A macrocyclic covalent organic polymer material for suppressing π-π stacking effect and its application

Abstract

This invention discloses a macrocyclic covalent organic polymer material for suppressing the π-π stacking effect and its applications, belonging to the field of biomedical technology. In a protective atmosphere, 5,10,15,20-tetra(4-ethynylphenyl)copper porphyrin and a macrocyclic monomer are added to an organic solvent, and a solvothermal reaction is carried out in the presence of a catalyst and a base to obtain the macrocyclic covalent organic polymer material. This invention utilizes the porous polymer MCP-CU-POP formed by 5,10,15,20-tetra(4-ethynylphenyl)copper porphyrin and the macrocyclic monomer. This material exhibits synergistic antibacterial activity by combining photothermal capabilities with the effects of photothermal (PTT), photodynamic (PDT), peroxidases (PODs), and glutathione peroxidase (GSH-Px). It can also induce bacterial death by utilizing the intrinsic characteristics of the infection microenvironment and significantly accelerate the healing of bacterial infection wounds by regulating the microenvironment at the bacterial infection site.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a macrocyclic covalent organic polymer material that inhibits the π-π stacking effect and its applications. Background Technology

[0002] In recent years, emerging nanozymes have shown a trend towards becoming a new generation of antibiotics due to their excellent characteristics such as broad-spectrum antibacterial activity, low toxicity, and lack of drug resistance. Typically, nanozymes with peroxidase-like activity specifically catalyze the conversion of hydrogen peroxide into highly toxic reactive oxygen species (ROS), such as hydroxyl radicals, singlet oxygen, and superoxide anions, to attack the bacterial membrane at weakly acidic infection sites, thereby achieving a bactericidal effect. ROS kill bacteria non-specifically, therefore requiring no specific target to exert their effect. Compared to antibiotics, ROS-based bactericidal methods can kill bacteria by disrupting their cell membranes, DNA, and proteins; thus avoiding the development of bacterial resistance.

[0003] Covalent organic polymers are a class of high-molecular-weight compounds formed by covalent bonds. The atoms in their molecules form chemical bonds through shared electron pairs, exhibiting strong stability and chemical inertness. Porphyrins are large heterocyclic compounds formed by four pyrrole subunits with α-carbon atoms linked by methylene bridges (=CH-). Covalent organic polymers prepared from porphyrins possess photothermal and photodynamic effects, can prevent bacterial resistance, and are increasingly used in the preparation of antibacterial drugs. However, during porphyrin polymerization, porphyrin molecules are tightly packed together, and the large π-bond planes between them overlap, resulting in a π-π stacking effect. Therefore, the strong fluorescence of a single porphyrin is significantly weakened or even disappears after aggregation. π-π stacking leads to the rapid quenching of excited-state energy within the aggregate through "energy transfer" or "charge transfer," a process much faster than the transfer of energy to surrounding oxygen molecules. Although the aggregates can absorb light, they cannot effectively transfer energy to oxygen molecules to generate singlet oxygen, resulting in a significant decrease in the singlet oxygen quantum yield. The tight π-π packing structure may physically hinder the diffusion of oxygen molecules to the active centers of porphyrin molecules, further reducing the efficiency of reactive oxygen species generation and thus reducing photodynamic effects. Introducing a macrocyclic structure into porphyrin polymerization can suppress π-π packing. Patent application CN117327210A discloses a porphyrin microporous composite material based on a β-cyclodextrin-terephthalaldehyde inclusion complex, its preparation method, and its application. This method introduces ultra-high molecular weight cyclodextrin into porphyrin to form a macrocyclic structure, giving the reacted material excellent biocompatibility and phototherapy properties not found in traditional photosensitizers. However, since porphyrin and β-cyclodextrin polymerize to form a macrocyclic structure, cyclodextrin cannot be used directly; it must be encapsulated with terephthalaldehyde to form a β-cyclodextrin-terephthalaldehyde inclusion complex before it can polymerize with porphyrin, and its effect on suppressing π-π packing is limited. Therefore, there is a need to further develop covalent organic polymers that can inhibit porphyrin π-π stacking, possess multiple antibacterial modes, not only enhance photodynamic effects without affecting photothermal effects, but also utilize the intrinsic properties of the infection microenvironment (IME) to exert precise treatment, induce bacterial death, and significantly accelerate the healing of bacterial infection wounds by regulating the microenvironment of the bacterial infection site. Summary of the Invention

[0004] In view of the above-mentioned prior art, the purpose of this invention is to provide a macrocyclic covalent organic polymer material that suppresses the π-π stacking effect and its application. This invention uses a porous polymer MCP-CU-POP formed from 5,10,15,20-tetra(4-ethynylphenyl)copper porphyrin and a macrocyclic monomer. This material utilizes photothermal capabilities to synergistically antibacterially combine the effects of photothermal (PTT), photodynamic (PDT), peroxidases (PODs), and glutathione peroxidase (GSH-Px). It can also induce bacterial death by utilizing the intrinsic characteristics of the infection microenvironment and significantly accelerate the healing of bacterial infection wounds by regulating the microenvironment at the bacterial infection site.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides a macrocyclic covalent organic polymer material for suppressing the π-π stacking effect, wherein the macrocyclic covalent organic polymer material is a porous polymer formed by polymerizing 5,10,15,20-tetra(4-ethynylphenyl)copper porphyrin with a macrocyclic monomer.

[0007] The structural formula of the 5,10,15,20-tetra(4-ethynylphenyl)copper porphyrin is:

[0008] ;

[0009] The structural formula of the macrocyclic monomer is:

[0010] .

