Covalent organic framework antibacterial material with NO light-controlled release and preparation method and application thereof

Abstract

The application discloses a kind of covalent organic framework antibacterial materials with NO light control release and its preparation method and application, belong to biological medicine technical field.The covalent organic framework antibacterial material is the porous polymer with cyclodextrin inclusion compound and porphyrin metal coordination compound as structural unit;There are several porphyrin metal coordination compounds around cyclodextrin inclusion compound in each structural unit, and porphyrin metal coordination compound is not all on the same plane;The cyclodextrin inclusion compound is β-cyclodextrin inclusion BNN6;The porphyrin metal coordination compound is the coordination compound of porphyrin and copper.The application first prepares cyclodextrin porphyrin porous polymer, then it is cooperated with copper and is loaded BNN6 to obtain covalent organic framework antibacterial material, utilizes NO release and combines simulation photothermal, photodynamic, peroxidase and glutathione peroxidase synergistic antibacterial, significantly accelerates the recovery of bacterial infection wound.

Description

Technical Field

[0001] This invention relates to the field of technology, specifically to a covalent organic framework antibacterial material with NO photocontrolled release, its preparation method, and its application. Background Technology

[0002] The emergence of multidrug-resistant bacteria has significantly reduced the effectiveness of traditional antibiotic treatments, posing a severe challenge of "no drugs available" in clinical anti-infective therapy. Therefore, developing novel anti-infective strategies and functional materials has become an urgent priority. An ideal anti-infective system should possess highly efficient bactericidal capabilities, good biosafety, targeted targeting, and resistance to drug resistance, while also being able to cope with complex infection scenarios such as biofilms. In recent years, emerging technologies such as precision delivery systems based on functional carrier materials and responsive antimicrobial agents (such as NO-releasing materials) have shown great potential, providing new research directions for overcoming the bottlenecks of traditional anti-infective therapy and achieving highly efficient and low-toxicity interventions for bacterial infections.

[0003] Nitric oxide (NO) is a lipid-soluble small molecule free radical that can cross biological membranes through diffusion. As a novel antibacterial agent, NO can directly kill bacteria without inducing drug resistance by inducing lipid peroxidation, bacterial cell membrane rupture, and DNA deamination. NO is a highly reactive gas molecule with an extremely short half-life (a few seconds to a few minutes), making it difficult to control the release rate and dosage when used directly. Excessive local concentrations can easily lead to cytotoxicity (such as damage to normal tissues or induction of inflammatory responses), while insufficient concentrations may render it ineffective. BNN6, however, can slowly and continuously release NO under specific conditions (such as pH, phototriggered release, etc.), achieving "on-demand release," reducing side effects caused by instantaneous high concentrations of NO, and improving treatment safety. Patent application CN116099001A discloses a CD-Por-Cu-BNN6 photothermal antibacterial agent, its preparation method, and its application. This involves loading a NO-producing BNN6 donor into a porous CD-Por-Cu substrate. However, using only the loading method, BNN6 is prone to leakage, reducing NO production. Cyclodextrins, as a class of cyclic oligosaccharides with hydrophobic cavities, possess a unique "internal hydrophobic, external hydrophilic" structure, enabling them to encapsulate guest molecules through host-guest recognition. They can effectively encapsulate BNN6, thus enhancing NO release. However, encapsulating BNN6 solely with cyclodextrin offers a limited antibacterial mechanism. Currently, there are reports of combining cyclodextrins with porphyrins. For example, patent application CN117327210A discloses a porphyrin microporous composite material based on a β-cyclodextrin-terephthalaldehyde inclusion complex, its preparation method, and its application. This composite material, prepared using the β-cyclodextrin-terephthalaldehyde inclusion complex and porphyrins as structural units, is used as a photosensitizer. However, the small cavity structure of β-cyclodextrin significantly enlarges the interlayer spacing between porphyrins, making it difficult to overcome the π-π stacking problem between planes during porphyrin stacking. If a structure is to be designed that can combine porphyrin, cyclodextrin, and BNN6 to exert multiple antibacterial modes, and solve the problem of π-π stacking of porphyrin polymers, then high-efficiency antibacterial effect can be achieved by combining multiple modes. Summary of the Invention

[0004] To address the aforementioned limitations of existing technologies, the present invention aims to provide a covalent organic framework antibacterial material with controlled NO release, its preparation method, and its applications. The present invention first prepares a cyclodextrin porphyrin porous polymer, then combines it with copper and encapsulates BNN6 to obtain a covalent organic framework antibacterial material (CD-Por-Cu-BNN6). This material utilizes NO release combined with simulated photothermal (PTT), photodynamic (PDT), peroxidases (PODs), and glutathione peroxidase (GSH-Px) for synergistic antibacterial effects. Furthermore, it can induce bacterial death by utilizing the intrinsic characteristics of the infection microenvironment and significantly accelerate wound healing 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 covalent organic framework antibacterial material with NO light-controlled release, wherein the covalent organic framework antibacterial material is a porous polymer with cyclodextrin inclusion complexes and porphyrin metal coordination compounds as structural units; each structural unit contains a plurality of porphyrin metal coordination compounds surrounding the cyclodextrin inclusion complexes, and the porphyrin metal coordination compounds are not all on the same plane; the cyclodextrin inclusion complex is β-cyclodextrin inclusion complex BNN6; the porphyrin metal coordination compound is a coordination compound of porphyrin and copper.

[0007] Preferably, the β-cyclodextrin is hepta(6-deoxy-6-(4-formylphenyl))-β-cyclodextrin; and the porphyrin is 5,10,15,20-tetra(4-aminophenyl)porphyrin.

[0008] Preferably, the structural formula of the covalent organic framework antibacterial material is:

[0009] .

[0010] A second aspect of the present invention provides a method for preparing a covalent organic framework antibacterial material, comprising the following steps:

[0011] (1) Dissolve 4-hydroxybenzaldehyde and potassium carbonate in DMF and react at room temperature under a protective atmosphere. Then add hepta(6-iodo-6-deoxy)-β-cyclodextrin solution (prepared with N,N-dimethylformamide solvent) to react and obtain hepta(6-deoxy-6-(4-formylphenyl))-β-cyclodextrin.

[0012] (2) Hepta(6-deoxy-6-(4-formylphenyl))-β-cyclodextrin and 5,10,15,20-tetra(4-aminophenyl)porphyrin were dispersed in acetic acid and subjected to a solvothermal reaction under a protective atmosphere to obtain the cyclodextrin porphyrin porous polymer CD-Por-POP;

[0013] (3) Add the cyclodextrin porphyrin porous polymer and copper salt to a mixed solvent and carry out a solvothermal reaction under a protective atmosphere. After the reaction is complete, filter, wash and dry to obtain CD-Por-Cu.

