Preparation methods of gold nanoclusters / COF composite materials and their application in postoperative tumor diagnosis, treatment and recurrence monitoring.

Abstract

This invention discloses a method for preparing a gold nanocluster / COF composite material and its application in postoperative tumor diagnosis and recurrence monitoring, relating to the field of nanobiomaterials technology. The method involves using a porphyrin-based covalent organic framework as a carrier, loading the chemotherapeutic drug doxorubicin, and then using a bovine serum albumin template method to prepare gold nanoclusters as "nanovalve" to in-situ seal the framework pores. The gold nanoclusters and the covalent organic framework are tightly assembled through electrostatic and hydrogen bonding interactions, achieving zero drug leakage under physiologically neutral conditions; upon entering the acidic tumor microenvironment, the valve structure opens in response to pH changes, achieving targeted drug release. Simultaneously, under light irradiation, this composite material significantly enhances reactive oxygen species production through efficient interfacial electron transfer between the gold nanoclusters and porphyrin units, synergistically enhancing photodynamic therapy effects, and also possesses broad-spectrum antibacterial properties.

Description

Technical Field

[0001] This invention relates to the field of nanobiomaterials technology, specifically to a method for preparing a gold nanocluster / COF composite material and its application in postoperative tumor diagnosis and recurrence monitoring. Background Technology

[0002] Currently, the main clinical treatment for malignant tumors is surgical resection, supplemented by chemotherapy or radiotherapy. However, due to the complexity of the anatomical structure of the tumor's growth site, surgery often fails to completely remove tiny residual lesions or micrometastases already lurking in the surrounding area, directly leading to a high recurrence rate after tumor surgery. Simultaneously, surgical wounds are highly susceptible to bacterial infection during the healing process, which not only delays wound healing and triggers severe inflammatory responses but may also force the interruption of subsequent adjuvant therapy, seriously affecting the patient's prognosis and quality of life. Therefore, developing a multifunctional integrated diagnostic and therapeutic platform that can efficiently remove residual cancer cells, prevent postoperative infection, and provide real-time monitoring of tumor recurrence has become a research hotspot in the field of biomedical materials.

[0003] In existing research, covalent organic frameworks (COFs) are often used as drug delivery carriers or photosensitizer carriers due to their high specific surface area and good biocompatibility. Existing technology discloses a COF nanocarrier (COF@ICG@OVA) for synergistic phototherapy and immunotherapy. This technology utilizes COF loaded with the photosensitizer indocyanine green (ICG) and ovalbumin (OVA) to attempt to eliminate primary tumors and induce an immune response through photodynamic / photothermal therapy. However, this existing technology has certain drawbacks. In this approach, ICG and COF are adsorbed only through π-π stacking, lacking an effective gating mechanism. This leads to a high risk of leakage of the loaded small molecule drug before reaching the target site, which not only reduces the effective drug concentration at the lesion site but also causes serious systemic toxicity. Secondly, this approach neglects the critical complication of postoperative bacterial infection, and the materials used lack antibacterial activity, failing to address the complex clinical problem of coexisting postoperative wound infection and tumor recurrence.

[0004] To address the issues of drug leakage and limited functionality in existing technologies, those skilled in the art have explored improvements in previous research, disclosing a DOX / COF@AuNCs assembly. This research creatively introduces bovine serum albumin-protected gold nanoclusters (BSA-AuNCs) as nanovalve coatings on the surface of COF loaded with doxorubicin (DOX). This previous study demonstrated that the introduction of AuNCs effectively blocks COF pores, significantly reducing pre-release of the drug under physiological conditions, and utilizes the protonation and desorption of AuNCs in the acidic tumor microenvironment to achieve pH-responsive drug release. Simultaneously, the synergistic effect of AuNCs and COF achieves photodynamic / chemotherapy-assisted anticancer and antibacterial functions.

[0005] Although previous studies by those skilled in the art have addressed the issues of drug leakage and single anti-cancer function, this technology still has shortcomings in the specific clinical application scenario of postoperative tumor diagnosis and recurrence monitoring. While previous studies confirmed the material's fluorescence imaging capabilities, they did not optimize it for the highly sensitive scenario of postoperative recurrence monitoring. In the complex microenvironment where postoperative inflammation and tissue repair coexist, background fluorescence interference from biological tissues is severe, and the previously prepared composite materials still need improvement in long-term fluorescence stability and signal-to-noise ratio, making it difficult to achieve precise and long-term tracking of minute residual lesions. Summary of the Invention