[0011] Preferably, the 5,10,15,20-tetrakis(4-ethynylphenyl)copper porphyrin is prepared by the following method:

[0012] (1) 4-Trimethylsilylethynylbenzaldehyde was added to propionic acid under light-protected conditions and heated to reflux under argon protection. Pyrrole was added to the system and the mixture was stirred to react. After the reaction solution was cooled to room temperature, it was filtered and washed with methanol until the filtrate was colorless to obtain a purple solid 5,10,15,20-tetrakis(4-[(trimethylsilyl)ethynyl]phenyl)porphyrin;

[0013] (2) Under nitrogen protection, 5,10,15,20-tetrakis(4-[(trimethylsilyl)ethynyl]phenyl)porphyrin was reacted with copper acetate in a mixed solvent of N,N-dimethylformamide and chloroform to obtain 5,10,15,20-tetrakis(4-[(trimethylsilyl)ethynyl]phenyl)copper porphyrin;

[0014] (3) Dissolve 5,10,15,20-tetra(4-[(trimethylsilyl)ethynyl]phenyl)copper porphyrin in anhydrous tetrahydrofuran, and slowly add a tetrahydrofuran solution containing tetrabutylammonium bromide at -78°C. After the addition is complete, heat to room temperature and stir overnight to obtain the purified ligand 5,10,15,20-tetra(4-ethynylphenyl)copper porphyrin.

[0015] Preferably, the macrocyclic monomer is prepared by the following method:

[0016] (1) Add 4,4′-dibromotriphenylamine to tetrahydrofuran, add n-butyllithium dropwise at -78℃ and stir to react, then add a tetrahydrofuran solution of 9-fluorenone dropwise, continue stirring, then heat to room temperature and stir, and after purification, 9-(4-((4-bromophenyl)(phenyl)methyl)phenyl)-9H-fluoren-9-ol is obtained;

[0017] (2) In air, 9-(4-((4-bromophenyl)(phenyl)methyl)phenyl)-9H-fluorene-9-ol, dichloromethane and methanesulfonic acid were mixed and reacted with stirring. After purification, a macrocyclic monomer was obtained.

[0018] The structural formula of the 9-(4-((4-bromophenyl)(phenyl)methyl)phenyl)-9H-fluorene-9-ol is: .

[0019] Preferably, the macrocyclic covalent organic polymer material is prepared by the following method:

[0020] In a protective atmosphere, 5,10,15,20-tetra(4-ethynylphenyl)copper porphyrin and macrocyclic monomers were added to an organic solvent and subjected to a solvothermal reaction in the presence of a catalyst and a base. After the reaction was completed, the macrocyclic covalent organic polymer material was obtained by washing and drying.

[0021] Preferably, the molar ratio of 5,10,15,20-tetra(4-ethynylphenyl)copper porphyrin to the macrocyclic monomer is 3:4.

[0022] Preferably, the organic solvent is N,N-dimethylformamide; the catalyst includes cuprous iodide and Pd(PPh3)2Cl2 as catalysts; and the base is anhydrous diisopropylamine.

[0023] Preferably, the solvothermal reaction is carried out at a temperature of 90°C for 120 hours.

[0024] A second aspect of the present invention provides the use of macrocyclic covalent organic polymer materials in the preparation of antibacterial drugs.

[0025] Preferably, the antibacterial drug exerts its antibacterial effect through the combined action of photothermal, photodynamic, and enzyme activity.

[0026] Preferably, the enzyme activity includes peroxidase activity and glutathione peroxidase activity.

[0027] The beneficial effects of this invention are:

[0028] (1) The macrocyclic covalent organic polymer material (MCP-CU-POP) prepared in this invention utilizes photothermal capabilities to combine the treatment of PTT, PDT, PODs and GSH-Px. It can not only induce bacterial death by utilizing the intrinsic characteristics of the infection microenvironment, but also significantly accelerate the recovery of bacterial infection wounds by regulating the microenvironment of the bacterial infection site.

[0029] (2) The MCP-CU-POP prepared in this invention exhibits good photothermal conversion effect when irradiated with a 638nm wavelength laser. It can also convert endogenous H2O2 into hydroxyl radicals, and the enzyme activity is further increased after photothermal treatment. In addition, MCP-CU-POP has high biocompatibility, with a red blood cell lysis rate of less than 4% and minimal impact on the cell viability of 3T3 cells. It can also promote wound healing, which is beneficial for its application in the biological field. Attached Figure Description

[0030] Figure 1 (a) Infrared spectra of MCP, TEPP-Cu and MCP-CU-POP; (b) Low-temperature N2 absorption isotherm of MCP-CU-POP at 77 K; (c) Pore size distribution curve of MCP-CU-POP; (d) Thermogravimetric curve of MCP-CU-POP; (e) X-ray diffraction pattern of MCP-CU-POP.

[0031] Figure 2 Morphological characterization of MCP-CU-POP, including (a) SEM of MCP-CU-POP at a scale of 50 nm; (b) SEM of MCP-CU-POP at a scale of 100 nm; (c) SEM of MCP-CU-POP at a scale of 200 nm; (d) SEM of MCP-CU-POP at a scale of 1 μm; (e) TEM of MCP-CU-POP at a scale of 10 nm; (f) TEM of MCP-CU-POP at a scale of 20 nm; (g) TEM of MCP-CU-POP at a scale of 50 nm; (h) TEM of MCP-CU-POP at a scale of 200 nm. HR-TEM of MCP-CU-POP at nm scale; (i) HAADF-STEM of MCP-CU-POP; (j) Elemental mapping of MCP-CU-POP; (k) C elemental mapping in MCP-CU-POP; (l) N elemental mapping in MCP-CU-POP; (m) Cu elemental mapping in MCP-CU-POP.

[0032] Figure 3 EDS spectrum of MCP-CU-POP;

[0033] Figure 4 X-ray photoelectron spectra of MCP-CU-POP, including (a) XPS analysis spectrum; (b) C 1s spectrum; (c) N 1s spectrum; (d) Cu 2p spectrum;

[0034] Figure 5The photothermal properties of MCP-CU-POP were analyzed, including (a) the concentration-dependent photothermal effect of MCP-CU-POP under laser irradiation; (b) the photothermal effect of the same concentration of MCP-CU-POP (200 μg / mL) irradiated with different laser powers for 10 min; (c) the temperature change of MCP-CU-POP (200 μg / mL) after 5 cycles; and (d) a 638 nm laser (1.2 W / cm²) 2 (a) Heating and cooling temperature change curves of MCP-CU-POP (200 μg / mL) under irradiation and curves of cooling time versus the negative natural logarithm of temperature; (b) Infrared thermal images of MCP-CU-POP at different concentrations.

