A pheno-thiazine-based two-dimensional covalent organic framework material constructed by supramolecular inclusion complex and application thereof

Abstract

The application discloses a kind of phenthiazine-based two-dimensional covalent organic framework materials constructed by supramolecular inclusion complex and application thereof, belong to the field of biological medicine.The phenthiazine-based two-dimensional covalent organic framework material takes 4,4',4'',4'''-(anthracene-9,10-diylbis(10H-phenthiazine-10,3,7-triyl)) tetrakisbenzaldehyde and beta-CD dithylamine inclusion complex as structural unit, and is obtained by solvothermal reaction two-dimensional crystalline porous copolymer.The application utilizes supramolecular inclusion complex and copolymerization of phenthiazine derivative and beta-CD dithylamine to obtain PTz-CD-COF with negligible biological toxicity, the material can also realize the synergistic effect of photodynamic therapy and photothermal therapy simultaneously, and cyclodextrin forms "PDT-PTT-cyclodextrin mediated synergistic" multi-component synergistic treatment system through its unique molecular recognition and inclusion effect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a phenothiazine-based two-dimensional covalent organic framework material constructed using supramolecular inclusion complexation and its applications. Background Technology

[0002] Photothermal therapy (PTT) generates a localized high-temperature effect through a photothermal agent activated by near-infrared light (NIR). When the temperature exceeds 45°C, it can induce irreversible denaturation of heat shock proteins (HSPs) in bacteria, thereby effectively killing pathogens. This temperature-dependent antibacterial mechanism enables precise pathogen inactivation while maximizing the preservation of surrounding healthy tissue. Photodynamic antibacterial therapy (PDAT), as a novel antibacterial strategy, has a core mechanism where, under excitation by near-infrared light of a specific wavelength, photosensitizer molecules undergo a photochemical reaction, efficiently generating reactive oxygen species (ROS) through an energy transfer process. Studies have shown that the drug resistance mechanism of multidrug-resistant bacteria mainly stems from their biofilm-forming ability. A biofilm is a complex three-dimensional structure formed by bacterial communities irreversibly adhering to the surface of biological materials or tissues and encapsulating them with secreted extracellular polymeric matrix (EPS). This EPS matrix creates a unique microenvironment through the following mechanisms: 1) forming a physical barrier leading to local hypoxia; 2) promoting the activation of anaerobic glycolysis metabolic pathways; 3) interfering with the normal function of ion channels; and 4) significantly reducing the permeability and effectiveness of antibacterial drugs.

[0003] Crystalline covalent organic frameworks (COFs), as a representative class of materials, are assembled from pure organic structural units through dynamic covalent bonds. Among them, two-dimensional (2D) COFs show significant potential in fields such as luminescent materials, sensor development, and optoelectronic device applications due to their unique structural characteristics. This advantage mainly stems from two key structural features: firstly, the orderly channels formed by tightly packed organic layers, and secondly, the orderly arrangement of functional groups in the framework. The synergistic effect of these two features achieves an efficient columnar charge transport pathway. Phenothiazine derivatives (PTz) possess certain photosensitizing properties and can effectively generate reactive oxygen species (ROS) under ultraviolet or visible light, effectively killing bacteria. Cyclodextrins (CDs), with their unique hydrophobic cavity structure, can serve as an important class of cyclic host molecules, forming stable supramolecular complexes with various functional molecules through host-guest inclusion interactions. Furthermore, the inherent superbiocompatibility of CDs can effectively improve the biosafety of the final polymer materials. However, the single antibacterial mode of phenothiazine derivatives and cyclodextrin inclusion complexes has limited antibacterial effects. To improve the antibacterial effect, it is necessary to construct a multi-mode synergistic antibacterial mechanism. Therefore, it is necessary to utilize supramolecular inclusion complexation to construct phenothiazine-based two-dimensional covalent organic framework materials to endow copolymers with photothermal properties, which is of great significance for solving the key bottleneck of covalent organic frameworks (COFs) in biomedical applications. Summary of the Invention

[0004] To address the aforementioned limitations of existing technologies, the present invention aims to provide a phenothiazine-based two-dimensional covalent organic framework material constructed using supramolecular inclusion complexation and its applications. The present invention utilizes supramolecular inclusion complexation to copolymerize a phenothiazine derivative and β-CD benzidine to obtain a phenothiazine-based two-dimensional covalent organic framework material PTz-CD-COF with negligible biotoxicity. This material achieves a synergistic effect of both PDT and PTT dual antibacterial modes.

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

[0006] In a first aspect, the present invention provides a phenothiazine-based two-dimensional covalent organic framework material constructed by supramolecular inclusion complexation, wherein the phenothiazine-based two-dimensional covalent organic framework material uses phenothiazine derivatives and β-CD benzidine inclusion complexes as structural units, and obtains a two-dimensional crystalline porous copolymer through a solvothermal reaction.

[0007] Preferably, the phenothiazine derivative is 4,4',4'',4'''-(anthracene-9,10-dimethylbis(10H-phenothiazine-10,3,7-trimethyl))tetrabenzaldehyde, with the following structural formula:

[0008] .

[0009] Preferably, the 4,4',4'',4'''-(anthracene-9,10-dimethylbis(10H-phenthiazine-10,3,7-trimethyl))tetrabenzaldehyde is prepared by the following method:

[0010] (a) 4-Formylphenylboronic acid, neopentyl glycol and p-toluenesulfonic acid were dissolved in toluene and heated overnight under a protective atmosphere. After the reaction was completed, the product was purified to obtain a white needle-like solid, which is 2-(4-(5,5-dimethyl-1,3-dioxolane)phenyl)-5,5-dimethyl-1,3,2-dioxolane;

[0011] (b) 3,7-dibromo-10H-phenthiazine, 2-(4-(5,5-dimethyl-1,3-dioxolane)phenyl)-5,5-dimethyl-1,3,2-dioxoborane, cesium carbonate and tetratriphenylphosphine palladium were added to tetrahydrofuran and subjected to a solvothermal reaction under a protective atmosphere. The product was purified to obtain a yellow powder, which is 3,7-bis(4-(5,5-dimethyl-1,3-dioxolane-2-yl)phenyl)-10H-phenthiazine;

[0012] (c) 3,7-bis(4-(5,5-dimethyl-1,3-dioxacyclopentan-2-yl)phenyl)-10H-phenthiazine, 9,10-dibromoanthracene, sodium tert-butoxide, tri-tert-butylphosphotetrafluoroborate, 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl and tris(dibenzylideneacetone)dipalladium(0) were added to toluene and subjected to a solvothermal reaction under a protective atmosphere. The product was purified to obtain a red powder, which is 9,10-bis(3,7-bis(4-(5,5-dimethyl-1,3-dioxacyclopentan-2-yl)phenyl)-10H-phenthiazine-10-yl)anthracene;

[0013] (d) Under a protective atmosphere, 9,10-bis(3,7-bis(4-(5,5-dimethyl-1,3-dioxacyclopentan-2-yl)phenyl)-10H-phenthiazin-10-yl)anthracene was dissolved in dichloromethane, and then trifluoroacetic acid was added. The mixture was stirred at room temperature, and then the solvent was removed under reduced pressure to obtain a red powder, which is 4,4',4'',4'''-(anthracene-9,10-diylbis(10H-phenthiazin-10,3,7-triyl))tetrabenzaldehyde.

[0014] Preferably, the structural formula of the β-CD benzidine inclusion complex is:

[0015] .

[0016] Preferably, the β-CD benzidine inclusion complex is prepared by the following method:

[0017] Benzidine was added to anhydrous ethanol and completely dissolved to obtain a benzidine ethanol solution. β-Cyclodextrin was added to hot water and stirred until it was completely dissolved to obtain a β-Cyclodextrin solution. The benzidine ethanol solution was added dropwise to the β-Cyclodextrin solution, and the mixture was heated and stirred to carry out the reaction. After the reaction was completed, the ethanol was removed by rotary evaporation. The remaining mixture was placed in an ice bath and allowed to stand. The precipitate was filtered and vacuum dried to obtain a yellowish-white powder, which is the β-CD benzidine inclusion complex.

[0018] Preferably, the phenothiazine-based two-dimensional covalent organic framework material is prepared by the following method:

[0019] 4,4',4'',4'''-(anthracene-9,10-dimethylbis(10H-phenothiazine-10,3,7-trimethyl))tetrabenzaldehyde and β-CD benzidine inclusion complex were mixed, ground, and acetic acid solution and ethanol were added dropwise. The ground mixture was then added to n-butanol and o-dichlorobenzene for a first sonication, followed by a second sonication with acetic acid. The mixture was then heated under a protective atmosphere. After the reaction was completed, the mixture was filtered, washed, and vacuum dried to obtain a dark brown powder, which is the phenothiazine-based two-dimensional covalent organic framework material.