[0006] To address the shortcomings of the aforementioned background technologies, this invention aims to solve several key technical problems in the existing fields of cancer treatment and antibacterial therapy: Traditional mesoporous nanocarriers (such as simple COF) lack effective gating mechanisms, leading to non-specific leakage of loaded chemotherapeutic drugs (such as DOX) during blood circulation (pH 7.4), causing severe systemic toxicity (such as myelosuppression and cardiotoxicity); single chemotherapy is prone to drug resistance, while traditional photodynamic therapy (PDT) is limited by the instability of photosensitizers in vivo and the low conversion efficiency of reactive oxygen species (ROS), resulting in limited efficacy of single therapy; the wound microenvironment after cancer surgery is fragile, and the invasion of drug-resistant bacteria (such as MRSA) can easily lead to postoperative infection, treatment failure, and delayed healing; in addition, many synthetic nanomaterials also have potential immunogenicity or long-term toxicity, resulting in insufficient biocompatibility of the carrier.

[0007] This invention provides a method for preparing gold nanoclusters / COF composite materials and their application in postoperative tumor diagnosis and recurrence monitoring. The method uses a porphyrin-based covalent organic framework as a carrier, loads the chemotherapeutic drug doxorubicin onto it, and then uses gold nanoclusters prepared by a bovine serum albumin template method as "nanovalve" to seal the framework pores in situ. The gold nanoclusters and the covalent organic framework are tightly assembled through electrostatic and hydrogen bonding interactions, achieving zero drug leakage under physiologically neutral conditions; upon entering the acidic tumor microenvironment, the valve structure opens in response to pH changes, achieving targeted drug release.

[0008] The first objective of this invention is to provide a method for preparing gold nanoclusters / COF composite materials, comprising the following steps: The monomers 5,10,15,20-tetra(4-aminophenyl)porphyrin and 2,5-dihydroxy-1,4-benzenedicarboxaldehyde were dissolved in a mixed solvent of mesitylene and dioxane in a certain proportion. After adding a catalyst and sealing under vacuum, the mixture was reacted at 115-125℃ for 70-80 h. Subsequently, the reactants were subjected to ultrasonic exfoliation to obtain porphyrin-based COF nanosheets. After uniformly mixing bovine serum albumin solution and chloroauric acid solution, the pH was adjusted to 11-13, and the reaction was carried out at a constant temperature and speed of 36-38℃ for 10-18 hours. Then, the mixture was purified to obtain gold nanoclusters. Porphyrin-based COF nanosheets were dispersed in an aqueous solution of doxorubicin hydrochloride and stirred evenly in the dark. Then, gold nanoclusters were added. Under alkaline conditions, the gold nanoclusters / COF composite material was obtained by self-assembly using the electrostatic attraction between the negative charge on the surface of bovine serum albumin and the protonated groups on the porphyrin-based COF nanosheets.

[0009] Preferably, the mass ratio of the monomer 5,10,15,20-tetra(4-aminophenyl)porphyrin to 2,5-dihydroxy-1,4-benzenedicarboxaldehyde is 1.5~2.5:1.

[0010] Preferably, the volume ratio of mesitylene to dioxane in the mixed solvent is 1:1~2.

[0011] Preferably, the pH is adjusted to 11-13 using a 0.5-1.5 mol / L NaOH solution.

[0012] Preferably, the concentration of the bovine serum albumin solution is 45-55 mg / mL; and the concentration of the doxorubicin hydrochloride aqueous solution is 0.5-1.5 mg / mL.

[0013] Preferably, during self-assembly, the ratio of porphyrin-based COF nanosheets to gold nanoclusters is 1:10~15.

[0014] Preferably, during the purification process, a dialysis bag with a molecular weight cutoff of 8000-14000 Da is used for purification.

[0015] The second objective of this invention is to provide a gold nanocluster / COF composite material.

[0016] The third objective of this invention is to provide an application of gold nanoclusters / COF composite materials in the preparation of drugs for postoperative cancer treatment and recurrence monitoring.

[0017] Compared with the prior art, the beneficial effects of the present invention are: This invention provides a method for preparing a gold nanocluster / COF composite material and its application in postoperative tumor diagnosis and recurrence monitoring. Using a porphyrin-based covalent organic framework as a carrier, and loading the chemotherapeutic drug doxorubicin, gold nanoclusters prepared using a bovine serum albumin template method act as "nanovalves" to in-situ seal the framework pores. The gold nanoclusters and the covalent organic framework are tightly assembled through electrostatic and hydrogen bonding interactions, achieving zero drug leakage under physiologically neutral conditions. Upon entering the acidic tumor microenvironment, the valve structure opens in response to pH changes, achieving targeted drug release. Simultaneously, under illumination, the composite material significantly enhances reactive oxygen species production through efficient interfacial electron transfer between the gold nanoclusters and porphyrin units, synergistically enhancing photodynamic therapy effects, and also possesses broad-spectrum antibacterial properties. Furthermore, the gold nanoclusters serve as highly sensitive fluorescent probes, enabling long-term real-time monitoring of minimal postoperative residual lesions. This invention integrates precise drug release, synergistic therapeutic effects, long-lasting fluorescent tracing, and postoperative infection prevention, providing an efficient, safe, and multifunctional solution for postoperative tumor diagnosis and treatment.