[0035] Figure 6 The catalase-like activity of MCP-CU-POP was evaluated, including (a) the absorbance of different concentrations of MCP-CU-POP under 0.3% H2O2 conditions at pH 5.5; (b) the absorbance of MCP-CU-POP (200 μg / mL) in 0.3% H2O2 at different pH environments; and (c) the changes in UV absorption of TMB + H2O2 + MCP-CU-POP before and after 638 nm laser irradiation.

[0036] Figure 7 Electron spin resonance (ESR) spectra of MCP-CU-POP; where (a) superoxide anion signal detection, (b) singlet oxygen signal detection, and (c) hydroxyl radical signal detection.

[0037] Figure 8 MCP-CU-POP used a glutathione oxidase assay kit to detect the consumption of glutathione at different concentrations.

[0038] Figure 9 The sterilization effect of MCP-CU-POP is shown in the following diagrams: (a) Plate count photos of Staphylococcus aureus and Escherichia coli treated by different methods; (b) Quantitative diagram of plate count of Staphylococcus aureus treated by different methods; (c) Quantitative diagram of plate count of Escherichia coli treated by different methods.

[0039] Figure 10 : Bacterial TEM images, where (a) TEM images of Staphylococcus aureus after different treatments, and (b) TEM images of Escherichia coli after different treatments. The red tips in the images represent the locations of damage.

[0040] Figure 11 : Bacterial staining images, of which (a) fluorescence images of Staphylococcus aureus after different treatments incubated with SYTO-9 / PI live / dead staining agent; (b) fluorescence images of Escherichia coli after different treatments incubated with SYTO-9 / PI live / dead staining agent;

[0041] Figure 12 Hemolysis rate of different concentrations of MCP-CU-POP (n=3, error bars represent standard deviation);

[0042] Figure 13 Cell viability (%) after co-culturing 3T3 cells with different concentrations of MCP-CU-POP (n=3, error bars represent standard deviation);

[0043] Figure 14 Wound images of mice on days 1, 3, 5, 7, and 9 after treatment in different groups;

[0044] Figure 15 (a) Wound area percentage in mice (%); (b) Changes in body weight in mice during treatment;

[0045] Figure 16 Histological analysis was performed on H&E and Masson staining in each group.

[0046] Figure 17 Organ staining results for each treatment group;

[0047] Figure 18 Synthesis route diagram of MCP-CU-POP. Detailed Implementation

[0048] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0049] As described in the background section, the reported polymers based on 5,10,15,20-tetrakis(4-[(trimethylsilyl)ethynyl]phenyl)copper porphyrin have limited antibacterial activity, while cyclodextrin's effect on improving the π-π stacking effect of porphyrin is not significant enough, and it also requires the inclusion of small molecule compounds by cyclodextrin before it can polymerize with porphyrin.

[0050] Based on this, the purpose of this invention is to provide a macrocyclic covalent organic polymer material that suppresses the π-π stacking effect and its applications. This invention uses a porous polymer MCP-CU-POP formed from 5,10,15,20-tetra(4-ethynylphenyl)copper porphyrin and a macrocyclic monomer. The vertical structure of the fluorene ring in the macrocyclic monomer, after polymerization with the porphyrin, not only increases the spacing between porphyrins but also forms a unique three-dimensional structure, significantly improving the stacking effect (π-π stacking effect) of the planar polymer. This results in a polymer with an extremely high specific surface area, allowing for the loading of more drugs. Furthermore, the macrocyclic monomer does not need to encapsulate other compounds; after the polymer is prepared, its macrocyclic structure can still encapsulate small molecule drugs. Therefore, macrocyclic covalent organic polymer materials have extremely high application prospects, not only for antibacterial applications but also for loading and encapsulating different drugs, making them highly valuable for development and application.

[0051] The copper in MCP-CU-POP can convert endogenous H2O2 into highly toxic ·OH, thereby effectively regulating the infection microenvironment. MCP-CU-POP can achieve localized heating through photothermal conversion, rupturing bacterial membranes. In summary, the inherent photothermal and photodynamic capabilities of this material, combined with simulated peroxidases (PODs) and GSH-Px therapy, can serve as an intelligent platform. It can not only utilize the intrinsic characteristics of the infection microenvironment (IME) for precise treatment and induce bacterial death, but also significantly accelerate wound healing by regulating the microenvironment at the infection site. Furthermore, at optimal antibacterial concentrations, MCP-CU-POP exhibits almost no hemolytic effect and minimal impact on normal cell growth, making it a promising candidate for biological applications.

[0052] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0053] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels.

[0054] Example 1: Preparation of MCP-CU-POP

[0055] (1) Preparation of 5,10,15,20-tetrakis(4-[(trimethylsilyl)ethynyl]phenyl)copper porphyrin: 4-trimethylsilylethynylbenzaldehyde (4.6 g, 22.7 mmol) was added to propionic acid (150 mL) under light-protected conditions and heated to reflux under nitrogen protection. Subsequently, pyrrole (1.6 mL, 22.7 mmol) was added to the system, and the mixture was stirred at 140 °C for 2 hours. After the reaction solution was cooled to room temperature, it was filtered and washed with methanol until the filtrate was colorless, finally yielding a purple solid, 5,10,15,20-tetrakis(4-[(trimethylsilyl)ethynyl]phenyl)porphyrin.

[0056] Under nitrogen protection, 5,10,15,20-tetrakis(4-[(trimethylsilyl)ethynyl]phenyl)porphyrin (2 g, 2.0 mmol) and copper acetate tetrahydrate (3.62 g, 20.0 mmol) were dissolved in a mixed solvent of 150 mL N,N-dimethylformamide (DMF) and 50 mL chloroform, and reacted at 120 °C for 6 hours. After the reaction was completed, rotary evaporation was performed, and then a large amount of water was injected into the mixture after rotary evaporation. The precipitated solid was filtered to obtain 5,10,15,20-tetrakis(4-[(trimethylsilyl)ethynyl]phenyl)copper porphyrin.