[0020] Preferably, the molar ratio of the 4,4',4'',4'''-(anthracene-9,10-dimethylbis(10H-phenthiazine-10,3,7-trimethyl))tetrabenzaldehyde and the β-CD benzidine inclusion complex is 1:2; and the concentration of the acetic acid solution is 3M.

[0021] Preferably, the grinding time is 30 minutes; the first ultrasonic treatment time is 2 minutes; and the second ultrasonic treatment time is 2 minutes.

[0022] Preferably, the heating temperature is 150°C and the heating time is 3 days.

[0023] A second aspect of the present invention provides the use of phenothiazine-based two-dimensional covalent organic framework materials in the preparation of antibacterial drugs.

[0024] The beneficial effects of this invention are:

[0025] (1) This invention develops phenothiazine-based two-dimensional covalent organic framework materials (PTz-CD-COF and PTz-COF) with negligible biotoxicity for multimodal synergistic antibacterial therapy. Covalent organic frameworks (COFs) are artificial multifunctional porous materials with components similar to biological macromolecules, capable of generating reactive oxygen species, exhibiting superior photothermal and photodynamic properties, and demonstrating more significant bactericidal effects.

[0026] (2) The PTz-CD-COF material prepared by this invention exhibits superior antibacterial properties and in vivo wound repair potential. In in vitro antibacterial experiments, the material showed highly efficient killing activity against Gram-negative bacteria (such as Escherichia coli) and Gram-positive bacteria (such as Staphylococcus aureus), providing a new approach for the development of novel and highly efficient antibacterial materials.

[0027] (3) In this invention, after the PTz-CD-COF material targets diseased cells, it undergoes photophysical and photochemical transformation under near-infrared light irradiation, efficiently triggering the in-situ generation of reactive oxygen species (ROS). At the same time, by regulating the intracellular oxygen partial pressure level, it significantly improves the hypoxic microenvironment at the lesion site, providing favorable conditions for the therapeutic effect. This material can also simultaneously achieve the synergistic effect of photodynamic therapy (PDT) and photothermal therapy (PTT). The cyclodextrin (CD) component, through its unique molecular recognition and inclusion effect, further optimizes the distribution characteristics of the material in cells and its interaction with the target site, thereby significantly improving the synergistic bactericidal efficiency and forming a multi-functional synergistic therapeutic system of "PDT-PTT-cyclodextrin-mediated enhancement". Attached Figure Description

[0028] Figure 1 (a) Infrared absorption curves of PTz, CD-BZD and PTz-CD-COF, (b) ¹³C CP / MAS NMR spectrum of PTz-CD-COF, (c) X-ray diffraction pattern of PTz-CD-COF, (d) Low-temperature nitrogen adsorption isotherm of PTz-CD-COF at 77 K, (e) Pore size distribution curve of PTz-CD-COF, (f) Thermogravimetric analysis curve of PTz-CD-COF;

[0029] Figure 2Characterization of PTz-CD-COF: (a) Scanning electron microscopy (SEM) image of PTz-CD-COF, scale bar 500 nm; (b) Scanning electron microscopy (SEM) image of PTz-CD-COF, scale bar 200 nm; (c) Scanning electron microscopy (SEM) image of PTz-CD-COF, scale bar 100 nm; (d) Transmission electron microscopy (TEM) image of PTz-CD-COF, scale bar 200 nm; (e) TEM image of PTz-CD-COF, scale bar 100 nm; (f) TEM image of PTz-CD-COF, scale bar 50 nm; (g) TEM image of PTz-CD-COF, scale bar 50 nm; (h) High-resolution transmission electron microscopy (HR-TEM) image of PTz-CD-COF, scale bar 10 nm; (i) High-resolution transmission electron microscopy (HR-TEM) image of PTz-CD-COF, scale bar 5 nm; (j) Element-mapped image of PTz-CD-COF;

[0030] Figure 3 (a) Photothermal effect of PTz-CD-COF at different concentrations under 638 nm laser irradiation; (b) Photothermal effect of PTz-COF at different concentrations under 638 nm laser irradiation; (c) Infrared thermal images of PTz-CD-COF at different concentrations; (d) Comparison of photothermal effects of PTz-CD-COF and PTz-COF at 200 μg / mL; (e) Temperature change curves of PTz-CD-COF (200 μg / mL) under different laser powers; (f) Temperature change curves of PTz-COF (200 μg / mL) under different laser powers; (g) Temperature change curves of PTz-CD-COF and PTz-COF after 638 nm laser irradiation; (h) Photothermal curves of PTz-CD-COF (200 μg / mL) after five cycles; (i) Photothermal effect of PTz-CD-COF (200 μg / mL) at 638 nm laser irradiation. (j) Temperature change curve after irradiation with nm laser, and negative natural logarithm and temperature change curve during cooling period; (j) Comparison of temperature distribution of PTz-CD-COF after 30 days of incubation in water with that of 0 days (freshly prepared) under 638 nm laser irradiation.

[0031] Figure 4(a) UV-Vis absorption spectrum of pure DPBF under 638 nm laser irradiation; (b) UV-Vis absorption spectrum of PTz-CD-COF+DPBF under 638 nm laser irradiation; (c) UV-Vis absorption spectrum of PTz-COF+DPBF under 638 nm laser irradiation; (d) UV-Vis absorption spectrum of pure MB under 638 nm laser irradiation; (e) UV-Vis absorption spectrum of PTz-CD-COF + MB under 638 nm laser irradiation; (f) UV-Vis absorption spectrum of PTz-COF+MB under 638 nm laser irradiation; (g) Fluorescence spectrum of pure DHR-123 under 638 nm laser irradiation; (h) Fluorescence spectrum of PTz-CD-COF + DHR-123 under 638 nm laser irradiation; (i) Fluorescence spectrum of PTz-COF + DHR-123 under 638 nm laser irradiation; (j) Comparison of decay rates of PTz-CD-COF and PTz-COF-induced DPBF under 638 nm laser irradiation; (k) Comparison of decay rates of PTz-CD-COF and PTz-COF-induced MB under 638 nm laser irradiation; (l) Comparison of PTz-CD-COF and PTz-COF-induced ΔI (DHR-123) under 638 nm laser irradiation; (m) 1 EPR spectrum of O2; EPR spectrum of (n) •OH; (o) O2 •- EPR spectrum;

[0032] Figure 5 (a) Photographs of Staphylococcus aureus treated with different concentrations of PTz-CD-COF and PTz-COF; (b) Photographs of Escherichia coli treated with different concentrations of PTz-CD-COF and PTz-COF; (c) Bacterial survival rate of Staphylococcus aureus after different treatments; (d) Bacterial survival rate of Escherichia coli after different treatments;

[0033] Figure 6 (a) Transmission electron microscope image of Staphylococcus aureus; (b) Transmission electron microscope image of Escherichia coli; (c) SYTO9 / PI stained image of Staphylococcus aureus; (d) SYTO9 / PI stained image of Escherichia coli; (e) Quantitative analysis diagram of SYTO9 / PI staining of Staphylococcus aureus; (f) Quantitative analysis diagram of SYTO9 / PI staining of Escherichia coli.

[0034] Figure 7 (a) Cytotoxicity of different concentrations of PTz-CD-COF; (b) Hemolytic effect of different concentrations of PTz-CD-COF; (c) Cell migration as detected by wound healing assay; (d) Statistical graph of cell migration rate.

[0035] Figure 8 (a) Proportion of Staphylococcus aureus and Escherichia coli biofilms after PTz-CD-COF treatment; (b) Proportion of Staphylococcus aureus and Escherichia coli biofilms after PTz-COF treatment; (c) Quantitative analysis of Staphylococcus aureus and Escherichia coli biofilms using laser confocal fluorescence images; (d) Laser confocal fluorescence image of Staphylococcus aureus biofilm; (e) Laser confocal fluorescence image of Escherichia coli biofilm.

[0036] Figure 9 (a) Images of mouse wounds on days 1, 3, 5, 7, 9, and 11; (b) Changes in wound area from day 1 to day 11;

[0037] Figure 10 (a) H&E staining and Masson staining of wound skin in each group; (b) Schematic diagram of mouse organ sections;

[0038] Figure 11 : Blood routine analysis of mice after treatment;

[0039] Figure 12 Synthetic route diagram of PTz-CD-COF;

[0040] Figure 13 Synthesis route diagram of PTz-COF. Detailed Implementation

[0041] 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.

[0042] As described in the background section, severe aggregation of COFs' layered structure can disrupt intermolecular interactions and photophysical processes. Aggregation leads to a shortening of intermolecular distances, making it more difficult to separate photogenerated electrons and holes spatially, which can easily lead to rapid recombination, resulting in photoactivity quenching and an inability to effectively participate in subsequent photochemical reactions. Therefore, the antibacterial effect will decrease.