[0018] This invention achieves a series of significant technical effects: Regarding precise controlled-release performance, experimental data confirms that the assembly of this invention exhibits a drug release rate of less than 5% after 24 hours in a physiologically neutral pH 7.4 environment, demonstrating excellent drug encapsulation capabilities. Upon entering the acidic pH 5.4 microenvironment of a tumor, the drug release rate reaches 62.5% after 12 hours, achieving highly efficient tumor-specific targeted drug release. It also demonstrates a significant advantage in drug loading capacity. Thanks to the AuNCs valve structure, which greatly reduces drug loss during the washing process, its effective drug loading capacity can reach up to 47.2 µg / mg, significantly superior to conventional physical adsorption carriers. Furthermore, it exhibits outstanding synergistic therapeutic effects. Cellular experiments showed that the synergistic effect of chemotherapy and photodynamic therapy (PDT) resulted in a cell killing rate of up to 88.6% for B16-F10 cells, while the cell killing rate of the pure drug group at the same concentration was only 55%, demonstrating a significant synergistic effect. In vivo, the material exhibited excellent performance; in tumor-bearing mice, the combined light treatment resulted in a tumor growth inhibition rate approaching 100% within 14 days, with no significant fluctuation in mouse weight during the experiment, demonstrating extremely high biosafety. Furthermore, it possesses excellent broad-spectrum antibacterial properties, achieving a bactericidal rate of over 99.8% against Staphylococcus aureus and drug-resistant MRSA, showcasing strong potential for clinical application. Attached Figure Description

[0019] Figure 1 The X-ray diffraction (XRD) pattern of bulk COF; Figure 2The images show the scanning electron microscope (SEM) image (a) of the original bulk COF synthesized by solvothermal method, the scanning electron microscope (SEM) image (b) of the nano-sized Nano-COF, and the transmission electron microscope (TEM) image (c) of Nano-COF. Figure 3 shows the fluorescence emission spectrum (a) and UV-Vis absorption spectrum (b) of COF, DOX, BSA-AuNCs and DOX / COF@AuNCs assemblies. (a) confirms that the assemblies retain the fluorescence properties of each component, which is sufficient for fluorescence imaging applications. (b) verifies that DOX was successfully loaded and BSA-AuNCs were successfully modified, confirming the successful preparation of the DOX / COF@AuNCs assembly.

[0020] Figure 4 High-resolution TEM image (a) of DOX / COF@AuNCs composite material and elemental mapping image (b) of drug-loaded composite material; Figure 5 The drug release curves (a) and molecular mechanism diagram (b) of DOX / COF@AuNCs in buffer solutions of different pH values ​​are shown. Figure 6 This is a quadrant diagram of flow cytometry apoptosis analysis of B16-F10 cells after treatment under different conditions. In the diagram, DPBS+Xe represents Duchenne phosphate buffer and xenon gas, which can prevent cell clumping and maintain osmotic pressure balance; COF+Xe represents porphyrin-based COF nanomaterials combined with xenon lamp to simulate sunlight irradiation for photodynamic therapy in a single group; AuNCs+Xe represents gold nanoclusters and xenon gas; DOX / COF@AuNCs+Dark represents chemotherapy alone without light irradiation; DOX / COF@AuNCs+Xe represents chemotherapy-photodynamic therapy synergistic treatment with xenon lamp irradiation, where Xe represents the xenon lamp source; Figure 7 The graphs show the tumor volume changes in tumor-bearing mice under different treatment groups during the 14-day observation period. DPBS+Xe represents Duchenne phosphate buffer and xenon gas; COF+Xe represents porphyrin-based COF nanomaterials combined with photodynamic therapy using a xenon lamp to simulate sunlight; AuNCs+Xe represents gold nanoclusters and xenon gas; DOX / COF@AuNCs+Dark represents chemotherapy alone without light exposure; and DOX / COF@AuNCs+Xe represents chemotherapy-photodynamic therapy combined with xenon lamp irradiation, where Xe represents the xenon lamp source. Figure 8 Photographs of plate inhibition of different concentrations of DOX / COF@AuNCs assemblies against Escherichia coli and Staphylococcus aureus; Figure 9The images show the morphological comparison of Staphylococcus aureus (S. aureus) before and after damage, after being treated with different materials, using scanning electron microscopy (SEM). Detailed Implementation

[0021] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments and accompanying drawings. However, the embodiments described are not intended to limit the present invention.

