Photocatalyst based on nanometer covalent organic framework and preparation method and application thereof

Abstract

The present application relates to the field of photocatalysis, in particular to a photocatalyst based on nano covalent organic framework and a preparation method and application thereof. The preparation method of the photocatalyst based on nano covalent organic framework comprises the following steps: step S1, preparation of nano covalent organic framework: 1,3,5-tris(4-aminophenyl) triazine and 1,3,5-tris(4-formylphenyl) triazine are added into mixed organic solvents to be uniformly dispersed, to obtain a mixed suspension, wherein the mixed organic solvents are obtained by mixing mesitylene and 1,4-dioxane. The prepared photocatalyst can destroy the key metabolite (NAD + / NADH) balance in tumor cells under visible light excitation, induce proinflammatory cell pyroptosis of tumor cells, has high photocatalytic efficiency, and can still effectively play a therapeutic role in hypoxic tumors, solving the technical problems of traditional photodynamic therapy, such as dependence on oxygen, easy production of tolerance by tumor cells, and poor treatment effect.

Description

Technical Field

[0001] This invention relates to the field of photocatalysis technology, and particularly to photocatalysts based on nano-covalent organic frameworks, their preparation methods, and applications. Background Technology

[0002] Cancer poses a serious threat to human health and is one of the major challenges facing clinical medicine today. Although traditional therapies such as surgical resection, chemotherapy, and radiotherapy have made some progress, they still face many bottlenecks: surgery is difficult to completely remove micrometastases, resulting in a high postoperative recurrence rate; chemotherapy drugs often lack selectivity, leading to significant systemic toxic side effects (such as bone marrow suppression and organ damage); and radiotherapy inevitably causes damage to surrounding normal tissues. Therefore, there is an urgent need to develop novel cancer treatment strategies that are highly selective, have low toxicity, and can overcome drug resistance.

[0003] Traditional photodynamic therapy (PDT), as a minimally invasive, repeatable, and spatiotemporally controllable tumor treatment, has received widespread attention in recent years. Its basic principle is that under specific wavelength light excitation, a photosensitizer, in the presence of oxygen, undergoes energy transfer or electron transfer through type I or type II photochemical reactions, generating reactive oxygen species (ROS), which in turn induce tumor cell death. However, the hypoxic state is prevalent in the microenvironment of solid tumors, significantly limiting the efficacy of oxygen-dependent photodynamic therapy (PDT). Simultaneously, tumor cells exhibit strong metabolic plasticity, easily developing tolerance to this therapy, thus reducing treatment effectiveness. Summary of the Invention

[0004] To address the problems raised in the background art, the present invention aims to provide a method for preparing a photocatalyst based on a nano-covalent organic framework. The prepared photocatalyst, under visible light excitation, can disrupt key metabolites (NAD) within tumor cells. + NADH (Non-Anaerobic Hydrogen Deoxygenation) induces pro-inflammatory pyroptosis in tumor cells, activates anti-tumor immune responses, and has high photocatalytic efficiency. It can still play an effective therapeutic role in hypoxic tumors. Furthermore, based on metabolic intervention, it avoids the development of tolerance in tumor cells, ensuring therapeutic efficacy. It solves the technical problems of traditional photodynamic therapy, such as dependence on oxygen, easy development of tolerance in tumor cells, and poor therapeutic effect.

[0005] Another objective of this invention is to propose a photocatalyst based on a nano-covalent organic framework prepared by the above method, which exhibits good water dispersibility, biocompatibility, biosafety, and mitochondrial enrichment capacity. Under visible light excitation, it can disrupt key metabolites (NAD) in tumor cells. + NADH balance induces pro-inflammatory pyroptosis in tumor cells, activates anti-tumor immune responses, and has high photocatalytic efficiency.

[0006] Another objective of this invention is to propose the application of the above-mentioned photocatalyst based on a nano-covalent organic framework in the preparation of tumor therapeutic drugs.

[0007] The above-mentioned technical objective of the present invention is achieved through the following technical solution: A method for preparing a photocatalyst based on a nano-covalent organic framework includes the following steps: Step S1: Preparation of nano-covalent organic frameworks: 1,3,5-tris(4-aminophenyl)triazine and 1,3,5-tris(4-formylphenyl)triazine were added to a mixed organic solvent and dispersed evenly to obtain a mixed suspension. The mixed organic solvent was obtained by mixing mesitylene and 1,4-dioxane. Glacial acetic acid aqueous solution was added to the mixed suspension, and after stirring, centrifugation, precipitation washing, and freeze-drying, nano-covalent organic frameworks were obtained. Step S2: Preparation of dichloro(pentamethylcyclopentadienyl)rhodium(III) dimer-supported nano-covalent organic framework: Dichloro(pentamethylcyclopentadienyl)rhodium(III) dimer and the nano-covalent organic framework were added to a methanol solution. After stirring, centrifugation, precipitation washing and freeze drying, dichloro(pentamethylcyclopentadienyl)rhodium(III) dimer-supported nano-covalent organic framework was obtained. Step S3: Preparation of photocatalyst based on nano-covalent organic framework: The dichloro(pentamethylcyclopentadienyl)rhodium(III) dimer supported on nano-covalent organic framework, DSPE-PEG-2000 and PEG-TPP are added to a tetrahydrofuran solution and mixed evenly to obtain a tetrahydrofuran mixed solution. The tetrahydrofuran mixed solution is injected into deionized water and stirred to allow for nano-precipitation self-assembly. After dialysis, centrifugation, and freeze-drying, a photocatalyst based on nano-covalent organic framework is obtained.

[0008] To further clarify, in step S1, the molar ratio of 1,3,5-tris(4-aminophenyl)triazine to 1,3,5-tris(4-formylphenyl)triazine is 1:1, and the reaction time of the stirring reaction is 12-18 hours.

[0009] To further explain, in step S2, the mass ratio of the nano-covalent organic framework to the dichloro(pentamethylcyclopentadienyl)rhodium(III) dimer is 20:1, and the reaction time of the stirring reaction is 12-18 hours.

[0010] To further clarify, in step S3, the mass ratio of the dichloro(pentamethylcyclopentadienyl)rhodium(III) dimer-supported nano-covalent organic framework, DSPE-PEG-2000, and PEG-TPP is 5:3:2.

[0011] To further explain, in step S3, the volume ratio of the tetrahydrofuran solution to the deionized water is 1:5, and the stirring time for the self-assembly of the nanoprecipitate is 12-18 hours.

[0012] To further clarify, in step S3, the dialysis time is 24-26 hours, and the dialysis medium is deionized water.

[0013] To further explain, the preparation steps of the 1,3,5-tris(4-aminophenyl)triazine include: 4-Aminobenzonitrile was placed in a reaction vessel, and trifluoroacetic acid was added dropwise under 0°C and an inert atmosphere. The mixture was stirred and stirred until homogeneous, and then reacted at room temperature and under an inert atmosphere. After the reaction was completed, distilled water was added, and the pH was neutralized to 7.0 with NaOH solution. The solid was collected by filtration, and after washing and drying, 1,3,5-tris(4-aminophenyl)triazine was obtained. The molar ratio of 4-aminobenzonitrile to trifluoroacetic acid is 1:(3-4).

[0014] To further explain, the preparation steps of the 1,3,5-tris(4-formylphenyl)triazine include: Synthesis of 1,3,5-tris(4-methylphenyl)triazine: p-Toluenenitrile was added dropwise to trifluoroacetic acid at 0 °C under nitrogen protection to obtain a reaction mixture. The reaction mixture was treated with ice water and neutralized with ammonia water, then filtered, washed and dried under vacuum to obtain 1,3,5-tris(4-methylphenyl)triazine. Synthesis of geminal diacetate intermediate: The 1,3,5-tris(4-methylphenyl)triazine was suspended in acetic anhydride, concentrated sulfuric acid was added and stirred, and a CrO3 solution in acetic anhydride was added dropwise. The reaction solution was poured into ice water, filtered, washed and purified by column chromatography to obtain the geminal diacetate intermediate; the molar ratio of the 1,3,5-tris(4-methylphenyl)triazine to CrO3 was 1:(8-9). Synthesis of 1,3,5-tris(4-formylphenyl)triazine: The geminal diacetate intermediate was dispersed in a mixture of ethanol and deionized water, concentrated sulfuric acid was added, the mixture was refluxed and cooled to room temperature, the precipitate was collected by filtration, and the precipitate was washed and dried to obtain 1,3,5-tris(4-formylphenyl)triazine.

[0015] A photocatalyst based on a nano-covalent organic framework is prepared using the aforementioned method for preparing a photocatalyst based on a nano-covalent organic framework.

[0016] The application of the aforementioned photocatalyst based on a nano-covalent organic framework in the preparation of tumor therapeutic drugs.

[0017] Compared with the prior art, the embodiments of the present invention have the following beneficial effects: The aforementioned photocatalyst based on a nano-covalent organic framework constructed a triazine-based covalent organic framework material with high crystallinity and stability. Further loading with dichloro(pentamethylcyclopentadienyl)rhodium(III) dimer improved photocatalytic efficiency. Surface modification with the amphiphilic polymer DSPE-PEG-2000 (distearate phosphatidylethanolamine-polyethylene glycol 2000) and the mitochondrial targeting group triphenylphosphine (TPP) enhanced biocompatibility and mitochondrial targeting, forming a nano-photocatalyst with good water dispersibility, biocompatibility, and mitochondrial enrichment capacity. Under visible light excitation, this photocatalyst can catalyze NAD in situ within tumor cells. + It is reduced to NADH, by disrupting key metabolites (NAD) within tumor cells. + The balance of NADH (NaH2O) induces pyroptosis in tumor cells, activating anti-tumor immune responses. This breaks through the bottleneck of traditional photodynamic therapy that relies on oxygen, and can still play an effective therapeutic role in hypoxic tumors. Furthermore, based on metabolic intervention, it avoids the development of tolerance in tumor cells, ensuring the therapeutic effect. Attached Figure Description

[0018] Figure 1 This is a material characterization diagram of Embodiment 1 of the present invention. Figure 1 (a1) is a transmission electron microscope (TEM) image of COF (scale bar is 200 nm). Figure 1 (a2) is a transmission electron microscope (TEM) image of COF (scale bar is 100 nm). Figure 1 (a3) in the figure is the EDS distribution diagram of C element in COF. Figure 1 (a4) in the diagram is the EDS distribution of the O element in COF. Figure 1 (a5) in the figure is the EDS distribution diagram of the N element in COF. Figure 1 (a6) in the diagram is a superimposed distribution diagram of the C, O, and N elements in COF. Figure 1 (a7) in the diagram is the EDS energy spectrum of COF. Figure 1 (b) shows the UV-Vis absorption spectra of different materials. Figure 1 (c) in the figure shows the DLS particle size distribution of different materials. Figure 1 (d) in the figure represents the Zeta potential characterization diagrams for different materials. Figure 1 (e) in the figure is the PXRD (powder X-ray diffraction) characterization spectrum of COF. Figure 1 In the image (f), the Fourier transform infrared (FT-IR) spectra of different materials are shown. Figure 1 (g) in the figure represents the change in zeta potential of COF / M / TPP at different time points.