[0014] (4) CD-Por-Cu was added to the ethanol solution of BNN6 and the reaction was carried out in the dark under a protective atmosphere. After the reaction was completed, the mixture was centrifuged, washed and dried to obtain the covalent organic framework antibacterial material CD-Por-Cu-BNN6 with NO light-controlled release.

[0015] Preferably, in step (1), the molar ratio of 4-hydroxybenzaldehyde, potassium carbonate and hepta(6-iodo-6-deoxy)-β-cyclodextrin is 10:10:1; and the reaction time at room temperature is 2 hours.

[0016] Preferably, in step (2), the molar ratio of hepta(6-deoxy-6-(4-formylphenyl))-β-cyclodextrin and 5,10,15,20-tetra(4-aminophenyl)porphyrin is greater than or equal to 4:7; the temperature of the solvothermal reaction is 120°C and the time is 3 days.

[0017] Preferably, in step (3), the copper salt is copper chloride; the molar ratio of the cyclodextrin porphyrin porous polymer and the copper salt is less than or equal to 1:7; the mixed solvent is DMF and pH 5.5 phosphate buffer mixed at a volume ratio of 5:1; the temperature of the solvothermal reaction is 30°C and the time is 2 days.

[0018] Preferably, in step (4), the mass ratio of CD-Por-Cu to BNN6 is 5:1; the BNN6 solution is prepared by dissolving N,N'-disec-butyl-N,N'-dinitroso-1,4-phenylenediamine in 70wt% ethanol solution, with a concentration of 200 μg / mL; the temperature of the light-protected reaction is 30℃ and the time is 24h.

[0019] A third aspect of the present invention provides the use of covalent organic framework antimicrobial materials in the preparation of antimicrobial drugs.

[0020] Preferably, the covalent organic framework antibacterial material synergistically inhibits bacteria through five modes: photothermal, photodynamic, peroxidase, glutathione peroxidase, and NO release.

[0021] The beneficial effects of this invention are:

[0022] (1) The CD-Por-Cu-BNN6 of the present invention is based on a cyclodextrin-porphyrin porous polymer with a pocket structure. By adding copper salt to form a complex with porphyrin and adding BNN6, it can not only be loaded into the porous polymer, but also be encapsulated by cyclodextrin, thus loading a covalent organic framework antibacterial material. This material utilizes NO release combined with simulated PTT, PDT, PODs and GSH-Px treatment. It can not only induce bacterial death by utilizing the intrinsic characteristics of the infection microenvironment, but also significantly accelerate the healing of bacterial infection wounds by regulating the microenvironment of the bacterial infection site.

[0023] (2) The CD-Por-Cu-BNN6 of the present invention produces a good photothermal conversion effect when irradiated with a laser of 638 nm wavelength. At the same time, it can convert H2O2 into hydroxyl radicals, and the enzyme activity is further increased after photothermal treatment. In addition, CD-Por-Cu-BNN6 has high biocompatibility, with a red blood cell lysis rate of less than 4%, little impact on the cell viability of 3T3 cells, and can promote wound healing, which is beneficial for its application in the biological field.

[0024] (3) The copper contained in CD-Por-Cu-BNN6 of the present invention can convert H2O2 into •OH, which is highly toxic to bacteria, thereby effectively killing bacteria. CD-Por-Cu-BNN6 can achieve local heating through photothermal conversion, so as to rupture the bacterial membrane. The photodynamic effect of CD-Por-Cu-BNN6 also has a strong effect on killing bacteria. In summary, the inherent photothermal and photodynamic capabilities of this material, combined with the simulation of peroxidase (PODs) and GSH-Px treatment, can serve as an intelligent platform. It can not only utilize the intrinsic characteristics (IME) of the infection microenvironment to perform accurate treatment and induce bacterial death, but also significantly accelerate the recovery of bacterial infection wounds by regulating the microenvironment of the bacterial infection site. Furthermore, CD-Por-Cu-BNN6 has almost no hemolytic effect at the optimal antibacterial concentration and hardly affects the growth of normal cells, making it a promising candidate for biological applications. Attached Figure Description

[0025] Figure 1 (a) Infrared spectra of TAPP, 7F-β-CD, CD-Por-POP, CD-Por-Cu, CD-Por-Cu-BNN6 and BNN6; (b) Low-temperature N2 absorption isotherms of CD-Por-Cu and CD-Por-Cu-BNN6 at 77 K; (c) Thermogravimetric analysis of CD-Por-Cu-BNN6; (d) X-ray diffraction patterns of CD-Por-Cu and CD-Por-Cu-BNN6;

[0026] Figure 2Morphological characterization of CD-Por-Cu-BNN6, including (a) SEM of CD-Por-Cu-BNN6 at a scale of 20 μm; (b) SEM of CD-Por-Cu-BNN6 at a scale of 50 μm; (c) SEM of CD-Por-Cu-BNN6 at a scale of 500 nm; (d) TEM of CD-Por-Cu-BNN6 at a scale of 0.2 μm; (e) TEM of CD-Por-Cu-BNN6 at a scale of 50 nm; (f) HR-TEM of CD-Por-Cu-BNN6 at a scale of 5 nm; (g) HAADF-STEM of CD-Por-Cu-BNN6; (h) C elemental mapping; (i) N elemental mapping; (j) O elemental mapping; (k) Cu elemental mapping.

[0027] Figure 3 EDS spectrum of CD-Por-Cu-BNN6;

[0028] Figure 4 X-ray photoelectron spectra of CD-Por-Cu-BNN6, including (a) XPS analysis spectrum; (b) C1s spectrum; (c) Cu2p spectrum; (d) N1s spectrum; and (e) O1s spectrum.

[0029] Figure 5 Photothermal properties of CD-Por-Cu-BNN6, including (a) concentration-dependent photothermal effect of CD-Por-Cu-BNN6 under laser irradiation; (b) photothermal effect of CD-Por-Cu-BNN6 (200 μg / mL) at different laser powers for 10 min; (c) infrared thermographic images of CD-Por-Cu-BNN6 at different concentrations; (d) temperature change of CD-Por-Cu-BNN6 (200 μg / mL) after 5 cycles; and (e) photothermal effect of CD-Por-Cu-BNN6 at 1.2 W / cm². 2 Temperature curves under 638 nm laser irradiation and the negative natural logarithmic change of temperature during cooling process;

[0030] Figure 6 The catalase-like activity of CD-Pro-Cu-BNN6 was verified, including (a) the absorbance of CD-Pro-Cu-BNN6 (200 μg / mL) in 0.3% H2O2 at different pH environments; (b) the absorbance of CD-Pro-Cu-BNN6 at different concentrations in 0.3% H2O2 at pH 5.5; and (c) the changes in catalase-like activity under argon atmosphere, regardless of whether laser irradiation with a TMB probe was used.