[0022] This invention addresses the shortcomings of existing literature regarding drug leakage, poor photostability, insufficient sensitivity in the complex postoperative microenvironment, and poor long-term efficacy. It proposes an optimized method for preparing gold nanoclusters / COF composite materials and their applications. Through specific structural design, this invention further enhances the binding stability and fluorescence quantum yield of AuNCs and COF, aiming to provide a novel integrated diagnostic and therapeutic solution that utilizes AuNCs as a highly sensitive fluorescent probe for real-time monitoring of postoperative recurrence.

[0023] To achieve the above objectives, the first aspect of the present invention provides a method for preparing gold nanoclusters / COF composite materials, characterized by comprising the following steps: The monomers 5,10,15,20-tetra(4-aminophenyl)porphyrin and 2,5-dihydroxy-1,4-benzenedicarboxaldehyde were dissolved in a mixed solvent of mesitylene and dioxane in a certain proportion. After adding a catalyst and sealing under vacuum, the mixture was reacted at 115-125℃ for 70-80 h. Subsequently, the reactants were subjected to ultrasonic exfoliation to obtain porphyrin-based COF nanosheets (porphyrin-based Nano-COF). After uniformly mixing bovine serum albumin solution and chloroauric acid solution, the pH was adjusted to 11-13, and the reaction was carried out at a constant temperature and speed of 36-38℃ for 10-18 hours. Then, the mixture was purified to obtain gold nanoclusters (BSA-AuNCs). Porphyrin-based COF nanosheets were dispersed in an aqueous solution of doxorubicin hydrochloride and stirred evenly in the dark. Then, gold nanoclusters were added. Under alkaline conditions, the gold nanoclusters / COF composite material (DOX / COF@AuNCs) was obtained by self-assembly using the electrostatic attraction between the negative charge on the surface of bovine serum albumin and the protonated groups on the porphyrin-based COF nanosheets.

[0024] The core mechanism of this invention is mainly reflected in the following three dimensions: First, this invention constructs a pH-responsive "switch" mechanism based on the isoelectric point characteristics of BSA molecules. Under physiologically neutral conditions (pH 7.4), the BSA surface carries a negative charge and interacts with Nanoparticles containing protonated groups. The COF framework forms a strong electrostatic attraction, allowing AuNCs to act like a "valve" to tightly lock the orifice, achieving zero drug leakage. Upon entering the tumor microenvironment (pH 5.4), BSA protonation causes surface charge reversal, transforming electrostatic attraction into repulsion, rapidly opening the valve and enabling explosive drug release. Secondly, this invention constructs a highly efficient interfacial electron transfer channel through the close contact between AuNCs and porphyrin-based COFs. Under photoexcitation, AuNCs can effectively capture excited-state electrons generated by COFs, significantly inhibiting carrier recombination, thereby greatly improving the efficiency of triplet energy transfer to oxygen molecules. This increases singlet oxygen (1O2) production by approximately 1.5 times compared to pure COF, achieving a photodynamic synergistic enhancement effect. Finally, this invention achieves integrated biological functions. AuNCs not only act as physical sealing components, but their exposed metal active sites can also synergistically destroy the phospholipid bilayer of bacterial cell membranes with the highly active ROS generated by COFs, thus exhibiting broad-spectrum killing ability against both Gram-positive and Gram-negative bacteria, effectively solving the integration challenge of cancer treatment and postoperative infection prevention.

[0025] The mass ratio of the monomer 5,10,15,20-tetratetra(4-aminophenyl)porphyrin to 2,5-dihydroxy-1,4-benzenedicarboxaldehyde is 1.5~2.5:1. The volume ratio of mesitylene to dioxane in the mixed solvent is 1:1~2.

[0026] To adjust the pH to 11-13, a 0.5-1.5 mol / L NaOH solution is used, preferably a 1 mol / L NaOH solution.

[0027] The concentration of the bovine serum albumin solution is 45-55 mg / mL, preferably 50 mg / mL; The concentration of the aqueous solution of doxorubicin hydrochloride is 0.5~1.5 mg / mL, preferably 1 mg / mL.