[0043] Based on this, the purpose of this invention is to provide a phenothiazine-based two-dimensional covalent organic framework material constructed using supramolecular inclusion complexation and its applications. This invention selects a copolymer of 4,4',4'',4'''-(anthracene-9,10-dimethylbis(10H-phenothiazine-10,3,7-trimethyl))tetrabenzaldehyde (PTz) and β-CD benzidine inclusion complex. This is because the phenothiazine group, as a typical electron-rich donor, has sulfur and nitrogen atoms in its heterocycles that possess lone pairs of electrons, which can delocalize electrons to the connected aromatic rings through a p-π conjugated system. Furthermore, the oxygen-rich cycloalkyl groups of cyclodextrin molecules, due to the strong electronegativity of oxygen atoms, can induce electron-deficient regions (electron acceptor sites) with reduced local electron cloud density in the benzidine ring through an inductive effect. 4,4',4'',4'''-(anthracite-9,10-dimethylbis(10H-phenthiazine-10,3,7-trimethyl))tetrabenzaldehyde contains two phenthiazine groups and one anthracene. The introduction of the two phenthiazine groups significantly enhances the overall electron-donating ability of the molecule, while the large π-conjugated skeleton of the anthracene ring not only lowers the electronic transition energy barrier but also greatly enhances the degree of conjugation of the molecule. After binding with β-CD benzidine inclusion complex, a DA-type strongly conjugated structure is formed. When the polymer absorbs photons, electrons are excited from the donor (D) part and transferred to the acceptor (A) part, forming an excited state. The energy of the excited state is mainly dissipated through non-radiative means (such as molecular vibration and rotation) rather than being released in the form of fluorescence emission. This efficient non-radiative transition process is the fundamental reason for achieving high photothermal conversion efficiency, thus giving the material photothermal properties. In addition, the structural design of PTz-CD-COF successfully solved the problem of photoactive quenching caused by excessive interlayer aggregation in COF materials, significantly improving the photoresponse efficiency. Compared with PTz-COF prepared without the addition of cyclodextrin, PTz-CD-COF not only has better biocompatibility, but also forms a locally strongly electron-deficient structure, which enhances the conjugation effect with PTz.

[0044] 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.

[0045] Unless otherwise specified, all PBS used in this invention is neutral with a pH of 7.4.

[0046] 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.

[0047] Example 1: Preparation of PTz-CD-COF

[0048] (1) Preparation of 4,4',4'',4'''-(anthracene-9,10-dimethylbis(10H-phenthiazine-10,3,7-trimethyl))tetrabenzaldehyde (PTz)

[0049] (a) 3.0 g of 4-formylphenylboronic acid (20 mmol), 4.56 g of neopentyl glycol (44 mmol), and 69 mg of p-toluenesulfonic acid were dissolved in 30 mL of toluene. The solution was heated overnight at 115 °C under nitrogen atmosphere. After cooling to room temperature, dichloromethane was added, and the mixture was washed with saturated sodium bicarbonate solution, dried over anhydrous magnesium sulfate, and the solvent was removed by evaporation under reduced pressure. The solution was then recrystallized from anhydrous ethanol to give a white needle-like solid powder. This powder was named 2-(4-(5,5-dimethyl-1,3-dioxacyclopentanediyl)phenyl)-5,5-dimethyl-1,3,2-dioxaborane, and the synthetic route is as follows:

[0050] .

[0051] (b) 3.57 g of 3,7-dibromo-10H-phenthiazine (CAS: 21667-32-3, 10 mmol), 6.06 g of 2-(4-(5,5-dimethyl-1,3-dioxacyclopentan-2-yl)phenyl)-5,5-dimethyl-1,3,2-dioxaborane (22 mmol), 13.03 g of cesium carbonate (40 mmol), and 1.15 g of Pd(pph3)4 (10 mmol) were placed in a 250 mL three-necked flask. Under nitrogen protection, 100 mL of dry tetrahydrofuran was added, and the mixture was heated at 80 °C for 24 hours. After cooling to room temperature, the solvent was removed under reduced pressure. The resulting solid was washed with methanol and purified by silica gel column chromatography to obtain a yellow powder. Named 3,7-bis(4-(5,5-dimethyl-1,3-dioxacyclopentan-2-yl)phenyl)-10H-phenthiazine, the synthetic route is as follows:

[0052] .

[0053] (c) 1.16 g of 3,7-bis(4-(5,5-dimethyl-1,3-dioxacyclopentan-2-yl)phenyl)-10H-phenthiazine (2 mmol), 336 mg of 9,10-dibromoanthracene (1 mmol, CAS: 523-27-3), 577 mg of sodium tert-butoxide (6 mmol, CAS: 865-48-5), 195 mg of tri-tert-butylphosphotetrafluoroborate (0.67 mmol, CAS: 131274-22-1), 53.4 mg of 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (0.13 mmol, CAS: 564483-18-7) and 91.6 mg of tris(dibenzylacetone)dipalladium(0) (10 mmol (CAS: 51364-51-3) was added to a 100 mL three-necked flask and mixed thoroughly. 40 mL of toluene was added under argon atmosphere, and the mixture was heated at 105 °C for 72 hours. After cooling to room temperature, the solvent was removed under reduced pressure. The solid was washed with methanol, dissolved in dichloromethane, extracted with water, dried over MgSO4, and then evaporated by rotary evaporation to obtain the crude product. Further purification by column chromatography yielded a pure red powder. Named 9,10-bis(3,7-bis(4-(5,5-dimethyl-1,3-dioxacyclopentan-2-yl)phenyl)-10H-phenthiazin-10-yl)anthracene, the synthetic route is as follows:

[0054] .

[0055] (d) Under a nitrogen atmosphere, 770 mg of 9,10-bis(3,7-bis(4-(5,5-dimethyl-1,3-dioxacyclopentan-2-yl)phenyl)-10H-phenthiazin-10-yl)anthracene was dissolved in 50 mL of dichloromethane. 5 mL of trichloroacetic acid (TFA) was added, and the mixture was stirred for 12 hours at room temperature in the dark. The solvent was removed under reduced pressure to obtain a pure red powder. This powder was named 4,4',4'',4'''-(anthracene-9,10-diylbis(10H-phenthiazin-10,3,7-triyl))tetrabenzaldehyde (PTz), and the synthetic route is as follows:

[0056] .

[0057] (2) Preparation of β-CD benzidine inclusion complex (CD-BZD)

[0058] 0.92 g of benzidine was added to a reaction flask, followed by 50 mL of anhydrous ethanol. The mixture was sonicated until the solid was completely dissolved to obtain a benzidine ethanol solution. 5.675 g of β-cyclodextrin was added to 150 mL of hot water (55 °C) and stirred continuously until the β-cyclodextrin was completely dissolved to obtain a β-cyclodextrin solution. The benzidine ethanol solution was slowly added dropwise to the β-cyclodextrin solution (1-2 seconds / drop) and stirred at 55 °C for 5 hours. The ethanol was removed using a rotary evaporator, and the remaining mixture was placed in an ice bath for 12 hours. The precipitate was filtered, and the filter cake was vacuum dried to obtain a yellowish-white powder, which is the β-CD benzidine inclusion complex (CD-BZD). The synthetic route is as follows:

[0059] .

[0060] (3) Preparation of PTz-CD-COF:

[0061] 52.76 mg CD-BZD (0.04 mmol) and 19.8 mg PTz (0.02 mmol) were added to a clean mortar. After grinding the mixture, 3 M acetic acid solution (3 drops) was added and the mixture was ground until dry, followed by anhydrous ethanol (5 drops) and grinding until dry. After grinding repeatedly for 30 minutes, the mixture was transferred to a reaction vessel, and 0.33 mL of n-butanol (n-BuOH) and 0.67 mL of o-dichlorobenzene (DCB) were added. The mixture was sonicated for 2 min, and then 0.1 mL of 6 M acetic acid solution was added, followed by sonication for another 2 min. After degassing, nitrogen was purged, and the reactor was sealed and placed in a constant temperature oven at 150 °C for 3 days. After cooling, the precipitate was collected by filtration and washed successively with DMF, DMSO, THF, DCM, distilled water, and methanol / ethanol until the filtrate was colorless. The precipitate was collected and dried under vacuum at 60 °C to obtain a dark brown powder, named PTz-CD-COF. The synthetic route is shown in [link to synthetic route]. Figure 12 .

[0062] Comparative Example: Preparation of PTz-COF

[0063] The difference from Example 1 is that CD-BZD was not prepared; instead, benzidine was used to replace CD-BZD. The final product was a dark orange-red powdery solid, named PTz-COF. The synthetic route is shown below. Figure 13 .