[0057] 5,10,15,20-tetrakis(4-[(trimethylsilyl)ethynyl]phenyl)copper porphyrin (1.5 g, 1.4 mmol) was dissolved in 120 mL of anhydrous tetrahydrofuran, and tetrabutylammonium bromide (2.26 g, 7 mmol) was slowly added at -78 °C. The mixture was heated to room temperature and stirred overnight. After quenching with a large amount of water, the reaction was extracted with dichloromethane, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to obtain 5,10,15,20-tetrakis(4-ethynylphenyl)copper porphyrin TEPP-Cu.

[0058] (2) Preparation of macrocyclic monomers: In a nitrogen-filled glove box, 4,4′-dibromotriphenylamine (2.01 g, 5.0 mmol) and anhydrous tetrahydrofuran (15 mL) were added to a 40 mL glass vial equipped with a stir bar. The vial was capped with polytetrafluoroethylene, removed from the glove box, and stirred at -78 °C. Butyllithium (2.5 M, 2.0 mL, 5.0 mmol) was added dropwise via syringe, and the mixture was stirred for 2 h. Then, at -78 °C, 15 mL of anhydrous tetrahydrofuran solution containing 9-fluorenone (900 mg, 5.0 mmol) was added via syringe, and the mixture was stirred at -78 °C for 0.5 h. The mixture was then slowly heated to 25 °C and stirred for 12 h. The reaction was quenched with deionized water (5 mL). The aqueous layer was extracted with ethyl acetate (20 mL × 3). The combined organic layer was washed with saturated brine and dried on sodium sulfate. The solvent was removed under reduced pressure, and the product was purified by silica gel column chromatography to obtain 9-(4-((4-bromophenyl)(phenyl)methyl)phenyl)-9H-fluorene-9-ol.

[0059] In air, 1.51 g of 9-(4-((4-bromophenyl)(phenyl)methyl)phenyl)-9H-fluorene-9-ol, 300 mL of CH2Cl2, and 10 μL of CH3SO3H were sequentially added to a 500 mL round-bottom flask equipped with a stir bar. The flask was covered with a rubber diaphragm. The reaction mixture was stirred at 25 °C for 6 h and quenched with triethylamine (20 μL). The solvent was removed under reduced pressure, and the mixture was purified by silica gel column chromatography (eluent: petroleum ether / CH2Cl2 v / v = 3 / 1) to give the macrocyclic monomer MCP.

[0060] (3) 1.5 g of 5,10,15,20-tetra(4-ethynylphenyl)copper porphyrin and 2 g of macrocyclic monomer MCP were added to a reaction vessel, followed by 33 mL of N,N-dimethylformamide, 200 mg of cuprous iodide nanoparticles, 600 mg of Pd(PPh3)2Cl2, and 33 mL of anhydrous diisopropylamine. The reaction was carried out at 90 °C for 120 h under nitrogen protection. After the reaction, the mixture was washed with N,N-dimethylformamide and dichloromethane, and dried in a vacuum drying oven to obtain the covalent organic framework antibacterial material MCP-CU-POP based on tetraethynylphenyl porphyrin. The synthetic route is as follows: Figure 18 As shown.

[0061] Example 2: Characterization of MCP-CU-POP

[0062] (1) Using infrared spectroscopy Figure 1 (a) The structure of MCP-CU-POP was determined, and it can be clearly seen that MCP-CU-POP is located at 3000-3300 cm⁻¹. -1 The disappearance of the C≡CH peak belonging to 5,10,15,20-tetra(4-ethynylphenyl)copper porphyrin confirms the occurrence of the coupling reaction.

[0063] (2) The pore distribution of MCP-CU-POP was determined by the N2 adsorption-desorption curves and pore size distribution curves. The BET specific surface area of ​​MCP-CU-POP is 565.69 m². 2 g -1 .from Figure 1 (b) It can be seen that the adsorption curve of MCP-CU-POP exhibits typical IV isotherm characteristics, with a significant 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 MCP-CU-POP is 2.4854 nm. The pore size distribution curve also directly reflects its porosity characteristics.

[0064] (3) The thermal stability of MCP-CU-POP was determined by thermogravimetric analysis. The thermal stability of the synthesized material under N2 atmosphere was studied by thermogravimetric analysis (TGA). Figure 1 (d) shows that the first part of the mass loss of MCP-CU-POP is less than 5.07% (<200℃), which is due to the evaporation of water absorbed in the highly polar porous material; the second part of the mass loss is above 200℃, that is, the weight is maintained at 74.2% at 800℃, which is attributed to the decomposition of the porous network, indicating that the prepared MCP-CU-POP has excellent thermal stability.

[0065] (4) The crystallinity and porosity of MCP-CU-POP were estimated using powder X-ray diffraction (PXRD). Powder X-ray diffraction (PXRD) analysis was performed. Figure 1 (e) shows a large broad peak at around 25°, which can be attributed to the formation of amorphous coordination polymers.

[0066] (5) Use Figure 2 (a) ~ Figure 2 (d) SEM and Figure 2 (e)~ Figure 2 (h) TEM observation of the morphology of MCP-CU-POP. Scanning electron microscopy (SEM) showed that MCP-CU-POP is a bulk material composed of irregular particles with a uniformly distributed, interconnected macroporous structure, and its rough surface is conducive to bacterial adhesion. Transmission electron microscopy (TEM) further revealed that MCP-CU-POP has a continuous hierarchical pore structure, which is composed of loosely packed particles and exhibits clear pore characteristics.

[0067] Figure 2 (i) The elemental composition and distribution of MCP-CU-POP were determined using high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM), combined with... Figure 3 The energy-dispersive X-ray spectroscopy (EDS) pattern shows that C, N, and Cu elements are uniformly distributed on the MCP-CU-POP porous framework.

