a mass ratio of 30:1; and the light-proof stirring reaction time is 12 hours.

8. The bimetallic organic framework multifunctional enzyme according to claim 1, characterized in that In step S3, the molar ratio of Cu-CTC-NH2 to Au-CTC-CHO is 1:1; and the concentration of acetic acid is 6M.

9. Use of the bimetallic organic framework multifunctional enzyme according to any one of claims 1 to 8 in the preparation of antibacterial drugs, characterized in that: The antibacterial drug has catalase activity, glutathione oxidase activity, peroxidase activity and photothermal activity at the same time.

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