[0028] During self-assembly, the molar ratio of porphyrin-based COF nanosheets to gold nanoclusters is 1:10~15; it should be noted that the ratio of porphyrin-based COF nanosheets to gold nanoclusters is based on a Tph to Au molar ratio of 1:10~15.

[0029] During the purification process, dialysis bags with a molecular weight cutoff of 8000-14000 Da were used for purification.

[0030] An exemplary method for preparing a gold nanocluster / COF composite material includes: Synthesis and morphology regulation of porphyrin-based nano-COFs This study employed a solvothermal method to construct the crystal framework. First, monomers 5,10,15,20-tetratetra(4-aminophenyl)porphyrin (Thp) and 2,5-dihydroxy-1,4-benzenedicarboxaldehyde (Dha) were dissolved in a mixed solvent of mesitylene and dioxane (volume ratio 1:1). After adding 0.5 mL of 3 mol / L acetic acid catalyst and vacuum sealing, the mixture was reacted at 120 °C for 72 hours. Subsequently, the resulting purple blocky COF was subjected to high-power ultrasonic exfoliation (400 W, 30% duty cycle) using physical shear force to overcome interlayer interactions, ultimately yielding well-dispersed ultrathin nanosheets (Nano-COF) under ice bath conditions.

[0031] Preparation of BSA-AuNCs “Nanovalve” The synthesis of gold nanoclusters employed a biomolecular template method. A 50 mg / mL bovine serum albumin (BSA) solution was mixed with a 25 mM chloroauric acid solution under stirring at room temperature. After 2 minutes, a 1 mol / L NaOH solution was added to maintain the system at a strongly alkaline state (approximately pH 12), denaturing the protein and fully exposing its reducing groups. The mixture was reacted at a constant temperature and speed of 37 °C for 12 hours, followed by purification using a dialysis bag with a molecular weight cutoff of 8000-14000 Da to obtain red gold nanoclusters (BSA-AuNCs) exhibiting stable fluorescence.

[0032] Self-assembly of the intelligent drug delivery system DOX / COF@AuNCs The construction of the drug delivery system involves two key steps: 1) Drug loading: Utilizing the abundant pore structure of Nano-COF, nanosheets are dispersed in a 1 mg / mL aqueous solution of doxorubicin hydrochloride (DOX). After stirring in the dark for 24 hours, the drug is pumped into the pores through physical adsorption. 2) Valve encapsulation: BSA-AuNCs are added at a preferred molar ratio of AuNCs to COF of 10:1. Under slightly alkaline conditions, self-assembly is achieved using the electrostatic attraction between the negative charge on the BSA surface and the protonated groups on the COF framework. Finally, repeated washing removes residual free drug (DOX / COF@AuNCs).

[0033] pH-responsive release experiment: To evaluate the intelligent response performance of the system, the assembly was placed in a dialysis bag and then immersed in buffer solutions at pH 7.4 (simulating the blood circulation environment) and pH 5.4 (simulating the lysosomal environment of tumor cells), respectively. The drug release pattern of this drug delivery system under different pH conditions was investigated by taking samples at regular intervals.

[0034] A second aspect of the present invention provides a gold nanocluster / COF composite material.

[0035] A third aspect of this invention provides the application of a gold nanocluster / COF composite material in the preparation of drugs for postoperative cancer treatment and recurrence monitoring.

[0036] It should be noted that, unless otherwise specified, the experimental methods used in this invention are all conventional methods; and the reagents and materials used, unless otherwise specified, are all commercially available.

[0037] Example 1 A method for preparing a gold nanocluster / COF composite material, comprising: Synthesis and morphology control of porphyrin-based COF nanosheets: 40.5 mg of monomer 5,10,15,20-tetratetra(4-aminophenyl)porphyrin (Thp) and 19.9 mg of monomer 2,5-dihydroxy-1,4-benzenedicarboxaldehyde (Dha) were added to a 1:1 volume ratio of mesitylene and dioxane. 0.5 mL of 3 mol / L acetic acid catalyst was then added, and the mixture was vacuum-sealed. The reaction was carried out at 120 °C for 72 hours. Subsequently, the resulting purple blocky COF was subjected to high-power ultrasonic exfoliation (400 W, 30% duty cycle) using physical shear force to overcome interlayer interactions. Finally, well-dispersed ultrathin porphyrin-based COF nanosheets (Nano-COF) were obtained under ice bath conditions and a 0.4 mM Nano-COF aqueous solution was prepared for later use.