[0019] Figure 2 This is a graph from an in vitro photocatalytic NADH regeneration performance evaluation test. Figure 2 (a) represents NAD under different control conditions. + The UV-Vis absorption spectrum of the reduction system Figure 2 (b) shows NAD under different catalytic configurations. + The UV-Vis absorption spectrum of photoreduction to generate NADH. Figure 2 (c) in the figure shows the normalized comparison of the absorbance of the system at 340 nm when different biomolecules replace triethanolamine as electron donors.

[0020] Figure 3 It is a diagram representing intracellular performance. Figure 3 (a) in the figure shows the change in COF / M / TPP uptake over incubation time. Figure 3 (b) in the figure is a comparison diagram of mitochondrial colocalization of COF / M / TPP and COF. Figure 3 (c) in the figure shows the L-cysteine ​​content in different cell lines. Figure 3 (d) in the figure shows the changes in ATP content in different treatment groups under photocatalysis. Figure 3 (e) represents the NADH / NAD ratio under photocatalysis in different treatment groups. + Ratio change graph Figure 3 (f) in the figure is a cytotoxicity characterization diagram of COF / M / TPP. Figure 3 (g) in the figure shows the staining of live and dead cells in different treatment groups. Figure 3 (h) in the figure represents the pyroptosis morphology of 4T1 cells after COF / M / TPP photocatalysis.

[0021] Figure 4 This is a graph evaluating the efficacy of tumor treatment in a mouse tumor-bearing model. Figure 4 (a) shows the EPR effect assessment of mice after tail vein injection of different materials. Figure 4 (b) in the figure shows the change in tumor size in mice 14 days after treatment. Figure 4 (c) in the figure shows the change in tumor weight in mice 14 days after treatment. Figure 4 (d) in the figure shows the changes in tumor volume in mice during the treatment process. Figure 4 (e) in the figure shows the weight changes of the mice during the treatment process.

[0022] Figure 5 The image shows the ELISA results of IL-18, IL-6, and IL-1β levels in the serum of mice in different treatment groups. Figure 5 Figure (a) shows the ELISA results of IL-18 levels in the serum of mice in different treatment groups. Figure 5 Figure (b) shows the ELISA results of IL-6 levels in the serum of mice in different treatment groups. Figure 5 (c) in the figure shows the ELISA results of IL-1β levels in the serum of mice in different treatment groups.

[0023] Figure 6 This is a graph evaluating the in vivo anti-tumor immune activation effect in a mouse tumor-bearing model. Figure 6 (a) in the figure shows a flow cytometry image of dendritic cell maturation in the draining lymph nodes of mice in different treatment groups. Figure 6 (b) in the middle is Figure 6 The quantitative statistical results of dendritic cell maturation rate for each group (a) are shown in the figure.

[0024] Figure 7 This is a graph evaluating the in vivo anti-tumor immune activation effect in a mouse tumor-bearing model. Figure 7 (a) in the figure shows the flow cytometry data of M1 macrophage polarization in tumor tissues of mice in different treatment groups. Figure 7 (b) in the middle is Figure 7 (a) Graph showing the quantitative statistical results of the proportion of M1 macrophages in each group.

[0025] Figure 8 This is a graph evaluating the in vivo anti-tumor immune activation effect in a mouse tumor-bearing model. Figure 8 (a) shows the CD3 concentration in tumor tissues of mice in different treatment groups. + CD8 + Flow cytometry images of T cell infiltration and activation. Figure 8 (b) in the middle is Figure 8 CD3 corresponding to each group in (a) + CD8 + A graph showing the quantitative statistical results of the T cell ratio. Detailed Implementation

[0026] A method for preparing a photocatalyst based on a nano-covalent organic framework includes the following steps: Step S1: Preparation of nano-covalent organic frameworks (COFs): 1,3,5-tris(4-aminophenyl)triazine (TAPT) and 1,3,5-tris(4-formylphenyl)triazine (TFPT) were added to a mixed organic solvent and dispersed evenly to obtain a mixed suspension. The mixed organic solvent was obtained by mixing mesitylene and 1,4-dioxane. Glacial acetic acid aqueous solution was added to the mixed suspension, and after stirring, centrifugation, precipitation washing, and freeze-drying, nano-covalent organic frameworks (COFs) were obtained. Step S2: Preparation of dichloro(pentamethylcyclopentadienyl)rhodium(III) dimer supported on nano-covalent organic frameworks (COF / M): Dichloro(pentamethylcyclopentadienyl)rhodium(III) dimer (referred to as M) and the nano-covalent organic frameworks were added to a methanol solution. After stirring, centrifugation, precipitation washing, and freeze-drying, dichloro(pentamethylcyclopentadienyl)rhodium(III) dimer supported on nano-covalent organic frameworks (COF / M) was obtained. Step S3: Preparation of photocatalyst (COF / M / TPP) based on nano-covalent organic framework: The dichloro(pentamethylcyclopentadienyl)rhodium(III) dimer supported on nano-covalent organic framework (COF / M), DSPE-PEG-2000 (distearate phosphatidylethanolamine-polyethylene glycol 2000) and PEG-TPP (triphenylphosphine-polyethylene glycol derivative) were added to a tetrahydrofuran solution and mixed evenly to obtain a tetrahydrofuran mixed solution. The tetrahydrofuran mixed solution was injected into deionized water and stirred to allow for nano-precipitation self-assembly. After dialysis, centrifugation, and freeze-drying, the photocatalyst (COF / M / TPP) based on nano-covalent organic framework was obtained.

[0027] The Warburg Effect states that even under oxygen-rich conditions, tumor cells preferentially use glycolysis for energy metabolism rather than the more efficient oxidative phosphorylation pathway. This metabolic pattern provides tumor cells with multiple survival advantages: rapid synthesis of adenosine triphosphate (ATP) to meet their high-speed proliferation needs; generation of numerous metabolic intermediates for biosynthetic processes; and maintenance of an acidic microenvironment, which is conducive to tumor cell invasion and metastasis.

[0028] Nicotinamide adenine dinucleotide (NAD) + NADH is the most important redox coenzyme pair in cells, playing a crucial role in core metabolic pathways such as glycolysis, the tricarboxylic acid cycle, and oxidative phosphorylation. Among these, oxidized NAD+... + As a coenzyme of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key enzyme in glycolysis, its depletion directly blocks the ATP supply to the glycolytic pathway; reduced NADH, on the other hand, is an electron donor in the mitochondrial electron transport chain. Damage to NAD+ in tumor cells... + A balanced NADH level not only cuts off its energy supply but also triggers significant reductive stress, leading to mitochondrial dysfunction and inducing cell death.

[0029] Covalent organic frameworks (COFs) are a class of crystalline porous organic polymers formed by covalently linking light elements (such as carbon, hydrogen, oxygen, nitrogen, and boron). COFs possess high specific surface area, regular and tunable pore structures, excellent photoelectric properties, and good biocompatibility, showing broad application prospects in drug delivery, biosensing, and phototherapy. However, most current COF photocatalysts still rely on the traditional reactive oxygen species (ROS) generation mechanism, failing to overcome their dependence on oxygen.

[0030] The core structure of the photocatalyst of this invention is a nano-covalent organic framework (COF). This COF is composed of 1,3,5-tris(4-aminophenyl)triazine (TAPT) and 1,3,5-tris(4-formylphenyl)triazine (TFPT) as monomers, which are synthesized through a condensation reaction to obtain a two-dimensional nanomaterial with a periodic pore structure. The COF uses the structure shown in formula (Ⅰ) as its structural repeating unit. Equation (Ⅰ); In formula (Ⅰ), the “~” connected to the benzene ring indicates an omitted structural repeating unit; The structural formula of the ligand TAPT of the nano-covalent organic framework (COF) is shown in Formula (II): Equation (II); The structural formula of the ligand TFPT of the nano-covalent organic framework (COF) is shown in formula (Ⅲ): Formula (Ⅲ).

[0031] After obtaining the COF, it was further modified by nano-sizing and functionalization. Specifically, COF / M was first prepared, with dichloro(pentamethylcyclopentadienyl)rhodium(III) dimer (M) serving as the electron mediator and catalytically active component. Since the COF framework can absorb light energy and generate photogenerated electron-hole pairs under visible light irradiation, but without effective electron transfer channels, photogenerated electrons easily recombine with holes, thus reducing photocatalytic utilization efficiency. After loading M onto the surface and / or near the pores of the COF framework, M can preferentially capture the photogenerated electrons generated by the COF and further transfer them to NAD. + Substrate, thereby promoting NAD + The reduction reaction towards NADH is carried out, improving electron utilization efficiency and overall photocatalytic efficiency.

[0032] The COF utilizes its excellent photogenerated electron separation capability and specific band structure to construct a photocatalytic reaction core within cells, mimicking the function of the photosynthetic center. Under visible light, the COF / M / TPP photocatalyst can convert NAD+ into NAD+. + Restored to NADH (NAD + + H + + 2e - →NADH), this process consumes NAD, a substrate required for glycolysis. + (Cutting off the energy source) and at the same time causing an overload of the reduction product NADH (inducing reduction stress). This dual metabolic attack can effectively induce pyroptosis (a pro-inflammatory programmed cell death mechanism) in tumor cells, thereby activating the body's anti-tumor immune response and achieving synergistic treatment of "metabolic intervention + immune linkage", extending the photocatalytic NADH regeneration capacity of COF to the field of tumor treatment.

[0033] To further illustrate, COF / M / TPP, driven by visible light, will reduce NAD... + The ability to be reduced to NADH stems from its specific chemical structure design. The COF of this invention is formed by the condensation of TAPT and TFPT, and its framework contains multiple triazine units and aromatic conjugated structures. The triazine units are electronically defective structures, serving as electron acceptor sites; the extended π-conjugated system formed between the aromatic rings facilitates light absorption and the delocalization migration of photogenerated carriers. Therefore, this COF can be effectively excited under visible light irradiation to generate photogenerated electrons with reducing capabilities.