[0064] Example 2: Characterization

[0065] (1) The structure of PTz-CD-COF was analyzed using infrared spectroscopy, such as... Figure 1As shown in (a), the FTIR of PTz-CD-COF exhibits characteristics of simultaneously combining 4,4',4'',4'''-(anthracene-9,10-dimethylbis(10H-phenthiazine-10,3,7-trimethyl))tetrabenzaldehyde (PTz) and β-CD benzidine inclusion complex (CD-BZD), and the properties of β-CD (3370 cm⁻¹) can be observed. -1 V at the location O-H and 1030 cm -1 V at the location C-O-C ) and benzidine (3320-3340 cm) -1 V at the location N-H The characteristic absorption peaks of N−H (3200−3400 cm⁻¹) can be clearly observed. -1 ) and C=O (1690 cm -1 The tensile vibration of ) is greatly attenuated at 1620 cm. -1 A significant new peak corresponding to the C=N tensile mode appeared at this point. This is attributed to the tensile vibration of C=N caused by the condensation of aldehydes and ammonia in PTz-CD-COF.

[0066] To further investigate the framework carbon composition of PTz-CD-COF, solid-state nuclear magnetic resonance (CP-MAS) was performed. Figure 1 As shown in (b), the spectrum reveals characteristic signals for the β-cyclodextrin saturated carbon (-CH and -CH2 peaks at 30 and 35 ppm) and the aromatic carbon of the phenothiazine unit (at 127 and 133 ppm). Notably, a dense peak corresponding to the imine bond at 152 ppm can also be detected in the solid-state NMR spectrum of PTz-CD-COF. All these results confirm the establishment of PTz-CD-COF.

[0067] The crystal structure of PTz-CD-COF was analyzed using powder X-ray diffraction (PXRD). Figure 1 As shown in (c), the material exhibits significant diffraction peaks at 21.4° and 43.8°. This characteristic diffraction pattern clearly confirms that PTz-CD-COF has a highly ordered layered stacked structure.

[0068] Figure 1 (d) The pore characteristics of PTz-CD-COF were systematically characterized by a low-temperature nitrogen adsorption-desorption test at 77 K. Figure 1 (e) Analysis of the pore size distribution shows that the PTz-CD-COF has a wide pore size distribution range, from 1.12 to 25 nm, with the main peak at 1.76 nm. The secondary macropores have a pore size of more than 10 nm, which together constitute a hierarchical pore system.

[0069] Figure 1(f) Thermogravimetric analysis (TGA) results show that PTz-CD-COF exhibits excellent thermal stability, and can still retain more than 57% of its initial mass at 800℃. This characteristic provides an important guarantee for its application as a photothermal agent.

[0070] (2) The microstructure of PTz-CD-COF was systematically studied by combining scanning electron microscopy (SEM) and transmission electron microscopy (TEM) characterization techniques. Figure 2 (a) ~ Figure 2 (c) SEM analysis clearly reveals the typical morphological characteristics of this material: PTz-CD-COF exhibits a spatial blocky stacked structure formed by the self-assembly of nanosheets, with obvious pits observed on its surface. Furthermore, Figure 2 (d)~ Figure 2 The transmission electron microscopy results of (g) provide direct evidence for the layered porous structure of PTz-CD-COF. Figure 2 (h)~ Figure 2 (i) High-resolution transmission electron microscopy (HR-TEM) revealed light and dark contrasts caused by differences in electron density, corresponding to uniformly distributed microporous channels in the material. Direct visualization of the microporous structure confirmed the results of the gas adsorption tests. More importantly, Figure 2 Elemental surface distribution analysis and energy-dispersive X-ray spectroscopy (EDS) of (j) confirmed that the four characteristic elements C, O, N and S were highly uniformly dispersed in the material framework, reflecting the complete transformation of the precursor and the uniformity of the structure.

[0071] Example 3: Performance Testing

[0072] (1) Photothermal conversion performance test

[0073] PTz-CD-COF and PTz-COF aqueous suspensions with concentrations of 100, 200, 300, 400, and 600 μg / mL were prepared. Each concentration sample was aliquoted into 1 mL EP centrifuge tubes. A PBS group was used as a blank control to eliminate interference from the inherent photothermal effect of the solvent. The samples were then irradiated with a 638 nm laser at a power density of 1.5 W / cm². 2 The treatment lasted for 10 minutes. Simultaneously, the laser wavelength (638 nm) and irradiation duration (10 min) were kept constant, and four different powers were applied sequentially: 0.5 W / cm². 2 1 W / cm 2 1.5 W / cm 2 and 1.8 W / cm 2A PTz-CD-COF aqueous suspension with a concentration of 200 μg / mL was irradiated. During the experiment, temperature data was read in real time using an infrared thermal imaging display to capture temperature change images at different time points. One mL sample of the PTz-CD-COF aqueous suspension with a concentration of 200 μg / mL was selected and irradiated with a 638 nm near-infrared laser (1.5 W / cm²). 2 The sample was irradiated with a laser for 10 minutes after laser activation, then the laser was turned off. The suspension was allowed to cool naturally to room temperature, completing one "laser on / off" cycle. This process was repeated to accumulate five complete cycles. The temperature changes throughout the five cycles were recorded to generate a complete periodic temperature curve. Cooling phase data were extracted from each complete cycle, and the photothermal conversion efficiency of PTz-CD-COF was calculated through heat loss analysis to evaluate its photothermal stability. Subsequently, PTz-CD-COF aqueous suspensions (concentration 200 μg / mL) stored for 0 days and 30 days were respectively heated with a 638 nm near-infrared laser (1.5 W / cm²). 2 Irradiation was used to compare the changes in photothermal properties after long-term storage.

[0074] like Figure 3 As shown in (a), unlike the negligible temperature changes observed in the PBS control group, PTz-CD-COF exhibited a concentration-dependent temperature increase. At concentrations of 100, 200, 300, and 600 μg / mL, the temperature increased to 45.7, 57.7, 67.4, and 71.4 °C, respectively. In contrast, Figure 3 The temperature changes in (b) show that for PTz-COF concentrations of 100, 200, 300 and 600 μg / mL, the temperature only increased to 42.3, 50.3, 56.3 and 61.2 °C, respectively. Figure 3 (c) The thermal imaging analysis results intuitively demonstrate the excellent photothermal conversion performance of the PTz-CD-COF material. Figure 3 (d) shows that PTz-CD-COF exhibits significantly better photothermal performance than PTz-COF. Furthermore, Figure 3 (e) The thermal performance test results show that the photothermal conversion efficiency of PTz-CD-COF (200 μg / mL) is significantly positively correlated with the laser power density. Under 638 nm near-infrared laser irradiation, as the power density increases from 0.5 W / cm², the efficiency of the photothermal conversion increases. 2 Gradually increase to 1.8 W / cm 2 The temperature rose (ΔT) from 12.3 o C rose to 27.7 o C, confirming its highly responsive and efficient photothermal behavior. In contrast, Figure 3(f) Under the same power irradiation, the temperature rise of PTz-COF is significantly lower. Figure 3 (g) Side-by-side comparisons under the same irradiation conditions further validated the performance improvement of PTz-CD-COF compared to PTz-COF. Quantitative analysis showed that the photothermal effect of PTz-CD-COF can be dually adjusted by concentration (45.7~71.4℃) or laser power modulation (ΔT = 12.3~27.7℃), highlighting its flexibility as a photothermal agent.

[0075] In order to systematically evaluate the photostability of PTz-CD-COF, Figure 3 (h)~ Figure 3 (i) Multi-cycle photothermal performance tests were conducted. PTz-CD-COF was irradiated with a 638 nm near-infrared laser (1.5 W / cm²). 2 It exhibits excellent cycle stability and highly overlapping heating / cooling curves over five consecutive switching cycles. This repeatable photothermal response characteristic demonstrates the excellent photostability of PTz-CD-COF. Its molecular structure remains intact under laser irradiation, making it suitable for photothermal applications requiring long-term stability.

[0076] To systematically evaluate the long-term stability of the material, a 30-day stability test was conducted on the PTz-CD-COF aqueous suspension, and then its photothermal properties were retested. Figure 3 (j). Under the same test conditions (1.5 W / cm²), 2 After 10 min of laser irradiation, PTz-CD-COF, after being placed in water for 30 days, still exhibited a temperature response curve highly consistent with that of 0 days (freshly prepared), with a maximum temperature difference not exceeding 0.5℃. This result fully confirms that PTz-CD-COF did not degrade after 30 days of immersion, highlighting its excellent water stability. The continuous photothermal conversion capability of PTz-CD-COF confirms its reliable recyclability.

[0077] The photothermal conversion efficiency of PTz-CD-COF is η (%) = 28.06%. The photothermal conversion efficiency was calculated using the formula published in application CN202310175497.X, entitled "A Photothermal-Fenton-like Artificial Nanoenzyme and Its Preparation Method and Application".