[0031] Figure 7 NO release activity of CD-Por-Cu-BNN6, wherein (a) different concentrations of CD-Por-Cu-BNN6 release NO in a 45℃ water bath; (b) different concentrations of CD-Por-Cu-BNN6 (100, 200 and 300 μg / mL) release NO in a laser (1.2 W / cm²) bath. 2 (c) NO release under irradiation; (d) Loading efficiency of BNN6 in CD-Por-Cu-BNN6 system at different concentrations;

[0032] Figure 8 Photodynamic activity of CD-Por-Cu-BNN6, including (a) the photometric absorption curves of CD-Por-Cu-BNN6 at different laser irradiation periods; (b) the photometric absorption curves of MB at different laser irradiation times; (c) the photometric absorption curves of CD-Por-Cu-BNN6 under laser irradiation at different times; and (d) the photometric absorption curves of DPBF under laser irradiation at different times.

[0033] Figure 9 The consumption of glutathione at different concentrations was detected using a glutathione oxidase assay kit for CD-Por-Cu-BNN6.

[0034] Figure 10 The bactericidal efficacy of CD-Por-Cu-BNN6 was verified, including (a) plate count images of Staphylococcus aureus and Escherichia coli treated with different concentrations of CD-Por-Cu-BNN6 + H2O2 + laser; (b) bactericidal effect diagram of CD-Por-Cu-BNN6 + H2O2 + laser plate count; (c) plate count images of Staphylococcus aureus treated with different treatment methods; (d) plate count images of Staphylococcus aureus treated with different treatment methods; (e) plate count quantification diagram of Escherichia coli treated with different treatment methods; and (f) plate count quantification diagram of Escherichia coli treated with different treatment methods.

[0035] 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;

[0036] Figure 12 Hemolysis rate of CD-Por-Cu-BNN6 at different concentrations (n=3, error bars represent standard deviation);

[0037] Figure 13Cell viability (%) after co-culturing 3T3 cells with different concentrations of CD-Por-Cu-BNN6 (n=3, error bars represent standard deviation);

[0038] Figure 14 Wound images of mice on days 1, 3, 5, 7, and 9 after treatment in different groups;

[0039] Figure 15 (a) Wound healing rate in mice (%); (b) Changes in body weight in mice during treatment;

[0040] Figure 16 Histological analysis was performed on H&E and Masson staining in each group.

[0041] Figure 17 Organ staining results for each treatment group;

[0042] Figure 18 Synthesis route diagram of CD-Por-Cu-BNN6. Detailed Implementation

[0043] 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.

[0044] As described in the background section, loading BNN6 into porous materials is prone to leakage; encapsulating BNN6 with cyclodextrin results in a single antibacterial mode; although the preparation of porous polymers by polymerizing cyclodextrin with porphyrin can enable the material to have photothermal and photodynamic effects, the small cavity structure of β-cyclodextrin makes it difficult to expand the interlayer spacing of porphyrin, and π-π stacking still occurs between the planes of porphyrin, which inhibits the photothermal and photodynamic effects.

[0045] Based on this, the purpose of this invention is to provide a covalent organic framework antibacterial material with NO light-controlled release. This invention uses cyclodextrin with a pocket structure as the main framework, coordinates with copper to form a porous polymer CD-Por-Cu, and then encapsulates BNN6 to form CD-Por-Cu-BNN6. In the cyclodextrin-porphyrin porous polymer (CD-Por-POP), one cyclodextrin needs to bind with seven porphyrins. However, the steric hindrance generated by the porphyrins is too large, so these seven porphyrins cannot bind with the cyclodextrin in a planar manner. Figure 18 The model shows that all porphyrins are not on the same plane or are twisted, thus overcoming the parallel packing of porphyrins, that is, overcoming π-π packing.

[0046] In this invention, the molar ratio of hepta(6-deoxy-6-(4-formylphenyl))-β-cyclodextrin to 5,10,15,20-tetra(4-aminophenyl)porphyrin should be 4:7, with hepta(6-deoxy-6-(4-formylphenyl))-β-cyclodextrin in excess to ensure complete reaction of 5,10,15,20-tetra(4-aminophenyl)porphyrin; therefore, the molar ratio of hepta(6-deoxy-6-(4-formylphenyl))-β-cyclodextrin to 5,10,15,20-tetra(4-aminophenyl)porphyrin is ≥4:7. The molar ratio of the cyclodextrin-porphyrin porous polymer to copper chloride should be 1:7, but to ensure that copper can coordinate on the cyclodextrin-porphyrin porous polymer, copper chloride needs to be in excess. Therefore, the molar ratio of CD-Por-POP to copper chloride is less than or equal to 1:7. BNN6 ultimately binds to CD-Por-Cu. The cavities of the cyclodextrin can accommodate BNN6. Therefore, BNN6 is not only adsorbed into the pores of the polymer but also encapsulated by the cyclodextrin. Compared with the adsorption of BNN6 by porous materials, BNN6 can be adsorbed into both the pores and encapsulated in the cavities of the cyclodextrin, forming a multi-encapsulation structure. The loading of BNN6 is greater, resulting in a higher concentration of released NO and a better antibacterial effect.

[0047] 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.

[0048] 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.

[0049] Example 1: Preparation of CD-Por-Cu-BNN6

[0050] (1) Preparation of hepta(6-deoxy-6-(4-formylphenyl))-β-cyclodextrin (7F-β-CD): 1.22 g of 4-hydroxybenzaldehyde and 1.38 g of potassium carbonate were dissolved in 60 mL of DMF and reacted at room temperature for 2 hours under nitrogen protection. Then, 20 mL of hepta(6-iodo-6-deoxy)-β-cyclodextrin solution (prepared by adding 1.904 g of hepta(6-iodo-6-deoxy)-β-cyclodextrin to 20 mL of deionized water; CAS of hepta(6-iodo-6-deoxy)-β-cyclodextrin: 30754-23-5) was added dropwise, and the reaction temperature was raised to 80 °C. o C. 60 mL of DMF was evaporated under reduced pressure, and the reaction was completed within 24 h. After evaporating 60 mL of DMF under reduced pressure, the solution was poured into 200 mL of cold water, filtered, and the precipitate was collected to obtain 7F-β-CD.