[0068] (6) XPS spectral analysis of MCP-CU-POP. As shown in Figure 4(a), similar to the EDS results, XPS also showed the presence of C, N and Cu. Figure 4 (b) indicates that the C 1s of MCP-CU-POP contains three types of carbon: SP2 (284.8 eV), SP3C (285.82 eV), and SP C (288.39 eV). Figure 4 (d) indicates that Cu2p in MCP-CU-POP is Cu 2+(934.46eV). Figure 4 (c) indicates that the two types of N in N1s of MCP-CU-POP are CN (399.65eV), C=N (398.33eV), and C=N-Cu (399.58eV).

[0069] (7) Photothermal properties of MCP-CU-POP

[0070] By varying the concentration of MCP-CU-POP (50, 100, 200, 300, 400 μg / mL, prepared with distilled water) or the laser power density (0.5, 1.0, 1.2, 1.5, and 1.8 W / cm²), 2 The photothermal conversion performance of MCP-CU-POP was studied in detail. Using phosphate buffer as a control, a 638 nm laser (1.2 W / cm²) was used. 2 Temperature changes of different concentrations of MCP-CU-POP were monitored for 10 min, and the photophysical properties of the synthesized samples were preliminarily studied.

[0071] like Figure 5 As shown in (a), unlike pure water where temperature changes are negligible, MCP-CU-POP exhibits a concentration-dependent warming behavior, which increases rapidly with increasing concentration. Specifically, concentrations of 50, 100, 200, 300, and 400 μg / mL... -1 The temperatures of the MCP-CU-POP solutions rose to 33.7, 43.3, 49.0, 52.1, and 58.2 °C, respectively. Furthermore, Figure 5 In (b), the laser power intensity was also found to be positively correlated with the dispersion temperature. Figure 5 The image in (e) obtained from a thermal imaging camera demonstrates the excellent photothermal properties of MCP-CU-POP. All these results indicate that red light can be effectively converted into localized heat by adjusting the concentration of MCP-CU-POP or the power of the laser. To evaluate the photostability of MCP-CU-POP, periodic irradiation experiments were conducted, such as... Figure 5 (c) shows that the MCP-CU-POP has a stable laser switching effect, and the temperature hardly fluctuates after 5 consecutive laser on / off cycles. Figure 5 (d) shows the values ​​of τS and θ in the formula for calculating photothermal conversion efficiency, which is 33.4%.

[0072] (8) Peroxidase activity of MCP-CU-POP

[0073] The peroxidase activity of MCP-CU-POP at different pH values ​​was investigated. The ROS generation capacity of MCP-CU-POP was evaluated using a two-substrate system: a colorimetric system of H₂O₂ and 3,3',5,5'-tetramethylbenzidine (TMB), with TMB as the chromogenic agent. TMB can be oxidized by ROS to form chromogenic ox-TMB. The presence of copper endows MCP-CU-POP with the ability to act as a highly efficient •OH generator in acidic media.

[0074] The TMB used in the experiment was prepared as follows: 3.606 mg of TMB (0.015 mmol / L) was dissolved in 10 mL of ethanol to prepare a 1.5 mmol / L TMB ethanol solution; the mass concentration of H2O2 used in the experiment was 30%. 50 mL of PBS with a pH of 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.

[0075] Preparation of MCP-CU-POP at different pH values: Weigh 1 mg of MCP-CU-POP and disperse it thoroughly in 1 mL of PBS with different pH values ​​using an ultrasonic apparatus to prepare 1 mg / mL stock solutions with pH values ​​of 1.5, 2.5, 3.5, 4.5, 5.5, and 6.5. Then, take 200 μL of each stock solution and add it to 550 μL of PBS with pH values ​​of 1.5, 2.5, 3.5, 4.5, 5.5, and 6.5. Add 75 μL each of TMB and H2O2 to prepare PBS dispersions of MCP-CU-POP with a concentration of 200 μg / mL and pH values ​​of 1.5, 2.5, 3.5, 4.5, 5.5, and 6.5.

[0076] Preparation of MCP-CU-POP at different concentrations: Weigh 1 mg of MCP-CU-POP and disperse it thoroughly in 1 mL of pH 5.5 PBS using an ultrasonic apparatus to prepare a stock solution of 1 mg / mL. Then, take 50, 100, 200, 300, 400, and 500 μL from the stock solution and add them to 800, 750, 650, 550, 450, and 350 μL of pH 5.5 PBS, respectively. Add 75 μL each of TMB and H2O2 to prepare PBS dispersions of MCP-CU-POP with concentrations of 50, 100, 200, 300, 400, and 500 μg / mL.

[0077] MCP-CU-POP can act as a POD-like enzyme, catalyzing the generation of highly toxic hydroxyl radicals (•OH) and effectively oxidizing TMB. Therefore, only in a medium containing both MCP-CU-POP and H2O2 can colorless TMB be oxidized to blue oxTMB, exhibiting a characteristic absorbance peak at 652 nm. Therefore, all experiments were conducted under the conditions of MCP-CU-POP + H2O2 + TMB. Experiments with different concentrations of MCP-CU-POP (conducted under 0.3% H2O2 at pH 5.5) and experiments with different pH values ​​of MCP-CU-POP (MCP-CU-POP concentration of 200 μg / mL) were conducted without laser irradiation. Figure 6 (a) and Figure 6 (b) shows that the enzymatic catalytic activity of MCP-CU-POP is highly dependent on the pH of the solution and the concentration of MCP-CU-POP.

[0078] like Figure 6 As shown in (c), 75 μL of H2O2 was added to a 200 μg / mL MCP-CU-POP solution at pH 5.5. After irradiation with a 1.2 W 638 nm laser for 10 minutes, 75 μL of TMB was quickly added. It can be seen that the characteristic peak of TMB under laser irradiation showed a significant increase, which proves that laser can promote the enhancement of peroxidase activity.