[0034] Furthermore, the COF of this invention has a two-dimensional layered ordered structure and periodic channels, which is beneficial for the substrate NAD. + On the one hand, diffusion towards the active sites of the material facilitates the directional migration of photogenerated electrons within the framework, thereby reducing local recombination losses. Furthermore, M supported on the COF framework acts as an electron mediator and catalytic active center, preferentially capturing photogenerated electrons generated after COF excitation and promoting electron migration to NAD. + Substrate transfer. Therefore, the COF backbone is not merely a carrier, but provides photoresponsiveness, electron separation, and transport functions; M is responsible for further electron enrichment and catalytic transfer; PEG modification endows the photocatalyst with good blood compatibility, while TPP allows the photocatalyst to accumulate near the mitochondria, thereby enhancing the absorption of intracellular NAD+. + Local photocatalytic reduction capability.

[0035] The aforementioned photocatalyst based on a nano-covalent organic framework was constructed using a triazine-based covalent organic framework material with high crystallinity and stability. Further loading with dichloro(pentamethylcyclopentadienyl)rhodium(III) dimer improved photocatalytic efficiency. Surface modification with the amphiphilic polymer DSPE-PEG-2000 and the mitochondrial-targeting group triphenylphosphine (TPP) enhanced biocompatibility and mitochondrial targeting, resulting in a nano-photocatalyst with good water dispersibility, biocompatibility, and mitochondrial enrichment capacity. Under visible light excitation, this nano-covalent organic framework-based photocatalyst can catalyze NAD in situ within tumor cells. + It is reduced to NADH, by disrupting key metabolites (NAD) within tumor cells. + The NAD+ balance induces pro-inflammatory pyroptosis in tumor cells, activating an anti-tumor immune response. Compared to existing photodynamic therapy systems that primarily rely on reactive oxygen species (ROS) generation, the photocatalyst of this invention provides a novel photocatalytic therapy strategy based on metabolic intervention: by interfering with NAD+... + / NADH metabolic balance induces pyroptosis in cancer cells and activates the immune system, achieving a synergistic anti-tumor effect of "pyroptosis-immune linkage"; proposes photocatalytic reduction of NAD + The novel therapeutic mechanism of →NADH overload→metabolic energy depletion→pyroptosis breaks through the bottleneck of traditional photodynamic therapy that relies on oxygen. It can still play an effective therapeutic role in hypoxic tumors and is expected to reduce treatment tolerance caused by hypoxic microenvironment or antioxidant defense.

[0036] It should be noted that in the photocatalyst of the present invention, the COF framework is composed of light organic elements (C, H, O, N) and does not contain heavy metals. PEG modification gives the photocatalyst good blood compatibility and good biosafety.

[0037] To further clarify, in step S1, the molar ratio of 1,3,5-tris(4-aminophenyl)triazine to 1,3,5-tris(4-formylphenyl)triazine is 1:1, and the reaction time of the stirring reaction is 12-18 hours.

[0038] In one embodiment of the present invention, in step S1, the amount of TAPT and TFPT added is 0.064 mmol each, the mixed organic solvent is obtained by mixing 0.5 mL of mesitylene and 0.5 mL of 1,4-dioxane; 200 μL of glacial acetic acid aqueous solution (12 mol / L) is added to the mixed suspension; the reaction time of the stirring reaction in step S1 is 16 h, and the reaction temperature is room temperature.

[0039] In step S1 of the preparation method of the present invention, a mesitylene / 1,4-dioxane / glacial acetic acid system is used for synthesis at room temperature. The preparation method is simple and the conditions are mild.

[0040] Specifically, in step S1, after the stirring reaction is completed, the system is centrifuged and the precipitate is collected. The collected precipitate is washed three times each with anhydrous ethanol, tetrahydrofuran, and acetone. After each wash, the solid is collected by centrifugation after thorough ultrasonic dispersion to completely remove unreacted monomers, oligomers, and solvent impurities, ensuring product purity. Anhydrous ethanol is used to remove polar small molecule impurities; tetrahydrofuran is used to remove unreacted organic monomers and oligomers; and acetone is used to further remove residual solvent.

[0041] To further explain, in step S2, the mass ratio of the nano-covalent organic framework to the dichloro(pentamethylcyclopentadienyl)rhodium(III) dimer is 20:1, and the reaction time of the stirring reaction is 12-18 hours.

[0042] Preferably, in step S2, the reaction time of the stirring reaction is 16 hours.

[0043] To further clarify, in step S3, the mass ratio of the dichloro(pentamethylcyclopentadienyl)rhodium(III) dimer-supported nano-covalent organic framework, DSPE-PEG-2000, and PEG-TPP is 5:3:2.

[0044] To further explain, in step S3, the volume ratio of the tetrahydrofuran solution to the deionized water is 1:5, and the stirring time for the self-assembly of the nanoprecipitate is 12-18 hours.

[0045] Preferably, in step S3, the reaction time of the stirring reaction is 16 hours.

[0046] To further clarify, in step S3, the dialysis time is 24-26 hours, and the dialysis medium is deionized water.

[0047] To further explain, the preparation steps of the 1,3,5-tris(4-aminophenyl)triazine (TAPT) include: 4-Aminobenzonitrile was placed in a reaction vessel, and trifluoroacetic acid was added dropwise under 0°C and an inert atmosphere. The mixture was stirred and stirred until homogeneous, and then reacted at room temperature and under an inert atmosphere. After the reaction was completed, distilled water was added, and the pH was neutralized to 7.0 with NaOH solution. The solid was collected by filtration, and after washing and drying, 1,3,5-tris(4-aminophenyl)triazine (TAPT) was obtained. The molar ratio of 4-aminobenzonitrile to trifluoroacetic acid is 1:(3-4).

[0048] Specifically, the synthetic route for TAPT is as follows: .

[0049] To further explain, the preparation steps of the 1,3,5-tris(4-formylphenyl)triazine (TFPT) include: Synthesis of 1,3,5-tris(4-methylphenyl)triazine (intermediate 1): p-Toluenenitrile was added dropwise to trifluoroacetic acid at 0 °C under nitrogen protection to obtain a reaction mixture. The reaction mixture was treated with ice water and neutralized with ammonia water, then filtered, washed and dried under vacuum to obtain 1,3,5-tris(4-methylphenyl)triazine (intermediate 1). The structural formula of intermediate 1 is shown in formula (Ⅳ): Formula (Ⅳ); Synthesis of geminal diacetate intermediate (intermediate 2): The 1,3,5-tris(4-methylphenyl)triazine was suspended in acetic anhydride, concentrated sulfuric acid was added and stirred, and acetic anhydride solution of CrO3 was added dropwise. The reaction was carried out to obtain a reaction solution, which was poured into ice water and purified by filtration, washing and column chromatography to obtain geminal diacetate intermediate (intermediate 2). The structural formula of intermediate 2 is shown in equation (V): Formula (V); The molar ratio of 1,3,5-tris(4-methylphenyl)triazine (intermediate 1) to CrO3 is 1:(8-9) to ensure that the methyl group is oxidized to geminal diacetate rather than carboxylic acid. Synthesis of 1,3,5-tris(4-formylphenyl)triazine (TFPT): The geminal diacetate intermediate was dispersed in a mixture of ethanol and deionized water, concentrated sulfuric acid was added, the mixture was refluxed and cooled to room temperature, the precipitate was collected by filtration, and the precipitate was washed and dried to obtain 1,3,5-tris(4-formylphenyl)triazine (TFPT).

[0050] Preferably, in the process of synthesizing intermediate 2: the molar ratio of 1,3,5-tris(4-methylphenyl)triazine (intermediate 1) to CrO3 is 1:8.8; the 1,3,5-tris(4-methylphenyl)triazine is suspended in acetic anhydride, concentrated sulfuric acid is added and stirred, and under the activation of concentrated sulfuric acid, an acetic anhydride solution of CrO3 is added dropwise at -20℃ to 0℃ to carry out the oxidation reaction. The dropping rate of the acetic anhydride solution of CrO3 is 1 drop / 3 seconds. Controlling the dropping rate can prevent local overheating and excessive oxidation. In addition, the molar ratio of intermediate 1 to CrO3, the reaction temperature and the dropping rate of CrO3 are strictly controlled to ensure that the methyl group is oxidized to geminal diacetate rather than over-oxidized to carboxylic acid.

[0051] Specifically, the synthesis route of TFPT is as follows: .

[0052] The synthesis methods of TAPT and TFPT of the present invention can improve monomer purity and functional group integrity, which is beneficial to the subsequent Schiff base condensation reaction, enhance the crystallinity, pore structure regularity and photocatalytic performance of COF, and at the same time reduce impurities and by-product residues, reduce the interference of impurities or by-product residues on metal coordination, electron transfer and evaluation of biological applications, and improve the structural controllability and performance reproducibility of the obtained photocatalyst.

[0053] A photocatalyst based on a nano-covalent organic framework is prepared using the aforementioned method for preparing a photocatalyst based on a nano-covalent organic framework.

[0054] The photocatalyst based on a nano-covalent organic framework prepared by the above method exhibits good water dispersibility, biocompatibility, biosafety, and mitochondrial enrichment capacity. Under visible light excitation, it can disrupt key metabolites (NAD) in tumor cells. + NADH balance induces pro-inflammatory pyroptosis in tumor cells, activates anti-tumor immune responses, and has high photocatalytic efficiency.

[0055] The application of the aforementioned photocatalyst based on a nano-covalent organic framework in the preparation of tumor therapeutic drugs.