[0078] (2) By detecting singlet oxygen in PTz-CD-COF ( 1 O2), hydroxyl radicals (•OH) and superoxide anions (O2) •- Evaluate its photodynamic performance

[0079] 1) A 1 mg / mL PTz-CD-COF stock solution was prepared using DMF as the solvent. 30 μL of this solution was diluted with 3 mL of DMF to adjust the absorbance to approximately 1.0. Subsequently, the solution was subjected to fluorescence sampling at 0, 2, 4, 6, 8, and 10 minutes at a power of 1.5 W / cm². 2 The DPBF probe was irradiated with a 638 nm laser. UV absorption measurements were performed after each irradiation to verify the stability of the DPBF probe under the experimental illumination conditions. Subsequently, a PTz-CD-COF dispersion with a concentration of 200 μg / mL was prepared using DMF, and 30 μL of the DPBF probe was added and thoroughly mixed. The mixture was placed in a cuvette and irradiated with the same laser parameters (638 nm, 1.5 W / cm²). 2 Irradiation for varying durations was conducted. Absorption spectra were measured immediately after each irradiation using a UV spectrophotometer. When calculating singlet oxygen yield, interference was eliminated by subtracting the absorbance of the PTz-CD-COF itself. Each experiment was repeated three times. Finally, the UV absorption spectrum of PTz-CD-COF was plotted based on the post-irradiation UV absorption detection results. The photodynamic performance evaluation of PTz-COF followed the exact same experimental procedure as for PTz-CD-COF. Figure 4 As shown in (a), after continuous irradiation with a 638 nm laser for 10 minutes, the absorbance of the characteristic absorption peak (415 nm) of the control group (pure DPBF solution) only decreased slightly, confirming that the system does not produce [catalytic oxidation] under non-catalytic conditions. 1 O2. Conversely. Figure 4 (b) PTz-CD-COF exhibited significant catalytic activity, with a decay rate as high as 54.99%, while Figure 4 (c) The PTz-COF system reduced the DPBF absorbance by only 39.58% within 10 minutes. Figure 4 The quantitative analysis results of (j) further confirm this trend: when PTz-CD-COF and PTz-COF are introduced into the DPBF system respectively, both show significant signal attenuation within a 10-minute laser irradiation cycle, and the attenuation amplitude of the PTz-CD-COF group is more significant. The observed absorbance attenuation and 1 O2-mediated DBB formation is directly related to the generation of reactive oxygen species (ROS) through type II photodynamic therapy (PDT).

[0080] 2) Methylene blue (MB) was used as an indicator to assess the hydroxyl radical (•OH) generation capacity of PTz-CD-COF by detecting its UV absorption change. First, 1 mg / mL MB stock solution and PTz-CD-COF aqueous solution were prepared using deionized water. In the control experiment, 30 μL of MB aqueous solution was added to an EP tube, diluted with 3 mL of deionized water, and thoroughly mixed. The mixture was then transferred to a 3 mL cuvette and purified using a 1.5 W / cm² cuvette.2 Irradiation with a 638 nm laser for 0, 2, 4, 6, 8, and 10 minutes was performed. UV absorbance was measured immediately after each irradiation to observe the intrinsic changes of MB under illumination. In the experimental group, a solution containing PTz-CD-COF (200 μg / mL) was first prepared, and 30 μL of MB indicator was added to adjust the total volume of the mixture to 3 mL. Subsequently, the same parameters (638 nm, 1.5 W / cm²) were used at the aforementioned time points. 2 The laser was used for irradiation. Absorption spectra were measured immediately after each irradiation using a UV spectrophotometer. When calculating the amount of hydroxyl radicals generated, the inherent absorbance of PTz-CD-COF had to be subtracted to eliminate interference. Finally, the UV absorption spectrum of PTz-CD-COF was plotted based on the UV absorption detection results after irradiation, and the amount of hydroxyl radicals (•OH) generated was assessed by the degree of absorbance reduction using an MB indicator. The photodynamic performance of PTz-COF was evaluated using the same method as for PTz-CD-COF, completing all experimental procedures. The hydroxyl radical (•OH) generation capacity of different material systems was systematically compared using a methylene blue (MB) oxidation and fading experiment. Figure 4 (d) Showing the blank control group (pure MB solution) under 638 nm laser (1.5 W / cm²) 2 After continuous irradiation for 10 minutes, the absorbance of its characteristic 665 nm absorption peak decreased by only 14.98%, indicating that spontaneous oxidation was negligible. Figure 4 (e) and Figure 4 (f) shows that the methyl blue fading rate of the PTz-CD-COF group reached 72.27%, while the methyl blue fading rate of PTz-COF was only 38.73%, and the efficiency of the PTz-CD-COF group was more than twice that of PTz-COF. Figure 4 As shown in the quantitative detection data (k), compared with the blank control group of pure MB solution, the detection values ​​decreased significantly within 10 minutes after laser irradiation after the introduction of PTz-CD-COF and PTz-COF into MB. The reduction effect induced by PTz-CD-COF was particularly significant.

[0081] 3) Using the dihydrorhodamine 123 (DHR123) fluorescent probe method, under 638 nm laser irradiation (1.5 W / cm²), 2 The quantitative evaluation of superoxide anion (O2) in different material systems was carried out. •- Generation capacity. Dihydrorhodamine 123 (DHR123) was used as a fluorescent probe to detect superoxide anion radicals (O2). •-The specific operating steps are as follows: Take a PBS solution (pH 5.5) containing PTz-CD-COF (concentration 200 μg / mL), add 3 μL of DHR123 solution (solvent DMSO, concentration 5 mM), and then adjust the total volume of the mixture to 3 mL with PBS of the same specification (pH 5.5); then use 1.5 W / cm 2 The mixture was irradiated with a 638 nm laser for 0, 2, 4, 6, 8, and 10 minutes. After each irradiation, fluorescence signals in the 500-600 nm wavelength range were detected using a fluorescence spectrometer to observe superoxide radicals (O2). •- The generation status was also examined. Furthermore, for comparative analysis, pure DHR123 solution and a mixture of DHR123 containing PTz-COF were tested under the same experimental conditions (consistent with the experimental conditions of the PTz-CD-COF group). Figure 4 (g)~ Figure 4 (i) DHR123 fluorescence detection showed that PTz-CD-COF induced a 2.85-fold increase in fluorescence intensity, significantly higher than the 1.74-fold enhancement of PTz-COF. Figure 4 (l) reflects its stronger O2 •- Generation capacity. All three assays showed a consistent ROS generation trend (PTz-CD-COF > PTz-COF > control group of pure DHR123 solution), verifying the synergistic effect mechanism of multiple ROS.

[0082] 4) To elucidate the photodynamic antibacterial mechanism of PTz-CD-COF, electron paramagnetic resonance (EPR) spectroscopy with a specific spin trapping agent was used to systematically study the reactive oxygen species (ROS) generation spectrum under 638 nm laser irradiation (1.5 W / cm², 10 min). EPR analysis clearly confirmed the simultaneous generation of multiple ROS. Figure 4 TEMP- (m) 1 The unique triplet signal presented in the O2 spectrum, with its three peaks exhibiting equal intensity (1:1:1), confirms the formation of singlet oxygen through energy transfer from the triplet state to molecular oxygen via photosensitizer excitation (type II pathway). 1 O2). Meanwhile... Figure 4 The typical peak shape with a 1:2:2:1 quadtt ratio in the (n) DMPO-•OH spectrum confirms the formation of hydroxyl radicals (•OH), which is likely generated through further reduction of O2. •– It generates, or through the strong oxidation of water by photogenerated holes in the valence band. Figure 4 (o) of DMPO-O2 •– The characteristic quartet signal (intensity ratio of 1:1:1:1) presented in the spectrum confirms the presence of superoxide anion (O2). •–As a major type I reactive oxygen species (ROS), O2 originates from the efficient photoinduced electron transfer from PTz-CD-COF to environmental oxygen molecules. •– / •OH / 1 The O2 ternary reactive oxygen system generates a powerful synergistic antibacterial effect by simultaneously attacking key targets of multiple microorganisms.

[0083] Test Example 1: In vitro antibacterial performance test

[0084] Staphylococcus aureus ( S. aureus ) and Escherichia coli ( E. coli As representatives of Gram-positive and Gram-negative bacteria, these two types of bacteria were used to test the antibacterial properties of PTz-CD-COF and PTz-COF. The experiment consisted of five concentrations: 100 μL of bacterial suspension containing Staphylococcus aureus or Escherichia coli (colony count 10⁻⁶) was used respectively. 8 CFU mL -1 Add 0, 50, 100, 150, or 200 μL of 1 mg / mL PTz-CD-COF suspension (prepared in PBS) or 1 mg / mL PTz-COF suspension (prepared in PBS) to each EP tube, then add sterile neutral PBS to each tube to a final volume of 1 mL. The concentrations of PTz-CD-COF and PTz-COF suspension are 0 (pure PBS), 50, 100, 150, and 200 μg / mL. The solution is then analyzed at 638 nm and 1.5 W·cm⁻¹. -2 After 10 minutes of laser irradiation, 80 μL of bacterial suspension was evenly spread onto a solid culture medium plate. After incubation at 37°C for 24 hours, bacterial growth morphology was observed, and relative bacterial activity was assessed by counting colonies and comparing them with a 0 μg / mL control group.