[0051] (2) Preparation of CD-Por-POP: 826 mg of 7F-β-CD and 468 mg of 5,10,15,20-tetra(4-aminophenyl)porphyrin (TAPP) (CAS: 22112-84-1, purchased from Shanghai Haohong Biomedical Technology Co., Ltd., purity 97%) were dispersed in acetic acid, purged with argon for 30 min, refluxed at 120 °C for three days, and washed with methanol and ethanol after the reaction was completed and dried to obtain CD-Por-POP.

[0052] (3) Preparation of CD-Por-Cu: 50 mg CD-Por-POP and 20 mg copper chloride were added to a mixed solution of 10 mL DMF and 2 mL distilled water, purged with argon, and reacted at 30 °C for 48 h. After the reaction was completed, the mixture was filtered and washed successively with tetrahydrofuran, dimethyl sulfoxide, water, methanol, and ethanol, and dried under vacuum at 25 °C overnight to obtain CD-Por-Cu.

[0053] (4) Preparation of CD-Por-Cu-BNN6: 3 mg of CD-Por-Cu was added to 3 mL of 200 μg / mL BNN6 solution (prepared with 70 wt% ethanol solution, CAS of BNN6: 106476-75-9), purged with argon, and reacted at 30 °C for 24 h. After the reaction was complete, the mixture was centrifuged, washed with water and ethanol, and then... o CD-Por-Cu-BNN6 was obtained by drying under vacuum at C. The synthetic route is shown in [reference needed]. Figure 18 .

[0054] Example 2: Characterization of CD-Por-Cu-BNN6

[0055] (1) Determination of infrared spectroscopy

[0056] The structures of TAPP, 7F-β-CD, CD-Por-POP, CD-Por-Cu, CD-Por-Cu-BNN6, and BNN6 were determined using infrared spectroscopy. Figure 1 As shown in (a), it can be observed that all synthetic polymers and composites retain the characteristics of both precursor units, with ~3365 cm⁻¹ being the most abundant. -1 The broad OH extension originates from the CD molecule, and ~3356 cm -1 The NH extension at the location is ~1629 cm. -1 The NH bending at that point originates from the porphyrin core. Importantly, each spectrum is at 1600 cm⁻¹. -1 A distinct new band was observed nearby, belonging to the C=N stretching vibration, while simultaneously, the amine (NH4+, 3356 cm⁻¹) of TAPP was observed. -1 ) and 7-F-β-CD aldehydes (C=O, 1681 cm⁻¹)-1 The characteristic peaks also almost disappeared. Compared with the initial framework CD-Por-POP, the spectrum of CD-Por-Cu at 1354 cm⁻¹... -1 A characteristic peak appeared at [location], which belongs to N-Cu oscillation, verifying the successful metallization and coordination of Cu(II) with the porphyrin center. This indicates that the free amine groups were also largely consumed after polymerization, and these results all indicate the successful progress of the polymerization reaction.

[0057] (2) The pore distribution of CD-Por-Cu-BNN6 was understood by N2 adsorption-desorption curves and pore size distribution curves. Figure 1 As shown in (b), the calculated BET surface area of ​​CD-Por-Cu is 32.5 m². 2 g -1 The cumulative pore volume is 0.144 cm³. 3 g -1 The cumulative pore volume is higher than that of CD-Por-Cu-BNN6 (4.27 m³). 2 g -1 and 0.0319 cm 3 g -1 This indicates that BNN6 was successfully loaded into the material.

[0058] (3) The thermal stability of CD-Por-Cu-BNN6 was determined by thermogravimetric analysis. The thermal stability of the synthesized material under N2 atmosphere was studied by thermogravimetric analysis (TGA). Figure 1 (c) shows that the thermogravimetric analysis (TGA) curves of CD-Por-Cu-BNN6 exhibit a distinct multi-stage decomposition characteristic. Initially, a slight mass loss of less than 4% occurs below 100 °C, which is related to the evaporation of physically adsorbed water and the release of thermally stimulated NO. Subsequent decomposition mainly occurs in two stages: a mass loss of approximately 13% below 200 °C, primarily attributed to the removal of residual solvent adsorbed on the surface. Above this temperature, the mass loss becomes more pronounced, with a cumulative loss of 30.85%, consistent with the thermal degradation of the porous organic framework. Notably, this material retains approximately 47% of its original mass at 800 °C, demonstrating good thermal stability and indicating its potential for thermochemical applications, such as high-temperature catalysis or energy conversion.

[0059] (4) The crystallinity and porosity of CD-Por-Cu-BNN6 were estimated using powder X-ray diffraction (PXRD). Powder X-ray diffraction (PXRD) analysis was performed. Figure 1 (d) Showing CD-Por-Cu through Cu 2+The main crystalline phase formed by coordination remains unchanged after the addition of BNN6, as can be seen from the almost identical PXRD patterns of CD-Por-Cu and CD-Por-Cu-BNN6. Both CD-Por-Cu and CD-Por-Cu-BNN6 exhibit strong reflections at 2θ = 15.67° and 21.49°, respectively, pointing towards the (010) and (200) planes of the layered COF. Furthermore, significant reflections are observed at 2θ = 34.9° and 37.4°, corresponding to the (002) and (111) crystal planes of CuO, respectively, confirming the formation of the crystalline CuO phase.

[0060] (5) The morphological and structural characteristics of CD-Por-Cu-BNN6 were studied using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Figure 2 (a) ~ Figure 2 The scanning electron microscope image in (c) shows a three-dimensional microstructure composed of coarse, irregular microparticles. These microparticles are aggregated from primary nanoparticles, forming a rough and porous surface morphology that facilitates bacterial adhesion. Figure 2(d) and Figure 2 (e) Low-magnification TEM images show that the secondary aggregates, loosely assembled from primary nanoparticles, have irregular spherical morphologies. This indicates that the present invention, through structural design, ensures that the porphyrins linked to a cyclodextrin are not on the same plane, overcoming the problem of porphyrin polymerization and stacking. The prepared polymer is no longer a layered structure, but a three-dimensional structure. Figure 2(f) High-resolution TEM further shows the obvious light and dark contrasts in these structures, indicating the presence of macropores.

[0061] Figure 2 (g)~ Figure 2 (k) The elemental composition and distribution of CD-Por-Cu-BNN6 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 (70.4%), N (12%), O (15.9%) and Cu (1.7%) elements are uniformly distributed on the CD-Por-Cu-BNN6 porous framework.