[0056] The tumor therapeutic drug is obtained through a photocatalytic reduction reaction (at an output power density of 10–200 mW / cm²). 2 Under visible light source excitation, NAD+ in tumor cells is... + The catalytic reaction equation for the conversion of NAD to NADH is: + + H + + 2e - →NADH can promote NAD + Exhaustion and NADH overload exert their effects by inducing pyroptosis. The aforementioned tumor therapeutic agent works by activating mitochondrial reductive stress, inducing pyroptosis, promoting dendritic cell maturation, promoting M1 polarization, and enhancing CD8. + T cell infiltration and the release of pro-inflammatory factors such as IL-1β and IL-18 exert anti-tumor effects. Specifically, the tumor therapeutic drug exerts its anti-tumor effect through the following synergistic mechanism: (1) Metabolic energy depletion: Photocatalysis consumes NAD + This directly inhibits the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key enzyme in glycolysis, cuts off the ATP source of tumor cells, and induces a state of energy depletion in cells. (2) NAD + Disruption of NADH redox balance: Intracellular NADH / NAD ratio in tumor cells after photocatalytic therapy + The ratio increased significantly, exhibiting characteristics of reducing stress and interfering with NAD.+ / NADH-dependent enzymatic reactions and mitochondrial electron transport function synergistically exacerbate metabolic disorders; (3) Induction of pyroptosis: Reductive stress activates the NLRP3 inflammasome, which in turn activates Caspase-3. The activated Caspase-3 cleaves the GSDME protein to produce an active N-terminal fragment (GSDME-N). This fragment forms pores on the cell membrane, ultimately leading to pyroptosis. (4) Anti-tumor immune activation: Pyroptosis cells release pro-inflammatory factors such as IL-1β and IL-18 and tumor-associated antigens, which activate the maturation of dendritic cells (DCs) and induce CD8. + T cell activation leads to a systemic anti-tumor immune response.

[0057] To further clarify, the tumor is a solid tumor, including but not limited to triple-negative breast cancer, liver cancer, colon cancer, lung cancer, pancreatic cancer, or melanoma.

[0058] To facilitate understanding of the present invention, a more comprehensive description is provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and complete understanding of the disclosure of the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with techniques or conditions described in the literature in the art or according to product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0059] p-Toluenenitrile was purchased from Aladdin; dichloro(pentamethylcyclopentadienyl)rhodium(III) dimer was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd. (CAS: 12354-85-7); DSPE-PEG-2000 was purchased from Aladdin (CAS: 147867-65-0); and PEG-TPP was purchased from Shanghai Tuoyang Biotechnology Co., Ltd.

[0060] Example 1 A method for preparing a photocatalyst based on a nano-covalent organic framework includes the following steps: Preparation of 1,3,5-tris(4-aminophenyl)triazine (TAPT) and 1,3,5-tris(4-formylphenyl)triazine (TFPT): The preparation steps of TAPT are as follows: In a 100 mL three-necked round-bottom flask equipped with a mechanical stirrer and thermometer, 0.772 g (6.53 mmol) of 4-aminobenzonitrile was added. The flask was placed in an ice-salt bath and cooled to 0 °C. Under nitrogen protection, 2.0 mL (22.6 mmol) of trifluoroacetic acid was slowly added dropwise using a syringe, with vigorous stirring during the addition. The temperature inside the three-necked flask was strictly controlled to not exceed 5 °C. The molar ratio of 4-aminobenzonitrile to trifluoroacetic acid was 1:3.5. After the addition was complete, the mixture was a viscous, dark red paste. The ice bath was removed, and the reaction system was allowed to rise naturally to room temperature. The reaction was continued for 16 h under room temperature and nitrogen protection with stirring. After the reaction was completed, the three-necked flask was placed in an ice-water bath, and 30 mL of distilled water was slowly added to quench the reaction, producing a large amount of dark orange precipitate. 2 M of distilled water was then slowly added dropwise with stirring. The pH was adjusted with NaOH solution. As the pH increased, the deep orange precipitate gradually dissolved, eventually turning into a light yellow suspension at pH=7.0. The solid was collected by vacuum filtration and washed with distilled water until the filtrate was neutral. The crude product was then washed three times with a mixture of dichloromethane and petroleum ether (volume ratio 1:2) and vacuum dried at 80℃ for 12 hours to obtain a grayish-white powder TAPT with a yield of approximately 85%. The structural formula of TAPT is shown in formula (II). Equation (II); Specifically, the synthetic route for TAPT is as follows: .

[0061] The preparation steps of TFPT are as follows: Synthesis of 1,3,5-tris(4-methylphenyl)triazine (intermediate 1): Under nitrogen protection at 0°C, 10 mL of p-toluenenitrile (83.7 mmol) was added dropwise to a 500 mL round-bottom flask containing 30 mL of trifluoroacetic acid. The solution changed from colorless to dark brown and gradually solidified. The reaction was maintained at 0°C for 2 h, followed by standing at room temperature for 16 h to obtain the reaction mixture. The reaction mixture was poured into 500 mL of ice water and stirred vigorously. The pH was adjusted to 9–10 by adding concentrated ammonia solution with a mass fraction of 25%–28%. The milky white precipitate was collected by filtration, washed with distilled water, and dried under vacuum at 80°C for 12 h to obtain a white solid intermediate 1 with a yield of approximately 95%. The structural formula of intermediate 1 is shown in formula (Ⅳ). Formula (Ⅳ); Synthesis of geminiacetic acid intermediate (intermediate 2): Intermediate 1 (1 g, 2.84 mmol) was suspended in 15 mL of acetic anhydride, cooled to -20 °C, and concentrated sulfuric acid (98%, 2 mL) was slowly added dropwise. After stirring for 20 min, the temperature was kept below 0 °C, and a solution of 25 mL of acetic anhydride containing 2.5 g of CrO3 (the molar ratio of intermediate 1 to CrO3 was approximately 1:8.8) was slowly added dropwise through a constant pressure dropping funnel at a rate of approximately 1 drop / 3 seconds to prevent local overheating and excessive oxidation. After the addition was complete, the reaction was stirred at room temperature for 2 h to obtain the reaction solution. The reaction solution was then slowly poured into 1.5 L of ice water at 600 °C. Excess acetic anhydride was hydrolyzed by stirring at rpm for 1 hour. The dark yellow solid was collected by filtration, washed with water, dried, and then purified by silica gel column chromatography (eluent: dichloromethane / ethyl acetate = 50:1, V / V). The solid was then dried by rotary evaporation to obtain a white powder intermediate 2 with a yield of approximately 20%. The structural formula of intermediate 2 is shown in formula (V). Formula (V); Synthesis of 1,3,5-tris(4-formylphenyl)triazine (TFPT): Intermediate 2 (240 mg, 0.34 mmol) was dispersed in a mixture of ethanol (5 mL) and deionized water (4 mL). Concentrated sulfuric acid (98%, 1 mL) was slowly added dropwise at 0 °C. The mixture was heated to 125 °C and refluxed for 12 h. After reflux, the mixture was cooled to room temperature, and the precipitate was collected by filtration. The precipitate was washed alternately with distilled water and ethanol until neutral (pH=7.0). The precipitate was dried under vacuum at 80 °C for 12 h to obtain a pale yellow powder of TFPT with a yield of approximately 96%. The structural formula of TFPT is shown in formula (Ⅲ). Formula (Ⅲ); The synthetic route for TFPT is as follows: .

[0062] Preparation of photocatalysts based on nano-covalent organic frameworks: Step S1: Preparation of nano-covalent organic frameworks (COFs): TAPT (0.064 mmol) and TFPT (0.064 mmol) were added to a 10 mL pressure-resistant glass tube. The molar ratio of TAPT to TFPT was 1:1. 1 mL of a mixed organic solvent was added to the glass tube. This mixed organic solvent consisted of 0.5 mL of mesitylene and 0.5 mL of 1,4-dioxane in a 1:1 volume ratio. The mixture was sonicated for 20 min to ensure complete dispersion, dissolution, and homogeneous mixing of the two monomers, resulting in a mixed suspension. 200 μL of a 12 mol / L glacial acetic acid aqueous solution was added to the mixed suspension as a Brønsted acid catalyst to activate the aldehyde groups, promote the Schiff base condensation reaction, and crystallize the framework. After adding the glacial acetic acid aqueous solution, the mixture was continuously stirred at room temperature for 16 h to allow the monomers to fully condense under acid catalysis. A yellow solid precipitate gradually appeared in the system. After the stirring reaction was completed, the mixture was centrifuged (8000 rpm). (rpm, 10 minutes), discard the supernatant, collect the yellow solid precipitate at the bottom, and wash the collected solid precipitate three times each with anhydrous ethanol, tetrahydrofuran, and acetone, respectively. After each wash, the solid is thoroughly dispersed by ultrasonication and collected by centrifugation to completely remove unreacted monomers, oligomers, and solvent impurities. Finally, the washed solid is placed in a freeze dryer and freeze-dried at a cold trap temperature of -50℃ for 15 hours to obtain a highly crystalline yellow powdered COF. The COF has the structure shown in formula (I) as the repeating unit: Equation (Ⅰ); In formula (Ⅰ), the “~” connected to the benzene ring indicates an omitted structural repeating unit.

[0063] Step S2: Preparation of dichloro(pentamethylcyclopentadienyl)rhodium(III) dimer-supported nano-covalent organic framework (COF / M): 0.3 mg of dichloro(pentamethylcyclopentadienyl)rhodium(III) dimer (M) and 6 mg of COF powder were added to 1.5 mL of methanol solution. The mixture was continuously magnetically stirred at room temperature for 16 h to ensure that M was fully loaded onto the pore structure or surface active sites of COF. After the reaction, the mixture was transferred to a centrifuge tube and washed three times each with deionized water and methanol. After each thorough ultrasonic dispersion, the mixture was centrifuged (8000 rpm, 10 min) to collect the solid and remove unloaded free M and solvent residue. The washed solid was freeze-dried at -45 °C for 24 h to obtain COF / M powder, which was then stored at -20 °C for later use.

[0064] Step S3: Preparation of photocatalyst (COF / M / TPP) based on nano-covalent organic framework: Step S31, Preparation of mixed solution: Add 5 mg of COF / M, 3 mg of DSPE-PEG-2000 and 2 mg of PEG-TPP to 2 mL of tetrahydrofuran solution. The mass ratio of COF / M, DSPE-PEG-2000 and PEG-TPP is 5:3:2. Dissolve by sonication and mix thoroughly to ensure that all components are completely dissolved, and obtain a homogeneous and transparent tetrahydrofuran mixed solution. Step S32, Nanoprecipitate self-assembly: Under magnetic stirring, the above tetrahydrofuran mixed solution was rapidly injected into 10 mL of deionized water. The volume ratio of the tetrahydrofuran mixed solution to deionized water was 1:5. At the moment of injection, the hydrophobic DSPE part rapidly self-assembled due to polarity abrupt change. The hydrophobic stearyl chain of DSPE-PEG-2000 wrapped around the COF / M surface to form a lipid layer, and the hydrophilic PEG chain extended outward to form a hydrophilic protective shell. PEG-TPP was simultaneously assembled on the surface of the nanoparticles, presenting the TPP cationic targeting group on the outer layer of the nanoparticles, thereby forming COF / M / TPP functionalized nanoparticles with a core-shell structure. The above mixed solution was continuously stirred at room temperature for 16 h to allow the tetrahydrofuran to fully evaporate naturally, and the assembled structure became more stable. Step S33, Dialysis purification: After stirring, the above solution is transferred to a dialysis bag (molecular weight cutoff MW: 3500Da). Deionized water is used as the dialysis medium. Dialyze thoroughly at room temperature for 24 hours, changing the dialysis medium every 8 hours for a total of three times to completely remove residual tetrahydrofuran organic solvent and free unassembled DSPE-PEG-2000 and PEG-TPP small molecules. Step S34, Centrifugation and Freeze-drying: After dialysis, transfer the solution in the dialysis bag to a centrifuge tube, centrifuge at 10,000 rpm for 10 minutes, discard the precipitate (to remove a small amount of aggregates and insoluble impurities), collect the supernatant, freeze-dry the supernatant at -45℃ for 24 hours to obtain a light yellow loose powder COF / M / TPP, and seal and store it at -20℃ for later use.