[0085] Irradiation with 638 nm laser (1.5 W / cm) 2 At 10 minutes, PTz-CD-COF and PTz-COF (0~200 μg / mL) both showed significant dose-dependent antibacterial effects after co-culturing with bacteria for 12 hours. Figure 5 (a) ~ Figure 5 As shown in (b), the number of surviving bacteria on the surfaces of PTz-CD-COF and PTz-COF treated with laser irradiation was significantly reduced compared to the control group (PBS). Notably, the antibacterial efficacy significantly increased with increasing concentrations of PTz-CD-COF and PTz-COF. At equivalent material concentrations, PTz-CD-COF exhibited superior bactericidal activity compared to PTz-COF, and this difference was evident in... Figure 5 (c)~ Figure 5(d) This can also be directly observed in the quantitative analysis. More notably, when the concentration of PTz-CD-COF was 200 μg / mL, its bactericidal rate against Staphylococcus aureus and Escherichia coli exceeded 99%, while PTz-COF required a higher concentration to produce the same bactericidal effect against Staphylococcus aureus and Escherichia coli. The difference in antibacterial effect clearly reveals the enhancing effect of cyclodextrin modification, which is mainly attributed to: the targeted binding of cyclodextrin cavities to bacterial membranes, the synergistic effect of photothermal and photodynamic processes, and the increased production of reactive oxygen species (ROS).

[0086] Experimental Example 2: Bacterial Staining Test

[0087] (1) Bacterial transmission electron microscopy (TEM)

[0088] The experiment was conducted in six groups: (I) PBS control group, (II) PTz-COF group, (III) PTz-CD-COF group, (IV) PBS + laser group, (V) PTz-COF + laser group, and (VI) PTz-CD-COF + laser group. The total volume of each mixture was 1 mL, containing 100 μL of a 10- concentration. 8 CFU / mL bacterial suspensions and corresponding groups of PTz-CD-COF and PTz-COF at concentrations of 200 μg / mL. Laser groups (IV), (V), and (VI) were used at a power density of 1.5 W / cm². 2 The samples were irradiated with a 638 nm infrared laser for 10 minutes. During the experiment, 2.5 wt% glutaraldehyde solution was added to each group of solutions, followed by fixation at 4°C for 24 hours. After fixation, the samples were washed three times with PBS, and then sequentially embedded and blocked in agar. Subsequent dehydration and deethanolification steps were as follows: the bacterial solutions were sequentially treated with 30 wt%, 50 wt%, 70 wt%, 90 wt%, 95 wt%, and 100 wt% ethanol solutions at room temperature, with each concentration treated for 10 minutes to achieve gradient dehydration. After dehydration, the samples were deethanolified with acetone at room temperature for 3 hours.

[0089] Figure 6 (a) ~ Figure 6Transmission electron microscopy (TEM) characterization results in (b) clearly revealed the bactericidal mechanism of PTz-CD-COF. In (I) the control group, (II) the PTz-COF group, (III) the PTz-CD-COF group, and (IV) the PBS+laser group, the bacteria maintained their intact morphological characteristics: the cell membrane structure was intact, the surface was smooth and undamaged, and the flagellar structure was clearly visible. In contrast, the laser irradiation treatment groups showed significant differences: the PTz-COF+laser group (V) only caused local membrane damage and flagellar breakage, while the PTz-CD-COF+laser group (VI) showed extensive membrane disintegration (damage rate >90%), resulting in a large amount of intracellular substances leaking out and organelle structure destruction. This progressive morphological change (intact → local damage → complete disintegration) not only confirmed the bactericidal advantage of photothermal-photodynamic synergy, but also revealed the mechanism by which β-cyclodextrin enhances the antibacterial mechanism at the ultrastructural level. By enhancing the affinity between the material and the bacterial membrane, the transmembrane transport of reactive oxygen species (ROS) and the local accumulation of heat energy are promoted, ultimately achieving highly efficient bactericidal activity.

[0090] (2) Staining of live / dead bacteria

[0091] Live and dead bacteria were distinguished using a dual-fluorescence staining method with SYTO-9 and PI. SYTO-9 can penetrate the cell membrane of all bacteria (regardless of membrane integrity), causing them to exhibit green fluorescence; while PI can only penetrate bacteria with damaged membranes, causing them to exhibit red fluorescence, and simultaneously weakening the green signal of SYTO-9. The experimental groups were the same as in (1). Groups (IV), (V), and (VI) were stained at a power density of 1.5 W / cm². 2 The bacteria were irradiated with a 638 nm infrared laser for 10 minutes. Then, 100 μL of bacterial solution was taken from each group. Under dark conditions at 37°C, 20 μL of a 1.5 × 10⁻⁶ concentration was added. -3 20 μL of SYTO-9 at a concentration of mol / L and PI at the same concentration were used. After staining for 15 minutes, excess dye was removed by centrifugation with PBS buffer. The bacteria were then resuspended in 50 μL of PBS and placed on a glass slide. Finally, images of Escherichia coli and Staphylococcus aureus were acquired using an inverted fluorescence microscope. Each experimental procedure was repeated three times to ensure the reliability of the results.

[0092] Figure 6 (c)~ Figure 6 (d) Confocal laser scanning microscopy (CLSM) live-dead staining analysis further confirmed the photoactivated antibacterial properties of PTz-CD-COF. Consistent with the plate counting results, (I) control group, (II) PTz-COF group, (III) PTz-CD-COF group and (IV) PBS+laser group mainly showed green fluorescence, indicating that these treatments had limited effect on bacterial activity. Figure 6 (e)~ Figure 6Quantitative analysis of live and dead cell staining in (f) showed that the PTz-COF+laser group (V) exhibited distinct coexistence of red and green fluorescence (mortality rate approximately 75%), while the PTz-CD-COF+laser group (VI) showed almost entirely red fluorescence (mortality rate >98%). The clear gradient changes in fluorescence signal indicate that the material itself has relatively weak phototoxicity, but laser activation significantly enhances the bactericidal effect, while CD modification optimizes the synergistic effect of photothermal / photodynamic therapy. Notably, the uniform distribution of red fluorescence in group VI confirms that the material can achieve spatially consistent antibacterial effects, providing important evidence for its uniform therapeutic application at infection sites.

[0093] Experimental Example 3: In vitro biocompatibility experiment

[0094] (1) Cytotoxicity test

[0095] 96-well plates were used as the culture medium for relevant assays of 3T3 cells (purchased from the Cell Bank of the Chinese Academy of Sciences). First, 100 μL of cell suspension was added to each well, with a cell density of 5,000 cells per well. Three replicate wells were set for each group to ensure the reliability of the results. To prevent excessive evaporation during culture, 100 μL of PBS buffer was added to each well on the periphery of the 96-well plate. After incubating the 96-well plate at a constant temperature for 24 hours, PTz-CD-COF suspension prepared in complete DMEM medium was added to each well. The suspension concentrations were set at 0, 100, 150, 200, 250, and 300 μg / mL. After incubation at a constant temperature for 24 hours, 10 μL of 5 mg / mL MTT solution was added to each well under dark conditions. After incubation for 4 hours, the supernatant was aspirated from each well, and 100 μL of dimethyl sulfoxide (DMSO) was added. The 96-well plate was then shaken for 10 minutes to ensure complete dissolution of the formamide crystals formed by the MTT reaction. Finally, the absorbance at 490 nm was measured using a microplate reader. To ensure data accuracy, this measurement was repeated three times. Cell viability was calculated based on the final absorbance data. This step used the MTT assay to quantitatively assess cell viability, with each group repeated three times. The calculation formula is as follows:

[0096] Cell viability (%) = (average value of treatment group) / (average value of control group) × 100%.

[0097] Figure 7 (a) The MTT assay data further confirmed that after PTz-CD-COF treatment for 24 hours, the survival rate of 3T3 fibroblasts remained above 80% (83.87±1.86% in the 300μg / mL group), indicating excellent cell compatibility.

[0098] (2) Material hemolysis test

[0099] Blood samples were collected from 5-week-old female KM mice (purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.). First, red blood cells (RBCs) were isolated and purified: blood was collected and centrifuged at 1500 rpm for 20 minutes, and the precipitated RBCs were collected. The RBCs were then washed with PBS buffer at a volume ratio of approximately 1:1, centrifuged again, and the supernatant was discarded. This washing and centrifugation step was repeated four times to remove blood impurities. After purification, RBC suspensions were prepared by adding PBS buffer at a volume ratio of 3:11. This suspension was then mixed with different concentrations of PTz-CD-COF solution (50, 100, 150, 200, 250, and 300 μg / mL) at a volume ratio of 1:9 and incubated at 37°C for 4 hours. After incubation, 100 μL of the supernatant from each mixture was transferred to a 96-well plate. The absorbance at 570 nm was measured using a microplate reader. A control group was set up: distilled water as a positive control and PBS buffer as a negative control for result comparison and analysis. The amount of hemolysis is calculated using the following formula:

[0100] Hemolysis rate (%) = (As-An) / (Ap-An)×100%.