[0062] (6) XPS spectral analysis of CD-Por-Cu-BNN6. As shown in Figure 4(a), similar to the EDS results, XPS also showed the presence of C, N, O and Cu. Figure 4 (b) indicates that CD-Por-Cu-BNN6 has four types of carbon in C 1s: C=C (284.2 eV), CC (284.8 eV), CO (286.42 eV), and C=N (287.7 eV). Figure 4(c) In the high-resolution Cu2p spectrum, Cu2p-related parameters were observed at 954 eV and 934 eV, respectively. 1 / 2 and Cu2p 3 / 2 Corresponding characteristic peaks. These binding energies are associated with previously reported Cu. 2+ The values ​​match very well, and no Cu was detected. + The characteristic signals confirmed that copper exists only in the divalent state in the material. The N1s XPS spectra of CD-Por-Cu-BNN6 in Figure 4(d) resolved two different nitrogen environments, including the ligand-associated C=N host at 398.6 eV and the copper ligand C=N-Cu molecule at 399.7 eV. Notably, copper coordination led to a positive shift of 1.1 eV in the binding energy of the C=N signal (398.6 to 399.7 eV), confirming the LMCT (ligand-to-metal charge transfer)-induced decrease in nitrogen electron density. Meanwhile, the O1s spectra in Figure 4(e) showed that CD-Por-Cu-BNN6 possesses two types of O, at 531.4 eV (O-Cu ligand bond) and 533 eV (OC phenolic group).

[0063] Example 3: Performance Testing

[0064] (1) Photothermal performance test

[0065] By varying the concentration of CD-Por-Cu-BNN6 (0, 100, 200, 300, 400 μg / mL) or the laser power density (0, 0.5, 0.8, 1.0, 1.2, 1.5, and 2.0 W / cm²), 2 The photothermal conversion performance of CD-Por-Cu-BNN6 was studied in detail. The preparation method for different concentrations of CD-Por-Cu-BNN6 was as follows: First, 1 mg of CD-Por-Cu-BNN6 was weighed and thoroughly dispersed in 1 mL of phosphate buffer solution (pH=5.5) using an ultrasonic apparatus to prepare a 1 mg / mL stock solution. Then, 100, 200, 300, and 400 μL of the stock solution were respectively added to 900, 800, 700, and 600 μL of distilled water to prepare CD-Por-Cu-BNN6 aqueous dispersions of 100, 200, 300, and 400 μg / mL. Pure water was used as a control, and a 638 nm laser (1.2 W / cm²) was used. 2 The temperature changes of CD-Por-Cu-BNN6 at different concentrations were monitored for 10 min, and the photophysical properties of the synthesized samples were preliminarily studied.

[0066] like Figure 5As shown in (a), unlike pure water where temperature changes are negligible, CD-Por-Cu-BNN6 exhibits a concentration-dependent heating behavior, which increases rapidly with increasing concentration. Specifically, concentrations of 0, 100, 200, 300, and 400 μg / mL... -1 The temperatures of the CD-Por-Cu-BNN6 solutions increased to 29.8, 37.9, 42.7, 50.4, 55.7, and 59 degrees Celsius, respectively. o C. In addition, Figure 5 In (b), the laser power intensity was also found to be positively correlated with the dispersion temperature. Figure 5 (c) The image obtained from the thermal imaging camera demonstrates the excellent photothermal properties of CD-Por-Cu-BNN6. All these results indicate that red light can be effectively converted into photothermal energy by adjusting the concentration of CD-Por-Cu-BNN6 or the power of the laser. To evaluate the photostability of CD-Por-Cu-BNN6, periodic irradiation experiments were conducted, such as... Figure 5 (d) shows that CD-Por-Cu-BNN6 exhibits a stable laser switching effect, with almost no temperature fluctuation after five consecutive laser on / off cycles. Furthermore, Figure 5 (e) shows the values ​​of τS and θ for the photothermal conversion efficiency, which is calculated to be 45.09%. The formula for calculating the photothermal conversion efficiency is based on the formula disclosed in application number CN115845086A, entitled "A Photothermal-Fenton-like Artificial Nanoenzyme and Its Preparation Method and Application".

[0067] (2) Peroxidase activity of CD-Por-Cu-BNN6

[0068] The peroxidase activity of CD-Por-Cu-BNN6 at different pH values ​​was investigated. The ROS generation capacity of CD-Por-Cu-BNN6 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 CD-Por-Cu-BNN6 with the ability to act as a highly efficient •OH generator in acidic media.

[0069] 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 H2O2 used in the experiment was 0.3 wt%. 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. Preparation of CD-Por-Cu-BNN6 at different pH values: Weigh 2 mg of CD-Por-Cu-BNN6 and disperse it thoroughly in 2 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 300 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 CD-Por-Cu-BNN6 with a concentration of 300 μg / mL and pH values ​​of 1.5, 2.5, 3.5, 4.5, 5.5, and 6.5. Preparation of PBS dispersions of CD-Por-Cu-BNN6 at different concentrations: Weigh 1 mg of CD-Por-Cu-BNN6 and disperse it thoroughly in 1 mL of PBS at pH 5.5 using an ultrasonic apparatus to prepare a stock solution of 1 mg / mL. Then, take 25, 50, 100, 150, 200, and 250 μL from the stock solution and add them to 825, 800, 750, 700, 650, and 600 μL of PBS at pH 5.5, respectively. Add 75 μL each of TMB and H2O2 to prepare PBS dispersions of CD-Por-Cu-BNN6 at concentrations of 25, 50, 100, 150, 200, and 250 μg / mL. Finally, use a UV-Vis spectrophotometer to measure the concentrations across the entire wavelength range.

[0070] like Figure 6 As shown in (a), the absorbance changes with pH, ​​indicating that the enzymatic catalytic activity of CD-Por-Cu-BNN6 depends on the pH of the solution, and its activity is highest at pH=2.5. Even in a microenvironment with bacterial infection (pH=5.5), it still exhibits enzymatic activity and can be used to kill bacteria; furthermore, as... Figure 6 As shown in (b), the absorbance increases with increasing CD-Por-Cu-BNN6 concentration, indicating that the enzymatic catalytic activity of CD-Por-Cu-BNN6 depends on its concentration; Figure 6 As shown in (c), the absorbance increases with the addition of laser light, indicating that the laser has a positive effect on the enzyme catalytic activity of CD-Por-Cu-BNN6.