[0065] Characterization and analysis of Example 1: 1. Characterization of COF.

[0066] (1) FT-IR (infrared spectroscopy) characterization: such as Figure 1 As shown in (f), the infrared spectrum is at 1620 cm⁻¹ -1 A characteristic absorption peak for an imine bond (C=N) appears nearby; simultaneously, at 3300–3450 cm⁻¹... -1 No N-H stretching vibration absorption of free amino groups (-NH2) was observed within the range of 1700 cm⁻¹. -1No C=O stretching vibration absorption of the aldehyde group (-CHO) was observed nearby, indicating that a full Schiff base condensation reaction occurred between the amino group of the TAPT monomer and the aldehyde group of the TFPT monomer, successfully constructing an ordered covalent skeleton linked by C=N bonds.

[0067] (2) PXRD (Powder X-ray Diffraction) characterization: such as Figure 1 As shown in (e), the powder X-ray diffraction pattern shows multiple sharp characteristic diffraction peaks in the low-angle region, indicating that the obtained COF material has good long-range ordered crystallinity and forms a periodic two-dimensional hexagonal network channel structure.

[0068] (3) TEM (Transmission Electron Microscopy) and EDS (Energy Dispersive X-ray Spectroscopy) characterization: Figure 1 As shown in (a1) and (a2), transmission electron microscopy reveals that the COF material exhibits a regular nanoscale morphology. Figure 1 The EDS surface scan results of (a3), (a4), (a5), (a6) and (a7) show that C, N, O and other elements are uniformly distributed in the COF framework, and no heavy metal elements were detected, proving that the material composition is pure.

[0069] (4) UV-vis spectrum (ultraviolet-visible absorption spectrum): such as Figure 1 As shown in (b), the COF material exhibits a broad and strong absorption band in the 400–600 nm visible light region, demonstrating its visible light response capability. The COF / M composite material simultaneously exhibits the characteristic absorption of both COF and M, and its absorption intensity in the visible light region is enhanced compared to COF alone. This indicates that M has been successfully introduced into the COF system, expanding the light absorption range of the composite material and improving its visible light utilization efficiency.

[0070] 2. Characterization of COF / M: such as Figure 1 As shown in (b), compared to COF, the UV-Vis absorption spectrum of COF / M shows the characteristic absorption peak of M at the corresponding wavelength, indicating that M has been successfully loaded into the COF framework. Figure 1 As shown in (f), the FT-IR spectrum of COF / M is basically consistent with that of COF, and the framework structure remains intact, proving that the loading of M did not destroy the chemical structure of the COF framework.

[0071] 3. Physicochemical characterization of COF / M / TPP.

[0072] (1) DLS (Dynamic Light Scattering) particle size analysis: such as Figure 1(c) DLS analysis showed that the hydrated particle size of COF / M / TPP was about 100~200 nm and the particle size distribution was uniform, indicating that the nanomaterials were well dispersed after PEG / TPP modification and met the size requirements for endocytosis (usually 100~300 nm).

[0073] (2) Zeta potential analysis: such as Figure 1 As shown in (d), the Zeta potential of COF / M is approximately negative. After modification with DSPE-PEG-2000 and PEG-TPP, the Zeta potential of COF / M / TPP further shifts towards the negative direction, indicating that PEG and TPP have been successfully coated on the COF surface. A good negative potential is beneficial for the material to maintain stable dispersion in physiological environments.

[0074] (3) Dispersion stability: such as Figure 1 As shown in (g), the Zeta potential of COF / M / TPP changes little at different time points, indicating that the material has good temporal and dispersion stability under aqueous conditions. This result demonstrates that PEG modification helps form a stable hydration layer on the particle surface, reducing particle aggregation and thus improving the material's stability in biological media.

[0075] (4) Infrared spectroscopy analysis: such as Figure 1 As shown in (f), FT-IR spectroscopy can be used to characterize the chemical bond composition and surface modification of materials. As mentioned earlier, framework-related characteristic absorption peaks can be observed in COF samples, around 1620 cm⁻¹. -1 The characteristic absorption peak of the imine bond C=N appears at approximately 1200–1350 cm⁻¹. -1 The presence of C-N or triazine / aromatic skeleton-related vibrational absorption peaks within the specified range indicates that the target COF structure has been successfully constructed. These characteristic peaks are also retained in COF / M and COF / M / TPP, suggesting that the COF skeleton structure was not destroyed after loading M and further PEG / TPP modification. Compared to COF and COF / M, the COF / M / TPP sample exhibits a higher absorption peak at approximately 2920 cm⁻¹. -1 and 2850 cm -1 Nearby aliphatic —CH2— stretching vibrations are absorbed more strongly, and this absorption is enhanced at approximately 1100 cm⁻¹. -1 The absorption of C—O—C stretching vibrations is observed or enhanced in the vicinity, a change consistent with the introduction of PEG segments; simultaneously, at approximately 1430–1450 cm⁻¹... -1 The nearby absorption changes can be attributed to the benzene ring or P-C related vibrations in the TPP.

[0076] The above results further demonstrate that the COF / M / TPP obtained in this invention is a composite nanosystem constructed while maintaining the integrity of the COF main framework.

[0077] 4. Evaluation of In Vitro Photocatalytic NADH Regeneration Performance. To verify the photocatalytic NADH regeneration capability of COF / M / TPP materials at the solution level, an in vitro photocatalytic reaction system was constructed. The specific operation is as follows: (1) COF / M / TPP in vitro photocatalytic NAD + Reduction performance test: In the presence of NAD + COF / M / TPP material was added to the reaction system, and different control groups were set up. The total volume of the reaction system in each group was 2.0 mL. NAD... + The concentration was 0.7 mg / mL; the COF / M / TPP material concentration was 1.0 mg / mL; triethanolamine was used as a sacrificial electron donor at a concentration of 15% (w / v). Unless otherwise specified, all groups were prepared under the same reaction system conditions.

[0078] Experimental grouping was set to NAD + Individual group (NAD) + ), NAD + + Illumination group (NAD) + +light), NAD + +COF / M / TPP Dark Processing Group (NAD) + +COF / M / TPP) and NAD + +COF / M / TPP+ Illumination Group (NAD) + +COF / M / TPP+light). The light-illuminated group reacted under white light irradiation for a certain time (100 mW / cm², 20 min). The changes in the absorption spectrum of each group of samples were detected using a UV-Vis spectrophotometer, with a focus on analyzing the intensity of the absorption peak at 340 nm.

[0079] The results are as follows Figure 2 As shown in (a) (verifying the catalytic effect of COF / M / TPP on NAD under illumination), + The ability to restore and generate NADH), and NAD + Individual group, NAD + + Illumination group and NAD + Compared to the COF / M / TPP dark treatment group, NAD +The +COF / M / TPP+ illumination group showed a significantly enhanced characteristic absorption peak at 340 nm. Since NADH has a characteristic absorption at 340 nm (NADH has a characteristic absorption peak near 340 nm, therefore, enhanced absorbance at 340 nm can be used as a characterization of NADH formation), while NAD... + The weak absorption at this wavelength indicates that COF / M / TPP can effectively catalyze NAD under illumination. + The reduction produces NADH. These results indicate that the combined effects of light and materials are necessary conditions for triggering this photocatalytic reduction reaction.

[0080] (2) Photocatalytic NAD of different materials + Comparison of restoration capabilities: To further evaluate the effect of material composite configuration on NAD + To investigate the effect of photocatalytic reduction performance, in vitro photocatalytic reaction systems with different catalytic configurations were constructed, and their NADH generation capacity under white light irradiation was compared. The total volume of each reaction system was 2.0 mL, containing NAD... + The concentration of the catalyst was 0.7 mg / mL, or 1 mmol / L; triethanolamine was used as a sacrificial electron donor at a concentration of 15% (w / v); and the material concentration was 1.0 mg / mL. For the physical mixture of COF and catalyst M, the concentration of M was 0.25 mmol / L, or 0.155 mg / mL. All groups reacted under white light irradiation at an intensity of 100 mW / cm² for 20 min. After the reaction, the changes in the absorption spectra of each sample were detected using a UV-Vis spectrophotometer, with a focus on analyzing the changes in the NADH characteristic absorption peak near 340 nm.

[0081] The experimental groups included: ① NAD + +COF + light group: NAD added to the system + COF and triethanolamine were used to evaluate the effect of COF alone on NAD under light irradiation. + The ability to restore; ② NAD + + COF + M + light group: NAD added to the system + COF, catalyst M, and triethanolamine were used to evaluate the effect of the physical mixture of COF and catalyst M on NAD50. + The effect of photocatalytic reduction ability; ③ NAD ++ COF / M / TPP + light group: NAD added to the system + COF / M / TPP composite material and triethanolamine were used to evaluate the NAD content of the composite nanocatalyst obtained by loading catalyst M onto COF and further modifying it with TPP under white light. + Photocatalytic reduction capability.

[0082] Wherein, COF + M represents a physical mixture system of COF and catalyst M; COF / M / TPP represents a composite material obtained by further modifying catalyst M with TPP after loading it onto COF.