[0101] Wherein, "As" represents the absorbance produced after adding different concentrations of PTz-CD-COF to the red blood cell suspension. "An" represents the absorbance produced after adding PBS (negative control) to the red blood cell suspension. "Ap" refers to the absorbance produced after adding distilled water (positive control) to the red blood cell suspension.

[0102] Figure 7 (b) The hemolysis test results showed that although the material exhibited concentration-dependent hemolysis (hemolysis rate of 0.12±0.04% to 0.89%±0.18% at 50-300 μg / mL), even at the highest test concentration (300 μg / mL), the hemolysis rate was still below the international safety threshold (<5%).

[0103] (3) Cell scratch test

[0104] First, 3T3 cells were seeded into 6-well plates and cultured under suitable conditions until the cell density exceeded 90%. Once the target density was reached, the central region of the 3T3 cell monolayer was gently and slowly scraped away using the tip of a 20 μL pipette, dividing each well into two halves to form a scratch. The culture plate was then washed with high-pressure PBS to remove any cells detached from the scratch. After washing, fresh DMEM basal medium containing different concentrations of PTz-CD-COF (0, 100, 200, and 300 μg / mL) was added to each well, with 0 μg / mL serving as the blank (control) group. Finally, photomicrographs of the 3T3 cells in each well were taken at predetermined time points, and cell migration ability was analyzed by observing changes in cell coverage in the scratched area.

[0105] Figure 7 (c) and Figure 7 (d) Cell scratch assays showed that, after 24 hours, the PTz-CD-COF treatment group (100–300 μg / mL) exhibited no abnormal proliferation or abnormal migration morphology compared to the PBS control group (55.09 ± 1.98%). These experimental results collectively demonstrate that PTz-CD-COF exhibits reliable blood compatibility within the therapeutic concentration range (≤200 μg / mL) without impairing normal cell proliferation and migration functions. Its biocompatibility meets the basic requirements for medical materials and shows broad potential for clinical translation.

[0106] Experiment Example 4: Bacterial Biofilm Experiment

[0107] (1) The inhibitory effects of PTz-CD-COF and PTz-COF on Staphylococcus aureus and Escherichia coli biofilms were quantitatively evaluated by crystal violet (CV) staining.

[0108] The experiment was divided into two main groups, such as Figure 8 (a) Groups: (I) PBS group, (II) PTz-CD-COF group, and (III) PTz-CD-COF + laser group; as shown Figure 8 (b) Grouping: (I) PBS group, (II) PTz-COF group, and (III) PTz-COF + laser group. Each group contained three replicate wells. The experimental procedure was as follows: First, 150 μL of Staphylococcus aureus and Escherichia coli second-generation stock solution was added to each well of each group, and then the 96-well plate was incubated under suitable conditions for 48 hours. After incubation, 50 μL of the solution from each treatment group was added to each well (ensuring that the concentration of PTz-CD-COF and PTz-COF was 200 μg / mL). For groups requiring light treatment, the light was applied at a wavelength of 638 nm and a wavelength of 1.5 W / cm². 2 Irradiate the sample under a strong light source for 10 minutes. After irradiation, wash each well three times with PBS to remove suspended bacteria and other impurities. Then, fixation, staining, and destaining are performed sequentially: first, fix with 150 μL of formaldehyde solution for 30 minutes, then wash three times with PBS; next, add 5 μL of 0.1% crystal violet staining solution dropwise. After staining for 30 minutes, wash three times again with PBS; finally, destain with 100 μL of acetic acid solution for 30 minutes. After destaining, wash three times with PBS. After completing all steps, measure the absorbance of each well at 490 nm using a microplate reader, and evaluate the experimental results based on the absorbance data.

[0109] Experimental results showed that, under conditions without laser irradiation, PTz-CD-COF and PTz-COF alone exhibited only limited anti-biofilm activity, resulting in over 90% of the biofilm biomass remaining intact. In contrast, under laser irradiation (638 nm, 1.5 W / cm²), [the remaining biofilm activity was significantly reduced]. 2 At 10 min, PTz-CD-COF (200 μg / mL) exhibited strong biofilm scavenging ability. Figure 8 As shown in (a), group III (PTz-CD-COF + laser) achieved a clearance rate of 94.30 ± 0.34% for Staphylococcus aureus and 93.11 ± 0.43% for Escherichia coli. Figure 8 As shown in (b), Group III (PTz-COF + laser) achieved a clearance rate of 74.32 ± 1.10% for Staphylococcus aureus and 76.52 ± 0.75% for Escherichia coli. In contrast, the PTz-CD-COF + laser group exhibited superior bacterial biofilm clearance efficiency. These values ​​were significantly higher than those of the PBS control group (retention rate approximately 100%) and the pure PTz-CD-COF control group (retention rate approximately 98-99%) (p < 0.01). Light irradiation enhanced the anti-biofilm effect through a complex mechanism: the photothermal effect physically disrupted the biofilm structure, while the photodynamic generation of reactive oxygen species (ROS) chemically degraded the extracellular matrix. Therefore, PTz-CD-COF, as a photoresponsive material with both antibacterial and anti-biofilm functions, provides a promising treatment strategy for the clinical treatment of refractory biofilm infections.

[0110] (2) Three-dimensional imaging and quantitative analysis of biomembrane structure were performed using SYTO 9 staining combined with laser confocal scanning microscopy (CLSM).

[0111] First, bacteria (Staphylococcus aureus and Escherichia coli) were inoculated into TSB medium and cultured in a 37°C shaking incubator until the logarithmic growth phase. Then, an appropriate amount of the bacterial solution was diluted with fresh TSB medium to a concentration of 1×10⁻⁶. 8 CFU / mL concentration. Under aseptic conditions, 400 μL of diluted bacterial solution was added to a glass-bottomed culture dish to establish a biofilm culture system. The culture dishes were incubated at 37°C for 48 hours to promote biofilm formation. Five treatment groups were established: I: PBS control group; II: PTz-CD-COF group; III: PTz-COF group; IV: PTz-CD-COF + laser group; V: PTz-COF + laser group. The concentration of PTz-CD-COF and PTz-COF in the culture dish was maintained at 200 μg / mL, while the PBS concentration was kept consistent across all groups. Irradiation was performed for 10 minutes using a 638 nm infrared laser at a power density of 1.5 W / cm². 2All experimental groups were then transferred back to a 37°C incubator for 24 hours of further incubation. After incubation, the bacterial solution in the culture dishes was gently poured off. Each culture dish was rinsed twice with PBS buffer preheated to 37°C, allowing it to stand for 1 minute after each rinse to thoroughly remove non-attached free bacteria. Then, 2 μL of propidium iodide (PI) dye was added to each culture dish, and the dishes were incubated at 37°C for 30 minutes in the dark to allow the dye to fully label the dead bacteria. Finally, biofilm samples were imaged and analyzed using confocal laser scanning microscopy (CLSM). By observing the distribution of dead bacteria stained with PI and changes in biofilm structure, the inhibitory activity of PTz-CD-COF alone and in combination with laser irradiation on bacterial biofilms was evaluated. SYTO 9 staining combined with confocal laser scanning microscopy (CLSM) was used for three-dimensional imaging and quantitative analysis of the biofilm structure.

[0112] Figure 8 (c) Quantitative fluorescence analysis confirmed the CV staining results, demonstrating that PTz-CD-COF has excellent biofilm disruption efficacy under laser activation. Figure 8 (d)~ Figure 8 (e) shows that groups I, II and III present intact and dense biofilms, while group IV, which was treated with PTz-CD-COF and irradiated with laser, shows a significantly thinned and fragmented structure, with only sporadic green fluorescence indicating residual live bacteria.