[0038] Preparation of BSA-AuNCs “nanovalve”: 250 mg of bovine serum albumin (BSA) was dissolved in 5 mL of ultrapure water under stirring at room temperature to prepare a 50 mg / mL BSA solution. 1 mL of a 50 mM chloroauric acid solution was added and diluted to 10 mM with 4 mL of ultrapure water. The diluted chloroauric acid solution was mixed with the BSA solution. After 2 minutes, 1 mol / L NaOH solution was added to adjust the pH of the system to 12, denaturing the protein and fully exposing the reducing groups. The mixture was stirred at a constant temperature and speed at 37°C for 12 hours. Purification was then performed using a dialysis bag with a molecular weight cutoff of 8000-14000 Da to obtain a 12.5 mM Au concentration red gold nanoclusters (BSA-AuNCs) solution, which was stored at 4°C for later use.

[0039] Self-assembly of the intelligent drug delivery system DOX / COF@AuNCs: Utilizing the abundant porous structure of Nano-COF, 5 mL of 0.4 mM Nano-COF aqueous solution was dispersed in 1 mg / mL doxorubicin hydrochloride (DOX) aqueous solution and stirred in the dark for 24 hours, allowing the drug to be pumped into the pores via physical adsorption. 0.32 mL of 12.5 mM BSA-AuNCs solution was added at a molar ratio of AuNCs to COF (Au to Thrp) of 10:1. Under slightly alkaline (pH≈8.0) NaOH solution conditions, self-assembly was achieved using the electrostatic attraction between the negative charge on the BSA surface and the protonated groups on the COF framework. After repeated washing to remove residual free drug, the precipitate was centrifuged at 14500 rpm for 15 min, washed twice with ultrapure water, and vacuum dried at 60℃ for 12 h to obtain the gold nanoclusters / COF composite material (DOX / COF@AuNCs).

[0040] pH-responsive release experiment: To evaluate the intelligent response performance of the system, the assembly was placed in a dialysis bag and then immersed in buffer solutions at pH 7.4 (simulating the blood circulation environment) and pH 5.4 (simulating the lysosomal environment of tumor cells), respectively. The drug release pattern of this drug delivery system under different pH conditions was investigated by taking samples at regular intervals.

[0041] Example 2 Same as Example 1, except that, Weigh 5 mg of doxorubicin hydrochloride (DOX) into a flask, add 5 mL of nano-sized COF (0.4 mM) solution, and stir in the dark for 24 h. Then add BSA. The AuNCs solution was prepared with an Au to Tph molar ratio of 10, and the mixture was stirred for 24 h. After centrifugation at 14500 rpm for 15 min, the precipitate was washed twice with ultrapure water and dried under vacuum at 60 °C for 12 h.

[0042] Example 3 Same as Example 2, except that, Adjust only BSA The amount of AuNCs solution added was adjusted to make the molar ratio of Au to Tph 15, and the subsequent centrifugation, washing and drying conditions remained unchanged.

[0043] Comparative Example 1 Same as in Example 1, except that the pure COF drug-loaded group was not coated with AuNCs; Preparation: Weigh 5 mg of doxorubicin hydrochloride (0.01 mmol) into a flask with a built-in magnetic stir bar. Add 5 mL of nano-sized COF (0.4 mM) solution to the flask, at which point the DOX concentration in the solution is 1 mg / mL. Stir in the dark for 24 h, then centrifuge at 14500 rpm for 15 min. Wash the resulting precipitate twice with ultrapure water and vacuum dry at 60 °C for 12 h for later use.

[0044] Comparative Example 2 Same as Example 1, except that, BSA-AuNCs and Nano-COF were simply mixed in a flask with stirring, without self-assembly.

[0045] To illustrate the relevant properties of the gold nanoclusters / COF composite material provided by the present invention, only Example 1 and Comparative Example 1 are used for illustration.

[0046] In the composite material provided in Example 1, which is a standard proportion group, the AuNCs:COF molar ratio is 10:1. 2.5 mL of BSA-AuNCs solution was added to 1 mg / mL Nano-COF loaded with DOX. The leakage rate at pH 7.4 was <5% within 24 hours, and the drug loading was 47.2 µg / mg.

[0047] Antitumor synergistic therapy (cellular / in vivo level): Using B16-F10 cells, administered at 0.5 W / cm² 2 The light.

[0048] Flow cytometry results showed an apoptosis rate of 88.6%, and in vivo tumor volume curves showed that tumor growth in the experimental group was almost completely inhibited after 14 days of treatment. Weight monitoring proved that the material had no systemic toxicity.