[0083] The results are as follows Figure 2 As shown in (b) (for comparing the NADH regeneration effects of a standalone COF system, a physical mixture of COF and catalyst M, and a COF / M / TPP composite system under white light irradiation), the standalone COF system significantly improves NADH regeneration. + The photocatalytic reduction ability of NADH is relatively limited; after introducing catalyst M, the characteristic absorption of NADH near 340 nm is enhanced, indicating that M can promote the photocatalytic reduction of NAD. + Reduction reaction. The COF / M / TPP group still exhibited significant NADH characteristic absorption under light conditions, indicating that the introduction of M enhanced the photocatalytic reduction ability of the material. After further modification with TPP, the material still maintained strong photocatalytic activity, providing a basis for subsequent intracellular mitochondrial-targeted photocatalysis.

[0084] (3) Screening of intracellular endogenous electron donors: To investigate endogenous reducing agents that can replace TEOA (triethanolamine) within cells, L-alanine, L-cysteine, L-glutamic acid, L-lysine, and L-aspartic acid were used in an in vitro photocatalytic system. The following amino acids, in sequence, replace TEOA as electron donors under the same conditions (white light, 100 mW / cm²): DL-histidine, DL-serine, glycine, L-proline, L-isoleucine, L-threonine, L-asparagine, L-valine, L-leucine, L-serine, L-glutamine, L-arginine, L-histidine, L-tyrosine, dihydropyridine, N-acetyl-L-cysteine, glycerol, and L-tryptophan. 2 The absorbance change at 340 nm was detected (30 min).

[0085] like Figure 2 Results (c) show (used to evaluate the effects of different electron donors on NAD) + (Effects on photoreduction and NADH generation efficiency): L-cysteine ​​can effectively replace TEOA to support COF / M / TPP photocatalytic NAD generation. + Reduction, while the effects of other endogenous substances are relatively weak. For example... Figure 3 As shown in (c), further detection of L-cysteine ​​content in different cell lines (4T1 breast cancer cells, HeLa cells and A549 cells) revealed that each tumor cell contained a high concentration of L-cysteine, indicating that the abundant L-cysteine ​​in tumor cells can serve as an endogenous electron donor to support intracellular photocatalytic reactions.

[0086] 5. Verification of cellular uptake and mitochondrial targeting.

[0087] The mouse triple-negative breast cancer cell line (4T1) was selected as the model cell line to verify the cellular uptake efficiency and mitochondrial targeting of COF / M / TPP.

[0088] (1) Cell culture.

[0089] 4T1 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C in a 5% CO2 incubator. Cells were passaged every other day, and cells in the logarithmic growth phase were selected for subsequent experiments.

[0090] (2) Cell uptake experiment.

[0091] 4T1 cells were fed at a rate of 1×10 5 Seeds were inoculated at a density of cells / well in confocal microscopy-specific glass-bottomed culture dishes (35 mm in diameter). After incubation at 37°C for 12 h, the culture medium was replaced with fresh medium containing FITC-labeled COF / M / TPP (COF / M / TPP-FITC, 50 μg / mL), and co-incubated for 2 h, 4 h, 6 h, and 8 h, respectively. After incubation, the medium was aspirated, and the cells were gently washed three times with pre-cooled PBS. The changes in FITC fluorescence signal intensity (excitation wavelength Ex = 488 nm) with incubation time were observed using a laser confocal microscope (CLSM).

[0092] like Figure 3 As shown in (a), the intracellular FITC fluorescence signal gradually increased with the extension of incubation time, and the fluorescence signal reached a strong level at 4h, indicating that COF / M / TPP can be effectively taken up by 4T1 cells in a time-dependent manner.

[0093] (3) Mitochondrial colocalization analysis.

[0094] After co-incubating 4T1 cells with FITC-labeled COF / M / TPP (COF / M / TPP-FITC, 50 μg / mL) for 4 h, mitochondrial red fluorescent probe MitoTracker Red (final concentration 200 nM) was added and incubated for 30 min to label mitochondria (red fluorescence). After washing three times with PBS, fluorescence images of the green channel (COF / M / TPP-FITC, Ex=488 nm) and the red channel (mitochondria, Ex=561 nm) were simultaneously acquired under a laser confocal microscope. The Pearson correlation coefficient (i.e., co-localization coefficient) of the two fluorescence images was calculated to quantitatively assess the degree of spatial co-localization between COF / M / TPP-FITC and mitochondria. In addition, a control group was set up. 4T1 cells were co-incubated with FITC-labeled COF / M / PEG (COF / M / PEG-FITC, 50 μg / mL) for 4 h and then subjected to the same treatment as above. The mitochondrial targeting effect of the TPP-modified material was compared. Among them, COF / M / PEG is a PEGylated COF / M control material without TPP targeting groups. Its preparation method differs from that of Example 1 in that PEG-TPP is not added in step S31, while the other preparation steps are the same.

[0095] like Figure 3 As shown in (b), the fluorescence images of COF / M / TPP-FITC (green) and MitoTracker Red (red) highly overlap, where Merged represents the fused image after superimposing the green channel (COF / M / TPP-FITC) and the red channel (MitoTracker Red). The Pearson colocalization coefficient is >0.85 (PCC represents the Pearson colocalization coefficient), confirming that TPP modification successfully guides the material to target mitochondria. In contrast, the Pearson colocalization coefficient of the unmodified TPP COF / M / PEG-FITC material with MitoTracker is significantly lower than that of COF / M / TPP-FITC, further validating the active mitochondrial targeting effect of TPP.

[0096] 6. Validation of in vitro cytotoxicity and intracellular metabolic intervention.

[0097] (1) Cytotoxicity (CCK-8 assay).

[0098] 4T1 cells were loaded at 5 × 10 3The COF / M / TPP particles were seeded at a density of [number] cells / well in 96-well plates and incubated at 37°C for 12 h to allow adhesion. Subsequently, different concentrations of COF / M / TPP dispersions were added to achieve final concentrations of 0, 2.5, 5, 10, 20, 40, and 80 μg / mL. The 0 μg / mL group served as the control group. The experiment was set up with a dark treatment group (COF / M / TPP-) and a white light irradiation group (COF / M / TPP-); the light irradiation group received white light at an intensity of 100 mW / cm². 2 The irradiation time was 30 min. After treatment, each group was cultured at 37℃ for 24 h, and CCK-8 reagent (10 μL / well) was added. After incubation for 1 h, the absorbance was measured at 450 nm using a microplate reader, and the cell viability (%) of each group was calculated.

[0099] like Figure 3 As shown in (f), even under dark conditions with a COF / M / TPP concentration as high as 80 μg / mL, the survival rate of 4T1 cells remained above 90%, indicating that the material itself has good biocompatibility and extremely low dark toxicity. After white light irradiation (100 mW / cm², 30 min), the cell survival rate in the phototherapy group decreased significantly, indicating that light irradiation is a necessary condition for triggering cytotoxicity, demonstrating the high selectivity of COF / M / TPP "light-controlled" therapy.

[0100] (2) Staining of live / dead cells.

[0101] 4T1 cells (1×10) 5 Cells were seeded and cultured at 100 cells / well, and then subjected to different treatments after cell adhesion. The experimental groups are as follows: ① Blank control group (Control group): treated with PBS, no materials added, no light exposure; ② COF / M / TPP(-) group: Add COF / M / TPP to a final concentration of 50 μg / mL, incubate for 6 h in the dark, without light; ③ COF / M(+) group: Add COF / M to a final concentration of 50 μg / mL, incubate for 6 h, and then irradiate with white light at an intensity of 100 mW / cm². 2 The irradiation time is 30 minutes; ④ COF / M / TPP(+) group: Add COF / M / TPP to a final concentration of 50 μg / mL, incubate for 6 h, and then irradiate with white light at an intensity of 100 mW / cm². 2 The irradiation time is 30 minutes.

[0102] After treatment, each group was cultured for another 4 hours and then stained with Calcein-AM / PI double staining kit (live cells showed green fluorescence, dead cells showed red fluorescence). Fluorescence images were observed and captured using a laser confocal microscope.

[0103] like Figure 3 As shown in (g), the cells in the Control group and the COF / M / TPP(-) group mainly exhibited green fluorescence, indicating that the material alone had a weak cell-killing effect in the dark. The COF / M(+) group showed a certain amount of red fluorescence after white light irradiation, indicating that COF / M has a certain photo-induced cell-killing ability. In contrast, the treatment group COF / M / TPP(+) (i.e., COF / M / TPP + white light irradiation) showed a large number of red fluorescence (dead cells) and very few green fluorescence (live cells), which directly demonstrated the highly efficient killing ability of photocatalytic therapy.

[0104] (3) Intracellular NAD + / NADH ratio detection.

[0105] 4T1 cells (1×10) 6 Cells were seeded and cultured at 100 cells / well, and then subjected to different treatments after cell adhesion. The experimental groups are as follows: ① Blank control group (Control group): treated with PBS, no materials added, no light exposure; ② COF / M / TPP(-) group: Add COF / M / TPP to a final concentration of 50 μg / mL, incubate for 6 h in the dark, without light; ③ COF / M / TPP(+) group: Add COF / M / TPP to a final concentration of 50 μg / mL, incubate for 6 h, and then irradiate with white light at an intensity of 100 mW / cm². 2 The irradiation time is 30 minutes.

[0106] Each group was cultured for another 4 hours after treatment. Then, it was cultured according to commercial NAD... + NADH Quantitative Detection Kit (Beyotime) Instructions: Lyse cells and detect NAD in the cell lysates. + And the concentration of NADH, calculate NADH / NAD + ratio.

[0107] like Figure 3 As shown in (e), compared with the control group, the treatment group (COF / M / TPP + light irradiation) had higher levels of intracellular NAD3. + The concentration of NADH decreased significantly, while the concentration of NADH increased significantly, and the NADH / NAD ratio increased. + The significant reversal of the ratio strongly demonstrates that COF / M / TPP successfully facilitated NAD intracellular NAD.+ →Photocatalytic reduction of NADH destroys intracellular NAD+. + / NADH metabolic balance.

[0108] (4) Detection of intracellular ATP content.

[0109] The ATP levels in cell lysates from the Control group, COF / M / TPP(-) group, and COF / M / TPP(+) group were quantitatively analyzed using an ATP luciferase assay kit. Figure 3 As shown in (d), compared with the Control group and the COF / M / TPP(-) group, the intracellular ATP content in the treatment group (COF / M / TPP(+)) was significantly reduced (by approximately 60%), demonstrating that NAD+ + Depletion directly inhibits the glycolysis pathway, leading to a significant reduction in ATP supply to tumor cells and inducing a state of cellular energy depletion.

[0110] 7. Validation of the pyroptosis-related inflammatory mechanism.

[0111] (1) Morphological observation of pyrolysis.