[0079] (9) Photodynamic verification of MCP-CU-POP

[0080] Hydroxyl radical detection: 2 mg MCP-CU-POP was added to 2 ml of ultrapure water and ultrasonically dispersed. 200 μl of the solution was transferred and 200 μl of 50 mM DMPO solution was added. The solution was drawn up with a capillary pipette, mixed and shaken well, and then placed in the instrument for testing. The laser wavelength was set to 638 nm, the power to 1.2 W, and the irradiation time to 10 min.

[0081] Singlet oxygen detection: 2 mg MCP-CU-POP was added to 2 ml of ultrapure water and ultrasonically dispersed. 200 μl of the solution was transferred and 200 μl of 50 mM TEMP solution was added. The solution was drawn up with a capillary pipette, mixed and shaken well, and then placed in the instrument for testing. The laser wavelength was set to 638 nm, the power to 1.2 W, and the irradiation time to 10 min.

[0082] Superoxide radical detection: 2 mg MCP-CU-POP was added to 2 ml of methanol and ultrasonically dispersed. 200 μl of the solution was transferred and 200 μl of 50 mM DMPO solution was added. The solution was drawn up with a capillary pipette, mixed and shaken well, and then placed in the instrument for testing. The laser wavelength was set to 638 nm, the power to 1.2 W, and the irradiation time to 10 min.

[0083] Capture characteristic signals and compare them with the dark control group.

[0084] like Figure 7 As shown, MCP-CU-POP can be more clearly seen from electron spin resonance (ESR) under laser irradiation. Figure 7 (a) It can be seen that MCP-CU-POP exhibits a typical superoxide anion signal peak under laser irradiation conditions. 7(b) is a singlet oxygen signal peak diagram. Figure 7 (c) is the hydroxyl radical signal peak, proving that superoxide anions, singlet oxygen, and hydroxyl radicals are generated under laser irradiation.

[0085] (10) Validation of GSH-Px activity of MCP-CU-POP

[0086] The ability of MCP-CU-POP to consume GSH was detected using the Yuanye R22075 reduced glutathione (GSH) assay kit, and the results were plotted using a UV-Vis spectrophotometer. Figure 8 It can be seen that as the concentration of MCP-CU-POP increases, the amount of GSH consumed also increases, and the GSH consumption capacity of MCP-CU-POP exhibits a concentration-dependent relationship.

[0087] Test Example 1: In vitro antibacterial test

[0088] (1) Bacterial culture

[0089] This experiment used two bacteria, *Staphylococcus aureus* and *Escherichia coli*, and utilized second-generation bacteria to complete the following experiments. The specific culture method for the second-generation bacteria was as follows: First, the frozen bacteria were thawed at 37°C. 100 µL of the bacterial culture was transferred to a shaker tube containing 5 mL of liquid culture medium and incubated on a shaker (110 rpm, 37°C) for 12 hours. Then, 100 µL of the cultured bacterial culture was transferred to a 2 mL EP tube containing 900 µL of the culture medium, and further diluted using a serial dilution method at 10-fold increments. -2Dilute 5-10 tubes, take 100 µL of bacterial suspension from each tube, and spread it evenly on a petri dish containing solid culture medium using a spreader. Incubate at 37°C for 24 hours, observe clonal morphology and colony count. The petri dish with approximately 1000 colonies is considered the first generation of bacteria. Use a pick to add one colony of the first generation to a shaker tube containing 5 mL of liquid culture medium. Culture the second generation of bacteria using the same method. The specific preparation method for the liquid culture medium is as follows: Disperse 5g of LB broth in 200 mL of distilled water, and then autoclave. The specific preparation method for the solid culture medium is as follows: Disperse 5g of LB broth and 3g of agar in 200 mL of distilled water, and then autoclave.

[0090] (2) In vitro antibacterial activity of MCP-CU-POP under different treatments

[0091] The antibacterial activity under different treatments was studied using the plate count method. The experiment was divided into eight groups: (I) PBS (laser off), PBS (laser on), (II) H2O2 (laser off), H2O2 (laser on), (III) MCP-CU-POP (laser off), MCP-CU-POP (laser on), (IV) MCP-CU-POP+H2O2 (laser off), MCP-CU-POP+H2O2 (laser on).

[0092] 10 µL of 10 was added to each of the above groups. 8 CFU mL -1 Bacterial solutions were prepared using PBS at pH 7.4. All solutions contained 10 μL of 30 wt% H2O2 in the H2O2-containing group and 200 μg / mL of MCP-CU-POP in the MCP-CU-POP-containing group. The laser-containing group used 1 mL of PBS. The laser parameters for the laser-containing group were λ = 638 nm and 1.2 W / cm². 2 10 minutes.

[0093] Then, according to the groups, each group was placed in a constant temperature shaker (110 rpm, 37℃) and incubated for 12 hours. The cultured bacterial solutions were then serially diluted 10⁻⁶ times using the same method as bacterial culture. 5 The bacterial culture was doubled, and 100 μL of the well-spread bacterial solution was transferred to a solid culture medium, spread evenly, and incubated at 37°C for 24 h. Colonies were counted and bacterial activity was compared with that of each group. Figure 9As shown, compared with the PBS (laser off) group and the PBS (laser on) group, the H2O2 (laser off) group and the H2O2 (laser on) group showed only slight antibacterial effects on bacteria. Groups III and IV show that laser irradiation enhances the bactericidal effect of MCP-CU-POP. Specifically, comparing the colony counts (CFU) of Staphylococcus aureus and Escherichia coli, the MCP-CU-POP (laser on) group significantly reduced the colony counts of Staphylococcus aureus and Escherichia coli to 49.8% and 1.8%, respectively, while the MCP-CU-POP (laser off) group showed colony counts of 93.3% and 92.7%, respectively. Compared with the MCP-CU-POP (laser off) group, due to synergistic effects, the colony counts of Staphylococcus aureus and Escherichia coli in the MCP-CU-POP + H2O2 (laser off) group were also significantly reduced to 13.65% and 17.0%, respectively. The sterilization efficiency of the MCP-CU-POP+ H2O2 (laser-activated) group is further improved, almost eliminating all bacteria, and reducing the number of Staphylococcus aureus and Escherichia coli colonies to 0.06% and 0.33%, respectively.