[0071] (3) Verification of NO release from CD-Por-Cu-BNN6

[0072] NO release from CD-Por-Cu-BNN6 was detected using the Beyotime S0021S Griess reagent kit. NO reacts with the Griess reagent (0.2% naphthylethylenediamine dihydrochloride and 2% sulfonamide in 5% phosphoric acid) to form nitrite (NO2). - The release of NO was indirectly quantified by generating an azo derivative with a pinkish-red characteristic. 2 mL of 1 mg / mL CD-Por-Cu-BNN6 suspension was added to a quartz tube and irradiated with a 638 nm laser at different power densities. After irradiation, the tube was removed and centrifuged (18000 rpm, 10 min) to remove CD-Por-Cu-BNN6, which interferes with NO detection. Then, 50 µL of Griess reagent I (S0021S-2) and 50 µL of Griess reagent II (S0021S-3) were added sequentially to 50 µL of the supernatant, and the mixture was incubated in the dark for 10 min. The absorbance of the mixture was then measured at 540 nm.

[0073] The encapsulation efficiency and loading rate of BNN6 in CD-Por-Cu-BNN6 were determined. The specific procedure was as follows: 1 mg of BNN6 was weighed and added to 70% ethanol to prepare solutions with concentrations of 0, 100, 300, 500, and 800 µg / mL. 3 mg of CD-Por-Cu-BNN6 was added to each solution, and the solutions were incubated at 25°C. o The mixture was stirred in a brown vial of C for 24 hours. Subsequently, the CD-Por-Cu-BNN6 coated with BNN6 was centrifuged and washed with deionized water. The supernatant was obtained after filtration and analyzed using UV-Vis absorption spectrometry at 265 nm. The coating loading rate (DL%) was calculated using the formula: DL% = W 包载 / W 载药纳米粒 ×100%; W 包载 The total mass of BNN6 carried by the carrier; W 载药纳米粒 The final total mass of drug-loaded nanoparticles (including carrier material + drug) is given. The encapsulation efficiency is calculated using the formula: EE% = (W 总 -W 游 ) / W 总 ×100%; W 总 W represents the total amount of drug added during preparation. 游 The amount of free drug in the system that is not encapsulated.

[0074] like Figure 7 As shown in (a), for CD-Por-Cu-BNN6 of different concentrations in a 45 ℃ water bath, the heating time was directly proportional to the NO release, and the concentration of the material was also directly proportional to the NO release; Figure 7As shown in (b), different concentrations of CD-Por-Cu-BNN6 at 1.2 W / cm²... 2 Under laser irradiation, the irradiation time is directly proportional to NO release, and the NO release increases more rapidly when heated in a 45°C water bath. For example... Figure 7 As shown in (c), the loading rate can reach up to 50.6% at 1 mg / mL. The calculated encapsulation efficiency (EE%) can reach up to 84.8%.

[0075] (4) Verification of photodynamic activity of CD-Por-Cu-BNN6

[0076] Methylene blue (MB) was used as a molecular probe to monitor the generation of hydroxyl radicals (•OH). Methylene blue has a characteristic absorption peak at a wavelength of 664 nm, which decreases after reacting with •OH. The specific operation was as follows: 2 mg of CD-Por-Cu-BNN6 was weighed and thoroughly dispersed in deionized water using an ultrasonicator to prepare a stock solution of 2 mg / mL. 1 mg of MB was weighed and added to deionized water to prepare a solution of 500 µg / mL (stored in the dark). For the experimental group, 300 μL of the stock solution was added to 2640 μL of deionized water, followed by 60 μL of MB. The solution was irradiated with a 638 nm laser for 0 s, 60 s, 120 s, 180 s, and 240 s, and the full wavelength was measured using a UV-Vis spectrophotometer. The control group consisted of 60 μL of MB added to 2940 μL of deionized water, which was then irradiated with a 638 nm laser for 0s, 60s, 120s, 180s, and 240s, and the full wavelength was measured using a UV-Vis spectrophotometer.

[0077] From Figure 8(a) and Figure 8 (b) It can be seen that under laser irradiation (638 nm, 1.5 W cm⁻¹), -2 In the presence of CD-Por-Cu-BNN6, compared to the control group, the absorption intensity of MB at 664 nm decreased rapidly. Based on normalized absorbance (A / A0), CD-Por-Cu-BNN6 rapidly generated •OH within 10 minutes, degrading 47.71% of methyl bromide, while the control group only degraded 18.21%. Similarly, 1,3-diphenylisobenzofuran (DPBF) was used as a molecular probe to monitor singlet oxygen (…). 1 The generation of O2, the typical absorption band of DPBF near 410 nm is related to... 1 O2 will be quenched after the reaction. Figure 8(c) and Figure 8 (d) It can be seen that under 638 nm laser irradiation (1.5 W cm⁻¹), -2Under certain conditions, CD-Por-Cu-BNN6 (200 μg / mL) induced rapid oxidation of DPBF. The oxidation degree of CD-Por-Cu-BNN6 was significantly higher than that of the control group. CD-Por-Cu-BNN6 achieved 58.35% degradation within 10 minutes, which was 1.71 times higher than the control group (34.12%) irradiated only by laser. This confirms that... 1 O2 is generated efficiently through the type I photodynamic pathway.

[0078] (5) Verification of glutathione activity of CD-Por-Cu-BNN6

[0079] The ability of CD-Por-Cu-BNN6 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 9 It can be seen that as the concentration of CD-Por-Cu-BNN6 increases, the amount of GSH consumed also increases, and the GSH consumption capacity of CD-Por-Cu-BNN6 exhibits a concentration-dependent relationship.

[0080] Test Example 1: In vitro antibacterial test

[0081] (1) Bacterial culture

[0082] Staphylococcus aureus (Staphylococcus aureus) was used S. aureus ) and Escherichia coli ( E. coli Two types of bacteria were used to complete the following experiments using second-generation bacteria. The specific culturing 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 constant-temperature shaker (110 rpm, 37°C) for 12 h. Then, 100 µL of the cultured bacterial culture was transferred to a 2 mL EP tube containing 900 µL of the culture medium, and then serially diluted 10 times. -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 h, observe clonal morphology and colony count. The petri dish with approximately 1000 colonies is considered the first generation of bacteria. Use a pick to pick one colony from the first generation and add it 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 5 g of LB broth in 200 mL of distilled water, and then autoclave to obtain the bacterial liquid culture medium. The specific preparation method for the solid culture medium is as follows: Disperse 5 g of LB broth and 3 g of agar in 200 mL of distilled water, and then autoclave to obtain the solid culture medium.