[0112] After incubating COF / M / TPP (50 μg / mL) with 4T1 cells for 6 h, the cells were irradiated with white light (100 mW / cm²). 2 Cells were cultured for 30 min and then incubated for another 4 hours. Subsequently, Annexin V-FITC and Hoechst 33342 were used to stain the cells. Annexin V-FITC was used to mark the eversion of phosphatidylserine residues in the cell membrane and any damage to membrane integrity, exhibiting green fluorescence; Hoechst 33342 was used to stain the cell nuclei, exhibiting blue fluorescence, to facilitate observation of nuclear morphology and cell localization. Cell morphological changes were observed under a laser confocal microscope after staining.

[0113] like Figure 3 As shown in (h), Annexin V FITC exhibits green fluorescence, while Hoechst 33342 exhibits blue fluorescence. The treated cells showed significant cell swelling, cell membrane blistering, and membrane rupture, which are typical morphological characteristics that distinguish pyroptosis from apoptosis.

[0114] 8. Evaluation of in vivo anti-tumor efficacy.

[0115] (1) Establishment of a mouse tumor-bearing model.

[0116] Female BALB / c mice aged 6-8 weeks were selected, and 4T1 cell suspension (1×10⁻⁶) was subcutaneously injected into the lower right side.6 (1 cell / 100 μL PBS), until the tumor volume grows to approximately 100 mm. 3 Treatment should begin at that time.

[0117] (2) Assessment of EPR effect in vivo.

[0118] Tumor-bearing mice were randomly divided into three groups (n=5): ①COF / M group: Mice were injected with COF / M via the tail vein; ②COF / M / PEG group: Mice were injected via tail vein with COF / M / PEG (COF / M / PEG is a PEGylated COF / M control material without TPP targeting groups; its preparation method differs from Example 1 in that PEG-TPP is not added in step S31, while the other preparation steps are the same; COF / M / PEG and COF / M / TPP have similar PEG surface modification and water dispersibility, but lack mitochondrial targeting ability. The purpose of this group is to exclude the influence of PEG modification and the photocatalytic effect of the material itself, thereby verifying that TPP-mediated mitochondrial targeting enrichment enhances NAD). + (Key role of NADH in metabolic intervention, induction of pyroptosis, and promotion of inflammatory cytokine release). ③COF / M / TPP group: Mice were injected with COF / M / TPP via the tail vein.

[0119] The dosage of each material was 5 mg / kg. Fluorescence imaging was performed using a small animal in vivo imaging system (IVIS) at 1 h, 2 h, 4 h, 8 h, 12 h and 24 h after injection to assess the enrichment of each material at the tumor site (EPR effect).

[0120] like Figure 4 As shown in (a), the fluorescence signal of the COF / M / TPP group at the tumor site increased over time and reached a peak at 8 hours, demonstrating that COF / M / TPP modified with PEG and TPP can be passively targeted and enriched at the tumor site through enhanced penetration and retention effect (EPR effect).

[0121] (3) In vivo anti-tumor therapy.

[0122] Tumor-bearing mice were randomly divided into the following 5 groups (n=5): Group I: PBS group: PBS was injected via tail vein, and no material treatment was given; this group served as a blank control. II: COF / M / PEG(-) group: COF / M / PEG was injected via tail vein at a dose of 5 mg / kg, and no white light irradiation was performed; III: COF / M / TPP(-) group: COF / M / TPP was injected via tail vein at a dose of 5 mg / kg, and no white light irradiation was performed; IV: COF / M / PEG(+) group: COF / M / PEG was injected via tail vein at a dose of 5 mg / kg, followed by white light irradiation; V: COF / M / TPP(+) group: COF / M / TPP was injected via the tail vein at a dose of 5 mg / kg, followed by white light irradiation.

[0123] In this study, "-" indicates no white light irradiation, and "+" indicates white light irradiation. The PBS group reflects the natural growth of tumors; the COF / M / PEG(-) group evaluates the dark toxicity and antitumor activity of the TPP-free targeted material under light-free conditions; the COF / M / TPP(-) group evaluates whether the TPP-targeted material has a significant antitumor activity under light-free conditions, thereby verifying the material's safety in the dark; the COF / M / PEG(+) group evaluates the phototherapy effect of the TPP-free targeted material under light-irradiation conditions; and the COF / M / TPP(+) group evaluates the complete therapeutic effect of materials with mitochondrial targeting capabilities under light-irradiation conditions. By comparing the COF / M / TPP(+) group with the COF / M / TPP(-) group, the necessity of light triggering for therapeutic effect can be verified; by comparing the COF / M / TPP(+) group with the COF / M / PEG(+) group, the contribution of TPP-mediated mitochondrial targeting to enhance anti-tumor efficacy can be verified; by comparing the COF / M / PEG(-) group, the COF / M / TPP(-) group with the PBS group, the biosafety and low dark toxicity of the material under light-free conditions can be evaluated.

[0124] For the light irradiation group, the tumor site was irradiated with white light at an intensity of 100 mW / cm² 8 hours after the material was injected via the tail vein. 2 The irradiation time is 30 minutes. During the treatment period, the long diameter (L) and short diameter (W) of the tumor are measured every 2 days using calipers, and the values ​​are calculated using the formula V = L × W. 2 / 2 The tumor volume was calculated, and the mouse weight was recorded simultaneously. After 14 days of treatment, mice in each group were sacrificed, and the tumors were completely removed, weighed, and photographed.

[0125] like Figure 4As shown in (b), (c), (d), and (e), after 14 days of treatment, tumors in the PBS group, the untreated COF / M / PEG(-) group, and the COF / M / TPP(-) group continued to grow, indicating that the material has limited inhibitory effect on tumor growth under light-free conditions and exhibits low dark toxicity. The COF / M / PEG(+) group showed a certain tumor-inhibiting effect after white light irradiation, indicating that COF / M / PEG has a certain photoresponsive anti-tumor ability. In contrast, the COF / M / TPP(+) light-treated group showed extremely significant inhibition of tumor growth, with a tumor inhibition rate exceeding 90%. Some mice exhibited crusting and necrosis of tumors, indicating that TPP-mediated mitochondrial targeting can significantly enhance the anti-tumor therapeutic effect of the material under light conditions. The body weight curves of mice in each group were stable, with no significant weight loss, demonstrating that there were no significant systemic toxic side effects during the treatment.

[0126] (4) Biosafety assessment.

[0127] After treatment, a comprehensive biosafety assessment was conducted on mice in each group: Orbital blood was collected from mice in each group for routine blood tests (white blood cells, red blood cells, platelets, etc.) and serum biochemical tests (alanine aminotransferase ALT, aspartate aminotransferase AST, creatinine Cr, blood urea nitrogen BUN, etc.). All indicators were within the normal reference range, demonstrating that the COF / M / TPP nanophotocatalyst has good in vivo biosafety at effective therapeutic doses, laying the foundation for its clinical translation.

[0128] 9. Evaluation of in vivo anti-tumor immune activation effect.

[0129] Pyroptosis is a typical form of immunogenic cell death (ICD). During pyroptosis, pyroptotic cells release a large number of inflammatory factors and tumor-associated antigens, thereby promoting antigen presentation and anti-tumor immune responses. To evaluate whether COF / M / TPP-induced tumor cell pyroptosis under white light irradiation can further activate in vivo anti-tumor immune responses, this invention studies the release of serum pro-inflammatory factors, dendritic cell maturation, tumor-associated macrophage polarization, and CD8+. + The study examined T-cell infiltration / activation and other aspects. In the in vivo immune activation evaluation experiment, corresponding experimental groups were set up according to different detection indicators. Among them, the detection of serum pro-inflammatory factors... Figure 5 The experimental groups were PBS(+), COF / M / PEG(+), and COF / M / TPP(+). Dendritic cell maturation assays, macrophage M1 polarization assays, and CD3+ assays were performed. + CD8 + T cell infiltration / activation assays correspond to Figure 6 , Figure 7 and Figure 8All three tests were performed using tumor-bearing mice from the same experimental batch, with groups defined as PBS(+), COF / M(+), COF / M / PEG(+), and COF / M / TPP(+). "+" indicates white light irradiation treatment. Figures 6 to 8 All mice were treated according to the same drug administration-lighting experimental procedure. The difference between the groups was the different formulations injected into the tail vein, while the light conditions, sampling time, and sample processing procedures remained the same.

[0130] After treatment, peripheral blood was collected from the mice for serum inflammatory factor detection; tumor draining lymph nodes were collected for dendritic cell maturation detection; and tumor tissue was collected for macrophage polarization status and CD8+ detection. + Analysis of T cell infiltration / activation.

[0131] (1) Detection of pro-inflammatory factor levels in serum.

[0132] After in vivo antitumor treatment, orbital blood was collected from mice in each group, and serum was separated by centrifugation. The concentrations of interleukin-18 (IL-18), interleukin-6 (IL-6), and interleukin-1β (IL-1β) in the serum were quantitatively detected using a commercially available enzyme-linked immunosorbent assay (ELISA) kit.

[0133] In this experiment, the treatment methods for each group shown in Figure 5 are as follows: ① PBS(+) group: Mice were injected with PBS via the tail vein and the tumor sites were irradiated with white light as a control group for simple light irradiation; ② COF / M / PEG(+) group: Mice were injected with COF / M / PEG via the tail vein. After administration, the tumor site was irradiated with white light to evaluate the immune activation effect of PEGylated materials without TPP mitochondrial targeting groups under light conditions. ③ COF / M / TPP(+) group: Mice were injected with COF / M / TPP via the tail vein. After administration, the tumor site was irradiated with white light to evaluate the ability of materials with TPP mitochondrial targeting capability to induce the release of inflammatory factors and immune activation under light conditions.

[0134] In this diagram, "+" indicates white light irradiation treatment. By setting up the PBS(+) group, the influence of light irradiation alone on inflammatory factor levels can be eliminated; by setting up the COF / M / PEG(+) group, the effect of the TPP-free targeted modification material under light conditions can be evaluated, and the contribution of TPP-mediated mitochondrial targeted modification to enhanced immune activation can be further demonstrated. Figure 5Images (a), (b), and (c) show that serum levels of IL-18, IL-6, and IL-1β were low in the PBS(+) and COF / M / PEG(+) groups; in contrast, serum levels of IL-1β, IL-18, and IL-6 were significantly elevated in the COF / M / TPP(+) group. IL-1β and IL-18, as direct downstream characteristic products of the pyroptosis pathway (Caspase-3 / GSDME) activation, were released in large quantities into the peripheral blood, confirming severe pyroptosis in tumor tissue at the in vivo level.