[0094] Experimental Example 2: Transmission Electron Microscopy of Bacteria

[0095] Following the method described in Example 1, the cultured bacterial suspensions were divided into eight treatment groups: the experimental groups were the same as in Example 1. Then, 100 μL of each of the eight groups 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 dehydrated by continuous treatment with ethanol solutions (30 wt%, 50 wt%, 70 wt%, 90 wt%, 95 wt%, and 100 wt%) at room temperature for 10 min, followed by treatment with acetone at room temperature for 3 h. The bacteria were then embedded in a gradient infiltration medium (epoxy resin) (impregnated with acetone and epoxy resin at mass ratios of 3:1, 1:1, and 1:3 for 1 hour each, and finally impregnated with pure epoxy resin overnight), negatively stained, and sectioned on a nickel grid. The nickel grid was placed under TEM to capture bacterial morphology. TEM was used to observe the integrity of the bacterial membranes in different treatment groups.

[0096] like Figure 10 As 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. However, bacteria treated under different MCP-CU-POP conditions showed cell membrane distortion and even rupture, indicating that MCP-CU-POP has a significant antibacterial effect. Among these groups, bacteria treated with MCP-CU-POP+H2O2 (laser on) showed the most significant morphological changes, leakage of cell contents, and complete disruption of the cell membrane.

[0097] Experimental Example 3: Bacterial Viability / Deadness Staining Test

[0098] 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 as green; PI only penetrates damaged bacterial membranes, marking the bacteria as red, while reducing the green color of SYTO-9.

[0099] The cultured bacterial suspension was divided into eight treatment groups according to the method in Experimental Example 1: the experimental groups were the same as in Experimental Example 1. Then, 100 µL of bacterial suspension from each group was mixed with 20 µL of SYTO-9 (1.0 × 10⁻⁶). -3 M) and 20 µL PI (1.5×10 -3 M) Incubate in the dark at 37°C for 15 min. After staining, centrifuge each group in PBS to remove excess SYTO-9 and PI. Then resuspend the bacteria in 50 µL PBS and place them on a glass slide. Images of Escherichia coli or Staphylococcus aureus are then captured using a fluorescence inverted microscope.

[0100] from Figure 11 The significant PTT / CAT synergistic antibacterial effect of MCP-CU-POP can be directly observed in both live and dead bacterial staining results. Bacteria treated with PBS (laser off), PBS (laser on), H2O2 (laser off), and H2O2 (laser on) groups showed strong green fluorescence, consistent with the plate count results. In contrast, bacteria treated with MCP-CU-POP (laser off), MCP-CU-POP (laser on), and MCP-CU-POP+H2O2 (laser off) groups showed obvious red fluorescence. Simultaneously, the MCP-CU-POP+H2O2 (laser on) group showed almost complete bacterial death, exhibiting the highest bactericidal efficiency; all bacteria were stained with red fluorescence, indicating a large number of dead bacteria.

[0101] Experimental Example 4: In vitro biocompatibility experiment

[0102] (1) Hemolysis test

[0103] Fresh blood was collected from 5-week-old female Kunming 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 volume of PBS until colorless, then the supernatant was discarded. Red blood cells were diluted with PBS at a volume ratio of 3:11, and then MCP-CU-POP solution at different concentration gradients (100, 200, 300, 400 μg / mL) was added (red blood cell solution:MCP-CU-POP solution volume ratio = 1:9). The mixture was incubated at 37℃ 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-labeled immunosorbent assay (ELISA). Distilled water was used as a positive control, and PBS as a negative control. The formula for calculating hemolysis volume is as follows:

[0104] Hemolysis volume (%) = (A-An) / (Ap-An) × 100%;

[0105] Where "A" represents the absorbance obtained by taking the supernatant after adding MCP-CU-POP to red blood cells. "An" represents the absorbance obtained by taking the supernatant after adding PBS to red blood cells (negative control). "Ap" represents the absorbance obtained by taking the supernatant after adding distilled water to red blood cells (positive control).

[0106] like Figure 12 As shown, MCP-CU-POP exhibits only very low hemolytic activity (below 4%) or no hemolytic activity within the concentration range where antibacterial activity is observed. The hemolysis rate of MCP-CU-POP varies with its concentration, remaining below 1% as the concentration increases from 100 to 400 μg / mL. This indicates that MCP-CU-POP has excellent blood compatibility and does not damage the erythrocyte membrane.

[0107] (2) Cytotoxicity test

[0108] In 96-well plates, mouse 3T3 fibroblasts (from the Cell Bank of the Chinese Academy of Sciences) were cultured at a density of 5 × 10⁶ cells per well. 3 Seed cells at a density of 180 µL per well, with 200 µL of PBS added to the surrounding replicate wells for liquid sealing to prevent excessive evaporation. After 24 h of incubation, 20 µL of MCP-CU-POP at different concentrations (100-500 μg / mL) was added and incubated for another 24 h. Then, 20 µL of MTT (4 mg / mL) solution was added to each well, and the cells were incubated for 4 h. The supernatant was then aspirated, and 150 µL of dimethyl sulfoxide was added to dissolve the MTT (tetramethylazazole blue). After dissolving on a shaker for 10 min, the absorbance of the 96-well plate was measured at 570 nm using a microplate reader. Each experiment was repeated three times. Figure 13As shown, after 24 h of culture, even at a high concentration (500 μg / mL), the survival rate of 3T3 cells treated with MCP-CU-POP was around 90%, indicating that MCP-CU-POP has low cytotoxicity.

[0109] Experimental Example 5: In vivo wound healing experiment

[0110] Five-week-old Kunming mice (purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.) were randomly divided into 6 groups: 3 mice in each group. (I) PBS group, (II) H2O2 group, (III) MCP-CU-POP group, (IV) MCP-CU-POP + laser group, (V) MCP-CU-POP + H2O2 group, and (VI) MCP-CU-POP + H2O2 + laser group. Normal mice without any treatment served as the control group. The control group received no treatment. For the other groups, the hind hair of each mouse was shaved before surgery to create a 5 mm diameter wound, which was then infected with Staphylococcus aureus (1×10⁻⁶). 6 A wound healing model was established by applying 200 μg / mL MCP-CU-POP solution to mice for 24 hours. Mice were treated according to the requirements of different group settings, with the wound covered by 200 μg / mL MCP-CU-POP solution. Wound appearance 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).