[0083] (2) Determination of the antibacterial activity of CD-Por-Cu-BNN6 by plate counting method

[0084] The preparation method for the dispersions of bacteria at different concentrations of CD-Por-Cu-BNN6 was as follows: 2 mg of CD-Por-Cu-BNN6 powder was thoroughly dispersed in 2 mL of PBS to prepare a 1 mg / mL CD-Por-Cu-BNN6 stock solution. 10 µL of this stock solution was then added to six 2 mL EP tubes. 8 CFU mL -1 Bacterial solution ( S. aureus or E. coli Then, 980, 960, 940, 920, 900, and 880 μL of PBS were added respectively. For each concentration, 10 μL of 0.3 wt% H2O2 was added, followed by 0, 20, 40, 60, 80, and 100 μL of 1 mg / mL CD-Por-Cu-BNN6 stock solution, respectively, to obtain CD-Por-Cu-BNN6 and 10 mg / mL solutions containing concentrations of 0, 20, 40, 60, 80, and 100 μg / mL. 8 CFU mL -1 The mixture of PBS dispersion is CD-Por-Cu-BNN6 solution.

[0085] CD-Por-Cu-BNN6 solutions were prepared at concentrations of 0, 50, 100, 150, and 200 μg / mL. 10 μL of 0.3 wt% H₂O₂ was added to each concentration, followed by laser irradiation (λ = 638 nm, 1.2 W / cm²). 2 The above solutions were incubated in a constant temperature shaker (110 rpm, 37℃) for 12 h, and then the cultured bacterial solutions were serially diluted 10 times according to the bacterial culture method. 5The 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 between groups. Figure 10 (a) ~ Figure 10 As can be seen in (b), CD-Por-Cu-BNN6 can achieve significant antibacterial effects at a concentration of 200 μg / mL, with antibacterial rates of 94.3% and 97.8% against Staphylococcus aureus and Escherichia coli, respectively.

[0086] (3) In vitro antibacterial activity of CD-Por-Cu-BNN6 under different treatments

[0087] The antibacterial activity under different treatments was studied using the plate count method. The experiment consisted of eight groups: PBS (laser off), PBS (laser on), H2O2 (laser off), H2O2 (laser on), CD-Por-Cu-BNN6 (laser off), CD-Por-Cu-BNN6 (laser on), CD-Por-Cu-BNN6+H2O2 (laser off), and CD-Por-Cu-BNN6 + H2O2 (laser on). In the groups containing CD-Por-Cu-BNN6, the concentration was 200 μg / mL; in the groups containing H2O2, the H2O2 concentration was 0.3 wt%; and in the groups containing laser, the laser parameters were λ = 638 nm and 1.2 W / cm². 2 10 min.

[0088] Then, according to the groups, each group was placed in a constant temperature shaker (110 rpm, 37℃) and incubated for 12 h. The cultured bacterial solutions were then serially diluted 10⁻⁶ times according to the bacterial culture method. 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 between groups. Figure 10 (c)~ Figure 10As shown in (f), compared with the PBS (laser off) group and the PBS (laser on) group, the bacteria in the H2O2 (laser off) group and the H2O2 (laser on) group showed only a slight antibacterial effect. From the CD-Por-Cu-BNN6 (laser off), CD-Por-Cu-BNN6 (laser on), CD-Por-Cu-BNN6+H2O2 (laser off), and CD-Por-Cu-BNN6 + H2O2 (laser on) groups, it can be seen that laser irradiation can enhance the bactericidal effect of CD-Por-Cu-BNN6. Specifically, comparing the colony counts (CFU) of Staphylococcus aureus and Escherichia coli, the CD-Por-Cu-BNN6 (laser-on) group significantly reduced the colony counts of Staphylococcus aureus and Escherichia coli to 59.3% and 52.4%, respectively, while the CD-Por-Cu-BNN6 (laser-off) group achieved colony counts of 95.1% and 93.7%, respectively. Compared to the CD-Por-Cu-BNN6 (laser-off) group, due to synergistic effects, the CD-Por-Cu-BNN6 + H2O2 (laser-off) group also significantly reduced the colony counts of Staphylococcus aureus and Escherichia coli to 92.2% and 83.8%, respectively. The CD-Por-Cu-BNN6 + H2O2 (laser-on) group further improved sterilization efficiency, almost eliminating all bacteria, and reducing the colony counts of Staphylococcus aureus and Escherichia coli to 5.4% and 3.7%, respectively.

[0089] Experimental Example 2: Bacterial Viability / Deadness Staining Test

[0090] 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.

[0091] Bacterial suspensions were prepared according to the method in Experimental Example 1 (3): PBS (laser off), PBS (laser on), H2O2 (laser off), H2O2 (laser on), CD-Por-Cu-BNN6 (laser off), CD-Por-Cu-BNN6 (laser on), CD-Por-Cu-BNN6+H2O2 (laser off), and CD-Por-Cu-BNN6+H2O2 (laser on). 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 -3M) 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.

[0092] from Figure 11 The significant synergistic antibacterial effect of CD-Por-Cu-BNN6 on PTT / PDT / PODs was also visually observed in the results of both live and dead bacterial staining. Bacteria treated with PBS (laser off), PBS (laser on), H2O2 (laser off), and H2O2 (laser on) groups showed strong green fluorescence, consistent with the results of the plate count method. In contrast, bacteria treated with CD-Por-Cu-BNN6 (laser off), CD-Por-Cu-BNN6 (laser on), and CD-Por-Cu-BNN6+H2O2 (laser off) groups showed obvious red fluorescence. Simultaneously, the CD-Por-Cu-BNN6+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.

[0093] Experimental Example 3: In vitro biocompatibility experiment

[0094] (1) Hemolysis test

[0095] Fresh blood was collected from 5-week-old female BALB / c 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 CD-Por-Cu-BNN6 solution at different concentration gradients (0, 100, 200, 300, 400 μg / mL) was added (red blood cell solution: CD-Por-Cu-BNN6 solution volume ratio = 1:9). The mixture was incubated at 37℃ for 3 h and centrifuged at 10,000 rpm for 10 min. 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:

[0096] Hemolysis volume (%) = (A-An) / (Ap-An) × 100%;

[0097] Wherein, A: the absorbance obtained by taking the supernatant after adding CD-Por-Cu-BNN6 to red blood cells;

[0098] An is the absorbance obtained by taking the supernatant after adding PBS to red blood cells (negative control).

[0099] Ap is the absorbance obtained by adding distilled water to red blood cells and taking the supernatant (positive control).

[0100] like Figure 12 As shown, CD-Por-Cu-BNN6 exhibited only very low hemolytic activity (below 4%) or no hemolytic activity within the concentration range exhibiting antibacterial activity. The hemolytic rate of CD-Por-Cu-BNN6 varied with its concentration, remaining below 4% as the concentration increased from 50 to 400 μg / mL. This indicates that CD-Por-Cu-BNN6 has good blood compatibility and does not damage the erythrocyte membrane.