[0113] Experiment 5: Wound Healing Experiment

[0114] A traumatic injury model was established using five-week-old female KM mice (purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.), with an initial weight of approximately 25g. Mice were randomly divided into six groups of three mice each (each group weighing between 20 and 25g). Experimental groups were: (I) PBS control group, (II) PTz-CD-COF, (III) PTz-COF, (IV) PTz-CD-COF + laser, (V) PTz-COF + laser irradiation, and (VI) blank control group. Laser irradiation parameters were: 638 nm, 1.5 W / cm². 2 (10 minutes); the concentrations of PTz-CD-COF and PTz-COF were both 200 μg / mL. Except for the blank control group, all other groups were prepared using PBS as the solvent, and the treatment volume was 50 μL. Except for the blank control group, the back hair of mice in the other groups was shaved after anesthesia. After disinfection of the back skin with 75% ethanol, a wound with a diameter of approximately 6 mm was created. Then, a 1×10⁻⁶ ppm PTz-CD-COF was applied. 6CFU / mL Staphylococcus aureus was inoculated into the wound. A back wound infection model was successfully established 24 hours after infection, and mice were then treated according to the grouping protocol. Changes in the back wound area were continuously recorded on days 1, 3, 5, 7, 9, and 11, and curves of back wound area change were plotted. After treatment on day 11, blood samples were first collected from the eyeballs of mice in each group. The mice were then euthanized by cervical dislocation. Back skin tissue and organs such as the heart, liver, spleen, lungs, and kidneys were removed and fixed in 4% formaldehyde solution to maintain their original morphology (this process can cause protein denaturation and coagulation). Simultaneously, 200 μL of blood was collected from each sample for routine hematological parameter testing. To directly and accurately assess the wound healing efficiency at the infection site, histological analysis and verification of new skin tissue were performed on the wounds of mice treated with different protocols. Specifically, after the 11-day treatment cycle, the repair and recovery status of the wounds in each group was observed and analyzed using hematoxylin and eosin (H&E) staining and Marzon staining.

[0115] Treatment monitoring data shows that Figure 9 (a) and Figure 9 (b) The wound area of ​​mice in each group gradually decreased over time, but there were significant differences in healing effects among different intervention regimens within the 11-day treatment period. Wound healing was monitored for 11 days. Although all groups showed a decrease in wound area over time, healing was significantly accelerated in group IV. By day 11, the wound area in group IV was only 7.75±0.51%, significantly smaller than that in group I (28.18±5.19%), group II (13.93±0.76%), group III (20.50±1.37%), and group V (13.03±1.51%) (p<0.01). Histological analysis was performed using hematoxylin-eosin (H&E) staining and Masson's trichrome staining techniques. The results are shown in [Figure 1]. Figure 10 (a) Images show that the PTz-CD-COF treatment group significantly promoted wound healing, with the PTz-CD-COF combined with laser treatment group (Group IV) being particularly outstanding. Clearly distributed new capillaries were observed in the subcutaneous tissue of this group, and the wound was almost completely closed. These findings confirm that PTz-CD-COF combined with laser treatment (Group IV) has a significant therapeutic effect in accelerating wound healing. These results fully validate the dual therapeutic advantages of PTz-CD-COF under photoactivated conditions: achieving highly efficient antibacterial effects through the CD-enhanced photothermal-photodynamic synergistic effect, while simultaneously accelerating tissue repair by promoting angiogenesis and collagen deposition. This provides experimental evidence for the development of intelligent dressings with both antibacterial and healing functions. More importantly, histological analysis of major organs such as the heart, liver, spleen, lungs, and kidneys was performed using H&E staining. Figure 10(b) The results show that all experimental groups exhibited intact organ structures, without pathological changes, inflammatory cell infiltration, or fibrosis, and cell morphology consistent with normal tissues. These multifaceted safety data collectively demonstrate that PTz-CD-COF does not induce systemic toxicity or cause organ-specific damage, fully meeting the stringent biosafety requirements for medical materials, thus providing a solid foundation for its clinical translation and application.

[0116] Systematic in vivo safety assessments confirmed that PTz-CD-COF possesses excellent biocompatibility. Routine blood test results ( Figure 11 The results showed that, regardless of whether the PTz-CD-COF treatment group used alone or the group used in combination with laser therapy, all key blood indicators were not significantly different from those of the control group, and all parameters were within the normal physiological range.

[0117] 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 phenothiazine-based two-dimensional covalent organic framework material constructed by supramolecular inclusion complexation, characterized in that, The phenothiazine-based two-dimensional covalent organic framework material uses phenothiazine derivatives and β-CD benzidine inclusion complexes as structural units, and is obtained as a two-dimensional crystalline porous copolymer through a solvothermal reaction. The phenothiazine derivative is 4,4',4'',4'''-(anthracene-9,10-dimethylbis(10H-phenothiazine-10,3,7-trimethyl))tetrabenzaldehyde, with the following structural formula: 。 2. The phenothiazine-based two-dimensional covalent organic framework material of claim 1, wherein The 4,4',4'',4'''-(anthracene-9,10-dimethylbis(10H-phenothiazine-10,3,7-trimethyl))tetrabenzaldehyde is prepared by the following method: (a) 4-Formylphenylboronic acid, neopentyl glycol and p-toluenesulfonic acid were dissolved in toluene and heated overnight under a protective atmosphere. After the reaction was completed, the product was purified to obtain a white needle-like solid, which is 2-(4-(5,5-dimethyl-1,3-dioxolane)phenyl)-5,5-dimethyl-1,3,2-dioxolane; (b) 3,7-dibromo-10H-phenthiazine, 2-(4-(5,5-dimethyl-1,3-dioxolane)phenyl)-5,5-dimethyl-1,3,2-dioxoborane, cesium carbonate and tetratriphenylphosphine palladium were added to tetrahydrofuran and subjected to a solvothermal reaction under a protective atmosphere. The product was purified to obtain a yellow powder, which is 3,7-bis(4-(5,5-dimethyl-1,3-dioxolane-2-yl)phenyl)-10H-phenthiazine; (c) 3,7-bis(4-(5,5-dimethyl-1,3-dioxacyclopentan-2-yl)phenyl)-10H-phenthiazine, 9,10-dibromoanthracene, sodium tert-butoxide, tri-tert-butylphosphotetrafluoroborate, 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl and tris(dibenzylideneacetone)dipalladium(0) were added to toluene and subjected to a solvothermal reaction under a protective atmosphere. The product was purified to obtain a red powder, which is 9,10-bis(3,7-bis(4-(5,5-dimethyl-1,3-dioxacyclopentan-2-yl)phenyl)-10H-phenthiazine-10-yl)anthracene; (d) Under a protective atmosphere, 9,10-bis(3,7-bis(4-(5,5-dimethyl-1,3-dioxacyclopentan-2-yl)phenyl)-10H-phenthiazin-10-yl)anthracene was dissolved in dichloromethane, and then trifluoroacetic acid was added. The mixture was stirred at room temperature, and then the solvent was removed under reduced pressure to obtain a red powder, which is 4,4',4'',4'''-(anthracene-9,10-diylbis(10H-phenthiazin-10,3,7-triyl))tetrabenzaldehyde.

3. The phenothiazine-based two-dimensional covalent organic framework material of claim 1, wherein The structural formula of the β-CD benzidine inclusion complex is: 。 4. The phenthiazine-based two-dimensional covalent organic framework material according to claim 3, characterized in that, The β-CD benzidine inclusion complex was prepared by the following method: Benzidine was added to anhydrous ethanol and completely dissolved to obtain a benzidine ethanol solution. β-Cyclodextrin was added to hot water and stirred until it was completely dissolved to obtain a β-Cyclodextrin solution. The benzidine ethanol solution was added dropwise to the β-Cyclodextrin solution, and the mixture was heated and stirred to carry out the reaction. After the reaction was completed, the ethanol was removed by rotary evaporation. The remaining mixture was placed in an ice bath and allowed to stand. The precipitate was filtered and vacuum dried to obtain a yellowish-white powder, which is the β-CD benzidine inclusion complex.

5. The phenothiazine-based two-dimensional covalent organic framework material of claim 1, wherein The phenothiazine-based two-dimensional covalent organic framework material was prepared by the following method: 4,4',4'',4'''-(anthracene-9,10-dimethylbis(10H-phenothiazine-10,3,7-trimethyl))tetrabenzaldehyde and β-CD benzidine inclusion complex were mixed, ground, and acetic acid solution and ethanol were added dropwise. The ground mixture was then added to n-butanol and o-dichlorobenzene for a first sonication, followed by a second sonication with acetic acid. The mixture was then heated under a protective atmosphere. After the reaction was completed, the mixture was filtered, washed, and vacuum dried to obtain a dark brown powder, which is the phenothiazine-based two-dimensional covalent organic framework material.

6. The phenothiazine-based two-dimensional covalent organic framework material of claim 5, wherein The molar ratio of the 4,4',4'',4'''-(anthracene-9,10-dimethylbis(10H-phenthiazine-10,3,7-trimethyl))tetrabenzaldehyde and β-CD benzidine inclusion complex is 1:2; the concentration of the acetic acid solution is 3M.

7. The phenothiazine-based two-dimensional covalent organic framework material of claim 5, wherein The grinding time is 30 minutes; the first ultrasonic treatment time is 2 minutes; the second ultrasonic treatment time is 2 minutes.

8. The phenothiazine-based two-dimensional covalent organic framework material of claim 5, wherein, The heating temperature is 150°C, and the heating time is 3 days.

9. The use of the phenothiazine-based two-dimensional covalent organic framework material according to any one of claims 1 to 8 in the preparation of antibacterial drugs.

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