[0111] like Figure 14 As shown, the wound area of ​​mice in different groups gradually decreased over time. The degree of healing varied considerably among the different groups after 9 days. Figure 15 (a) It can be seen that among the six groups, the MCP-CU-POP+H2O2+laser group showed the most significant advantage in wound healing and skin regeneration, with a wound healing rate of over 93%, far exceeding that of the PBS group (75.1%), H2O2 group (79.0%), MCP-CU-POP group (81.1%), MCP-CU-POP + laser group (88.3%), and MCP-CU-POP + H2O2 group (88.9%). Meanwhile, compared with the control group, Figure 15 (b) It can be seen that no significant weight change was detected during the entire treatment process, indicating that MCP-CU-POP has good biocompatibility.

[0112] Histological analysis was performed using hematoxylin and eosin (H&E) and Masson staining to directly assess the healing status of skin tissue 9 days after treatment. Figure 16As shown, the PBS-treated group exhibited significant inflammatory cells and incomplete skin epidermis. In contrast, the MCP-CU-POP-treated groups showed varying degrees of skin structure regeneration. The MCP-CU-POP+H2O2+laser group demonstrated the most significant effect in promoting wound healing, with visible collagen fibers and a naturally matured epidermal layer, indicating complete and good wound healing.

[0113] In addition, histological sections were collected, and H&E staining was used to assess the damage to internal organs (heart, liver, spleen, lungs, and kidneys) in different groups. Figure 17 As shown, no abnormal lesions or inflammation were observed in the major organs, and no histological changes were found. These results confirm that MCP-CU-POP has good biocompatibility in vivo.

[0114] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A macrocyclic covalent organic polymer material for suppressing π-π stacking effects, characterized in that, The macrocyclic covalent organic polymer material is a porous polymer formed by the polymerization of 5,10,15,20-tetra(4-ethynylphenyl)copper porphyrin with macrocyclic monomers. The structural formula of the 5,10,15,20-tetra(4-ethynylphenyl)copper porphyrin is: ； The structural formula of the macrocyclic monomer is: 。 2. The macrocyclic covalent organic polymer material according to claim 1, characterized in that, The 5,10,15,20-tetra(4-ethynylphenyl)copper porphyrin was prepared by the following method: (1) 4-Trimethylsilylethynylbenzaldehyde was added to propionic acid under light-protected conditions and heated to reflux under argon protection. Pyrrole was added to the system and the mixture was stirred to react. After the reaction solution was cooled to room temperature, it was filtered and washed with methanol until the filtrate was colorless to obtain a purple solid 5,10,15,20-tetrakis(4-[(trimethylsilyl)ethynyl]phenyl)porphyrin; (2) Under nitrogen protection, 5,10,15,20-tetrakis(4-[(trimethylsilyl)ethynyl]phenyl)porphyrin was reacted with copper acetate in a mixed solvent of N,N-dimethylformamide and chloroform to obtain 5,10,15,20-tetrakis(4-[(trimethylsilyl)ethynyl]phenyl)copper porphyrin; (3) Dissolve 5,10,15,20-tetra(4-[(trimethylsilyl)ethynyl]phenyl)copper porphyrin in anhydrous tetrahydrofuran, and slowly add a tetrahydrofuran solution containing tetrabutylammonium bromide at -78°C. After the addition is complete, heat to room temperature and stir overnight to obtain the purified ligand 5,10,15,20-tetra(4-ethynylphenyl)copper porphyrin.

3. The macrocyclic covalent organic polymer material according to claim 1, characterized in that, The macrocyclic monomer is prepared by the following method: (1) Add 4,4′-dibromotriphenylamine to tetrahydrofuran, add n-butyllithium dropwise at -78℃ and stir to react, then add a tetrahydrofuran solution of 9-fluorenone dropwise, continue stirring, then heat to room temperature and stir, and after purification, 9-(4-((4-bromophenyl)(phenyl)methyl)phenyl)-9H-fluoren-9-ol is obtained; (2) In air, 9-(4-((4-bromophenyl)(phenyl)methyl)phenyl)-9H-fluorene-9-ol, dichloromethane and methanesulfonic acid were mixed and reacted with stirring. After purification, a macrocyclic monomer was obtained.

4. The macrocyclic covalent organic polymer material according to claim 1, characterized in that, The macrocyclic covalent organic polymer material is prepared by the following method: In a protective atmosphere, 5,10,15,20-tetra(4-ethynylphenyl)copper porphyrin and macrocyclic monomers were added to an organic solvent and subjected to a solvothermal reaction in the presence of a catalyst and a base. After the reaction was completed, the macrocyclic covalent organic polymer material was obtained by washing and drying.

5. The macrocyclic covalent organic polymer material according to claim 4, characterized in that, The molar ratio of the 5,10,15,20-tetra(4-ethynylphenyl)copper porphyrin to the macrocyclic monomer is 3:

4.

6. The macrocyclic covalent organic polymer material according to claim 4, characterized in that, The organic solvent is N,N-dimethylformamide; the catalyst includes cuprous iodide and Pd(PPh3)2Cl2; and the base is anhydrous diisopropylamine.

7. The macrocyclic covalent organic polymer material according to claim 4, characterized in that, The solvothermal reaction was carried out at a temperature of 90°C for 120 hours.

8. The use of the macrocyclic covalent organic polymer material according to any one of claims 1 to 7 in the preparation of antibacterial drugs.

9. The application according to claim 8, characterized in that, The antibacterial drug exerts its antibacterial effect through the combined action of photothermal, photodynamic, and enzyme activity.

10. The application according to claim 9, characterized in that, The enzyme activity includes peroxidase activity.

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