[0101] (2) Cytotoxicity test

[0102] 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 CD-Por-Cu-BNN6 at different concentrations (50-200 μg / mL) were 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 13 As shown, after 24 h of culture, even at a high concentration (400 μg / mL), the survival rate of 3T3 cells treated with CD-Por-Cu-BNN6 was above 80%.

[0103] Experiment Example 4: In vivo wound healing experiment

[0104] Five-week-old BALB / c mice (purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.) were randomly divided into six groups: PBS group (Ⅰ), H2O2 group (Ⅱ), CD-Por-Cu-BNN6 group (Ⅲ), CD-Por-Cu-BNN6 + H2O2 group (Ⅳ), CD-Por-Cu-BNN6 + laser group (Ⅴ), and CD-Por-Cu-BNN6 + H2O2 + laser group (Ⅵ), with six mice in each group. Normal mice without any treatment were used as a control group; no wound healing model was established in the control group. Before surgery, the hind hair of each mouse was shaved to create a 5 mm diameter wound, which was then infected with Staphylococcus aureus (1×10⁻⁶). 6A wound healing model was established by administering the treatment (CFU / mL) for 24 hours. In this study, the treatment agent was carefully dripped drop by drop to the bacterial infection site, ensuring complete coverage of the wound surface. Treatments were administered according to their respective groups: the CD-Por-Cu-BNN6 group had a concentration of 200 μg / mL; the H2O2 group had a concentration of 0.3 wt%; and the laser group had laser parameters of λ = 638 nm and 1.2 W / cm². 2 10 min.

[0105] Mice were treated according to the requirements of different group settings, and the wounds of mice in each group were recorded on days 1, 3, 6, and 9, while changes in body weight were monitored. Changes in wound size were measured using an image analysis program (Image.J).

[0106] 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 CD-Por-Cu-BNN6 + H2O2 + laser group mice showed the most significant advantages in wound healing and skin regeneration, with a wound healing rate of over 95%, far exceeding that of the PBS group (87.4%), CD-Por-Cu-BNN6 group (90.9%), CD-Por-Cu-BNN6 + laser group (92.3%), and CD-Por-Cu-BNN6 + H2O2 group (93.4%). Meanwhile, compared with the blank group (normal mice that did not receive any treatment), Figure 15 (b) It can be seen that no significant weight change was detected during the entire treatment process, indicating that CD-Por-Cu-BNN6 has good biocompatibility.

[0107] 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 16 As shown, the PBS-treated group exhibited significant inflammatory cells and incomplete epidermis. In contrast, the CD-Por-Cu-BNN6-treated groups showed varying degrees of skin structure regeneration. The CD-Por-Cu-BNN6 + 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.

[0108] 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 17As shown, no abnormal lesions or inflammation were observed in the major organs, and no histological changes were found. These results confirm that CD-Por-Cu-BNN6 has good biocompatibility in vivo.

[0109] 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 covalent organic framework antibacterial material with NO light-controlled release, characterized in that, The covalent organic framework antibacterial material is a porous polymer with cyclodextrin inclusion complexes and porphyrin metal coordination compounds as structural units. Each structural unit contains several porphyrin metal coordination compounds surrounding the cyclodextrin inclusion complexes, and the porphyrin metal coordination compounds are not all on the same plane. The cyclodextrin inclusion complex is β-cyclodextrin inclusion complex BNN6. The porphyrin metal coordination compound is a coordination compound of porphyrin and copper. The β-cyclodextrin is hepta(6-deoxy-6-(4-formylphenyl))-β-cyclodextrin; the porphyrin is 5,10,15,20-tetra(4-aminophenyl)porphyrin; the structural formula of the covalent organic framework antibacterial material is: 。 2. The method for preparing the covalent organic framework antibacterial material according to claim 1, characterized in that, Includes the following steps: (1) Dissolve 4-hydroxybenzaldehyde and potassium carbonate in DMF and react at room temperature under a protective atmosphere. Then add hepta(6-iodo-6-deoxy)-β-cyclodextrin solution to react and obtain hepta(6-deoxy-6-(4-formylphenyl))-β-cyclodextrin. (2) Hepta(6-deoxy-6-(4-formylphenyl))-β-cyclodextrin and 5,10,15,20-tetra(4-aminophenyl)porphyrin were dispersed in acetic acid and subjected to a solvothermal reaction under a protective atmosphere to obtain a cyclodextrin porphyrin porous polymer. (3) Add the cyclodextrin porphyrin porous polymer and copper salt to a mixed solvent and carry out a solvothermal reaction under a protective atmosphere. After the reaction is complete, filter, wash and dry to obtain CD-Por-Cu. (4) CD-Por-Cu was added to the ethanol solution of BNN6 and the reaction was carried out in the dark under a protective atmosphere. After the reaction was completed, the mixture was centrifuged, washed and dried to obtain a covalent organic framework antibacterial material with NO light-controlled release.

3. The preparation method according to claim 2, characterized in that, In step (1), the molar ratio of 4-hydroxybenzaldehyde, potassium carbonate and hepta(6-iodo-6-deoxy)-β-cyclodextrin is 10:10:1; the reaction time at room temperature is 2 hours.

4. The preparation method according to claim 2, characterized in that, In step (2), the molar ratio of hepta(6-deoxy-6-(4-formylphenyl))-β-cyclodextrin to 5,10,15,20-tetra(4-aminophenyl)porphyrin is greater than or equal to 4:7; the temperature of the solvothermal reaction is 120°C and the time is 3 days.

5. The preparation method according to claim 2, characterized in that, In step (3), the copper salt is copper chloride; the molar ratio of the cyclodextrin porphyrin porous polymer and the copper salt is less than or equal to 1:7; the mixed solvent is DMF and pH 5.5 phosphate buffer mixed at a volume ratio of 5:1; the temperature of the solvothermal reaction is 30°C and the time is 2 days.

6. The preparation method according to claim 2, characterized in that, In step (4), the mass ratio of CD-Por-Cu to BNN6 is 5:1; the BNN6 solution is prepared by dissolving N,N'-disec-butyl-N,N'-dinitroso-1,4-phenylenediamine in 70 wt% ethanol solution, with a concentration of 200 μg / mL; the light-protected reaction is carried out at a temperature of 30℃ for 24 h.

7. The application of the covalent organic framework antibacterial material according to claim 1 in the preparation of antibacterial drugs.

8. The application according to claim 7, characterized in that, The covalent organic framework antibacterial material synergistically inhibits bacteria through five modes: photothermal, photodynamic, peroxidase, glutathione peroxidase, and NO release.

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