[0049] Xenon lamps were used to simulate sunlight as the light source, and the irradiation conditions depended on the slightly acidic environment (pH 5.4) at the tumor site. After the material is taken up by tumor cells, in the slightly acidic environment of the tumor, the BSA-AuNCs nanovalve on the surface will be protonated. The change in surface potential causes the AuNCs to detach from the assembly surface, triggering the opening of the pores and the slow and controllable release of the chemotherapy drug DOX (chemotherapy mechanism). Simultaneously, under xenon lamp irradiation simulating sunlight, COF and AuNCs synergistically generate large amounts of reactive oxygen species (ROS) to kill cancer cells (photodynamic therapy mechanism). The combination of these two approaches achieves synergistic anti-tumor therapy through chemotherapy and photodynamic therapy.

[0050] Sensitivity testing at different acidity gradients: The release gradients at pH 7.4, 6.4, and 5.4 were tested. Weigh 3 mg of DOX / COF@AuNCs sample, sonicate it in a small amount of phosphate-buffered saline (PBS), and transfer it to a dialysis bag with a molecular weight cutoff of 6000 Da. Place the dialysis bag into a centrifuge tube containing 3 mL of buffer solution, and set the pH gradient of the buffer solution to 5.4, 6.4 and 7.4 respectively; At 37°C, place the centrifuge tubes on a shaker and shake at 100 rpm; At different set time points, 300 μL of buffer solution was taken out to measure the UV-Vis absorbance, and an equal volume of fresh buffer solution was added to keep the total volume constant. The drug release curve was plotted by continuous monitoring for 3 days. The release rate increases linearly with increasing acidity, demonstrating that the material has a good stepwise response to both tumor stroma (medium acid) and lysosomes (strong acid).

[0051] Comparative Example 1: Pure COF drug-loaded group without AuNCs coating At pH 7.4, the DOX leakage rate exceeded 35% within 2 hours.

[0052] Without the protection of AuNCs nanovalve, drugs can cause severe systemic toxicity during transport, demonstrating the necessity of the sealing structure of this invention.

[0053] Comparative Example 2: Physical Mixing Group (AuNCs and COF were simply mixed without self-assembly) After centrifugation, most AuNCs remained in the supernatant, indicating a low drug loading (only 15 µg / mg).

[0054] To illustrate that the self-assembly process (electrostatic adsorption + pH adjustment) provided by this invention is key to the formation of functionalized composite materials, the following description is provided in conjunction with the accompanying drawings.

[0055] Figure 1 The image shows the X-ray diffraction (XRD) pattern of the bulk COF. XRD indicates the successful preparation of bulk COF with a crystalline structure, and that the nano-COF retained its original crystalline structure after ultrasonic exfoliation. Changes in the characteristic peaks of amino and aldehyde groups in the infrared spectrum confirm the successful condensation of the structural units. Figure 2(a) is a scanning electron microscope (SEM) image of the original bulk COF, 2(b) is a scanning electron microscope (SEM) image of the nano-COF after nano-sizing, and 2(c) is a transmission electron microscope (TEM) image of the nano-COF. SEM and TEM images show that the size of the ultrasonically treated nano-COF is reduced to approximately 200 nm, with good dispersion; elemental mapping confirms the uniform distribution of C, N, and O elements.

[0056] Figure 3 (a) and (b) are the fluorescence emission spectrum and UV-Vis absorption spectrum of COF, DOX, BSA-AuNCs and DOX / COF@AuNCs assemblies, respectively. (a) confirms that the assemblies retain the fluorescence properties of each component and have the basis for fluorescence imaging applications; (b) verifies the successful loading of DOX and the successful modification of BSA-AuNCs, confirming the successful preparation of the DOX / COF@AuNCs assembly.

[0057] Figure 4 (a) and (b) are high-resolution TEM images of the DOX / COF@AuNCs composite material and elemental mapping images of the drug-loaded composite material, respectively. The TEM and elemental mapping images show that the assembly is maintained at around 200 nm, and AuNCs are mainly distributed in the edge region of COF, confirming the co-existence of elements such as C, N, O, Au, and Cl.

[0058] Figures 5(a) and (b) show the drug release curves and molecular mechanism diagrams of responsive drug release of DOX / COF@AuNCs in phosphate-buffered saline (PBS) at different pH values, respectively. The release curves at different pH values ​​indicate that the material has significant pH responsiveness. The mechanism is demonstrated to be that protonation on the AuNCs surface under acidic conditions leads to the detachment of nanovalves, thereby accelerating drug release.