[0135] (2) Dendritic cell maturation detection.

[0136] To further evaluate whether COF / M / TPP photocatalytic therapy can enhance in vivo antigen presentation capacity, this invention follows... Figure 6 , Figure 7 and Figure 8 The groups shown were used to evaluate the immune activation of tumor-bearing mice. Figures 6 to 8 Tumor-bearing mice from the same experimental batch were used. All groups underwent the same drug administration, lighting, and tissue collection procedures, the difference being the formulation administered via tail vein injection. The experimental groups are as follows: ① PBS(+) group: Mice were injected with PBS via the tail vein and the tumor sites were irradiated with white light as a control group for simple light irradiation; ② COF / M(+) group: Mice were injected with COF / M via the tail vein and then exposed to white light to evaluate the effect of materials loaded only with catalyst M and without PEG and TPP surface modification on dendritic cell maturation under light conditions. ③ COF / M / PEG(+) group: Mice were injected with COF / M / PEG via the tail vein. After administration, the tumor sites were irradiated with white light to evaluate the effect of PEG-modified materials without TPP mitochondrial targeting groups on dendritic cell maturation under light conditions. ④ COF / M / TPP(+) group: Mice were injected with COF / M / TPP via the tail vein. After administration, the tumor site was irradiated with white light to evaluate the effect of materials with TPP mitochondrial targeting ability on inducing dendritic cell maturation under light conditions.

[0137] In this diagram, "+" indicates treatment with white light irradiation. The dosage of each material was 5 mg / kg, administered via tail vein injection. After the material accumulated at the tumor site, the tumor area was then irradiated with white light. Tumor drainage lymph nodes were collected from treated mice, and single-cell suspensions were prepared. These suspensions were stained with anti-CD80 and anti-CD86 fluorescent antibodies, and mature dendritic cells (CD80+) were detected by flow cytometry. +CD86 + )Proportion.

[0138] The results are as follows Figure 6 (a) and Figure 6 As shown in (b), compared with the PBS(+) group, the COF / M(+) group, and the COF / M / PEG(+) group, the CD80 concentration in the COF / M / TPP(+) group was significantly lower. + CD86 + The significantly increased proportion of mature dendritic cells indicates that the treatment can effectively promote dendritic cell maturation, enhance tumor antigen presentation capacity, and provide a basis for the activation of subsequent adaptive immune responses.

[0139] (3) Detection of macrophage M1 polarization.

[0140] To further evaluate the effect of COF / M / TPP photocatalytic therapy on macrophage polarization in the tumor immune microenvironment, tumor tissues from mice treated in the same batch and group were collected and single-cell suspensions were prepared. These suspensions were stained with anti-CD80 and anti-CD206 fluorescent antibodies, and macrophage polarization was analyzed by flow cytometry. CD80 can be used as a marker for pro-inflammatory M1-like macrophages, and CD206 can be used as a marker for immunosuppressive M2-like macrophages.

[0141] The results are as follows Figure 7 (a) and Figure 7 As shown in (b), compared with the PBS(+) group, the COF / M(+) group, and the COF / M / PEG(+) group, the proportion of M1 macrophages in the COF / M / TPP(+) group was significantly increased, suggesting that the COF / M / TPP of the present invention can promote the transformation of the tumor immune microenvironment from an immunosuppressive state to a pro-inflammatory and anti-tumor state. The increase in M1 macrophages is beneficial to the secretion of inflammatory factors, tumor antigen processing, and enhanced anti-tumor immune effects.

[0142] (4) CD8 + T-cell infiltration / activation detection.

[0143] To further evaluate whether COF / M / TPP photocatalytic therapy can enhance the local cytotoxic T lymphocyte immune response in tumors, tumor tissues from mice treated in the same batch and group were collected and single-cell suspensions were prepared. These suspensions were stained with anti-CD3 and anti-CD8 fluorescent antibodies, and CD3 levels were analyzed by flow cytometry. + CD8 + The proportion of T cells was used to evaluate the infiltration and activation of cytotoxic T lymphocytes.

[0144] The results are as follows Figure 8 (a) and Figure 8 As shown in (b), CD3 in the COF / M / TPP+ illumination group + CD8 + The proportion of T cells was significantly higher in the PBS(+) group, COF / M(+) group, and COF / M / PEG(+) group, indicating that this treatment can effectively enhance the local cytotoxic T cell immune response in the tumor, thereby promoting tumor cell clearance.

[0145] (5) Results analysis.

[0146] Combining the above serum inflammatory factor detection results and flow cytometry analysis results, it can be seen that COF / M / TPP under light irradiation can not only induce pyroptosis-related inflammatory responses in tumor cells, but also further promote dendritic cell maturation, enhance macrophage polarization towards the M1 type, and increase CD8+. + T cell infiltration / activation levels. This indicates that the COF / M / TPP of the present invention can achieve a significant synergistic anti-tumor effect of "pyroptosis-immune linkage" through a cascade process of "pyroptosis induction - inflammation amplification - immune activation".

[0147] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for preparing a photocatalyst based on a nano-covalent organic framework, characterized in that, Includes the following steps: Step S1: Preparation of nano-covalent organic frameworks: 1,3,5-tris(4-aminophenyl)triazine and 1,3,5-tris(4-formylphenyl)triazine were added to a mixed organic solvent and dispersed evenly to obtain a mixed suspension. The mixed organic solvent was obtained by mixing mesitylene and 1,4-dioxane. Glacial acetic acid aqueous solution was added to the mixed suspension, and after stirring, centrifugation, precipitation washing, and freeze-drying, nano-covalent organic frameworks were obtained. Step S2: Preparation of dichloro(pentamethylcyclopentadienyl)rhodium(III) dimer-supported nano-covalent organic framework: Dichloro(pentamethylcyclopentadienyl)rhodium(III) dimer and the nano-covalent organic framework were added to a methanol solution. After stirring, centrifugation, precipitation washing and freeze drying, dichloro(pentamethylcyclopentadienyl)rhodium(III) dimer-supported nano-covalent organic framework was obtained. Step S3: Preparation of photocatalyst based on nano-covalent organic framework: The dichloro(pentamethylcyclopentadienyl)rhodium(III) dimer supported on nano-covalent organic framework, DSPE-PEG-2000 and PEG-TPP are added to a tetrahydrofuran solution and mixed evenly to obtain a tetrahydrofuran mixed solution. The tetrahydrofuran mixed solution is injected into deionized water and stirred to allow for nano-precipitation self-assembly. After dialysis, centrifugation, and freeze-drying, a photocatalyst based on nano-covalent organic framework is obtained.

2. The method for preparing a photocatalyst based on a nano-covalent organic framework according to claim 1, characterized in that, In step S1, the molar ratio of 1,3,5-tris(4-aminophenyl)triazine to 1,3,5-tris(4-formylphenyl)triazine is 1:1, and the reaction time of the stirring reaction is 12-18 h.

3. The method for preparing a photocatalyst based on a nano-covalent organic framework according to claim 1, characterized in that, In step S2, the mass ratio of the nano-covalent organic framework to the dichloro(pentamethylcyclopentadienyl)rhodium(III) dimer is 20:1, and the reaction time of the stirring reaction is 12-18 h.

4. The method for preparing a photocatalyst based on a nano-covalent organic framework according to claim 1, characterized in that, In step S3, the mass ratio of the dichloro(pentamethylcyclopentadienyl)rhodium(III) dimer supported on the nano-covalent organic framework, DSPE-PEG-2000, and PEG-TPP is 5:3:

2.

5. The method for preparing a photocatalyst based on a nano-covalent organic framework according to claim 1, characterized in that, In step S3, the volume ratio of the tetrahydrofuran solution to the deionized water is 1:5, and the stirring time for the self-assembly of the nanoprecipitate is 12-18 hours.

6. The method for preparing a photocatalyst based on a nano-covalent organic framework according to claim 1, characterized in that, In step S3, the dialysis time is 24-26 hours, and the dialysis medium is deionized water.

7. The method for preparing a photocatalyst based on a nano-covalent organic framework according to claim 1, characterized in that, The preparation steps of the 1,3,5-tris(4-aminophenyl)triazine include: 4-Aminobenzonitrile was placed in a reaction vessel, and trifluoroacetic acid was added dropwise under 0°C and an inert atmosphere. The mixture was stirred and stirred until homogeneous, and then reacted at room temperature and under an inert atmosphere. After the reaction was completed, distilled water was added, and the pH was neutralized to 7.0 with NaOH solution. The solid was collected by filtration, and after washing and drying, 1,3,5-tris(4-aminophenyl)triazine was obtained. The molar ratio of 4-aminobenzonitrile to trifluoroacetic acid is 1:(3-4).

8. The method for preparing a photocatalyst based on a nano-covalent organic framework according to claim 1, characterized in that, The preparation steps of the 1,3,5-tris(4-formylphenyl)triazine include: Synthesis of 1,3,5-tris(4-methylphenyl)triazine: p-Toluenenitrile was added dropwise to trifluoroacetic acid at 0 °C under nitrogen protection to obtain a reaction mixture. The reaction mixture was treated with ice water and neutralized with ammonia water, then filtered, washed and dried under vacuum to obtain 1,3,5-tris(4-methylphenyl)triazine. Synthesis of geminal diacetate intermediate: The 1,3,5-tris(4-methylphenyl)triazine was suspended in acetic anhydride, concentrated sulfuric acid was added and stirred, and a CrO3 solution in acetic anhydride was added dropwise. The reaction solution was poured into ice water, filtered, washed and purified by column chromatography to obtain the geminal diacetate intermediate; the molar ratio of the 1,3,5-tris(4-methylphenyl)triazine to CrO3 was 1:(8-9). Synthesis of 1,3,5-tris(4-formylphenyl)triazine: The geminal diacetate intermediate was dispersed in a mixture of ethanol and deionized water, concentrated sulfuric acid was added, the mixture was refluxed and cooled to room temperature, the precipitate was collected by filtration, and the precipitate was washed and dried to obtain 1,3,5-tris(4-formylphenyl)triazine.

9. A photocatalyst based on a nano-covalent organic framework, characterized in that, The photocatalyst based on a nano-covalent organic framework was prepared using the preparation method described in any one of claims 1 to 8.

10. The application of the photocatalyst based on the nano-covalent organic framework as described in claim 9 in the preparation of tumor therapeutic drugs.

Images