[0059] Figure 6 Quadrant diagrams of flow cytometry apoptosis analysis of B16-F10 cells after treatment under different conditions show that the synergistic therapy (chemotherapy and photodynamic therapy) system constructed by the DOX / COF@AuNCs assembly significantly improved the killing efficiency of cancer cells. Compared with the chemotherapy-only (DOX) group and the photodynamic therapy-only (light irradiation + carrier) group, the apoptosis rate of the "chemotherapy + photodynamic therapy" combined therapy group (Q2+Q3 quadrants) was the highest, usually reaching over 85%. This quantitatively demonstrates the therapeutic effect of 1+1>2.

[0060] Figure 7The graph shows the tumor volume changes in tumor-bearing mice under different treatment groups during the 14-day observation period. It can be seen from the graph that, compared to the control group, only the DOX / COF@AuNCs+Xe group showed a decrease in tumor volume, while the tumors in other groups continued to grow. This indicates that the synergistic treatment of controlled-release chemotherapy drugs and photodynamic therapy achieved excellent therapeutic effects.

[0061] Figure 8 Photographs of DOX / COF@AuNCs assemblies at different concentrations against Escherichia coli and Staphylococcus aureus show that the assemblies have good inhibitory performance on MRSA growth under light conditions.

[0062] Figure 9 This image shows a comparison of the scanning electron microscope (SEM) morphology of Staphylococcus aureus (MRSA) before and after treatment with different materials. MRSA remained morphologically intact after simple xenon lamp irradiation, AuNCs+Xe treatment, and COF+Xe treatment. Under dark conditions, the bacterial surface after DOX / COF@AuNCs treatment became rougher and more concave. Under light conditions, after assembly treatment, MRSA showed severe bacterial shrinkage, adhesion, and a very rough surface. Combined with the ROS content results, it can be inferred that the assembly induced bacterial cell membrane damage through ROS generated under light conditions, ultimately leading to bacterial death.

[0063] This invention describes preferred embodiments and their effects. However, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a gold nanocluster / COF composite material, characterized in that, Includes the following steps: The monomers 5,10,15,20-tetra(4-aminophenyl)porphyrin and 2,5-dihydroxy-1,4-benzenedicarboxaldehyde were dissolved in a mixed solvent of mesitylene and dioxane in a certain proportion. After adding a catalyst and sealing under vacuum, the mixture was reacted at 115-125℃ for 70-80 h. Subsequently, the reactants were subjected to ultrasonic exfoliation to obtain porphyrin-based COF nanosheets. After uniformly mixing bovine serum albumin solution and chloroauric acid solution, the pH was adjusted to 11-13, and the reaction was carried out at a constant temperature and speed of 36-38℃ for 10-18 hours. Then, the mixture was purified to obtain gold nanoclusters. Porphyrin-based COF nanosheets were dispersed in an aqueous solution of doxorubicin hydrochloride and stirred evenly in the dark. Then, gold nanoclusters were added. Under alkaline conditions, the gold nanoclusters / COF composite material was obtained by self-assembly using the electrostatic attraction between the negative charge on the surface of bovine serum albumin and the protonated groups on the porphyrin-based COF nanosheets.

2. The method for preparing the gold nanoclusters / COF composite material according to claim 1, characterized in that, The mass ratio of the monomer 5,10,15,20-tetra(4-aminophenyl)porphyrin to 2,5-dihydroxy-1,4-benzenedicarboxaldehyde is 1.5~2.5:

1.

3. The method for preparing the gold nanoclusters / COF composite material according to claim 1, characterized in that, The volume ratio of mesitylene to dioxane in the mixed solvent is 1:1~2.

4. The method for preparing the gold nanoclusters / COF composite material according to claim 1, characterized in that, To adjust the pH to 11-13, a 0.5-1.5 mol / L NaOH solution is used.

5. The method for preparing the gold nanoclusters / COF composite material according to claim 1, characterized in that, The concentration of the bovine serum albumin solution is 45-55 mg / mL; the concentration of the doxorubicin hydrochloride aqueous solution is 0.5-1.5 mg / mL.

6. The method for preparing the gold nanoclusters / COF composite material according to claim 1, characterized in that, During self-assembly, the molar ratio of porphyrin-based COF nanosheets to gold nanoclusters is 1:10~15.

7. The method for preparing the gold nanoclusters / COF composite material according to claim 1, characterized in that, During the purification process, dialysis bags with a molecular weight cutoff of 8000-14000 Da were used for purification.

8. A gold nanocluster / COF composite material prepared by the method according to any one of claims 1 to 7.

9. The use of the gold nanocluster / COF composite material according to claim 8 in the preparation of drugs for postoperative cancer treatment and recurrence monitoring.

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