Gold monatomic radiosensitizer with multienzyme activity as well as preparation method and application of gold monatomic radiosensitizer

Abstract

The invention belongs to the technical field of biological catalytic materials, and particularly relates to a gold monatomic radiosensitizer with multienzyme activity as well as a preparation method and application of the gold monatomic radiosensitizer. The gold monatomic radiosensitizer is a coordination complex formed by a gold source containing tetrachloro gold (III) acid radical ions and 2, 4, 6-tri (4-pyridyl)-1, 3, 5-triazine through a coordination substitution reaction of Au and pyridine nitrogen. The gold monatomic radiosensitizer disclosed by the invention is a biocatalyst which is newly designed, has multi-enzyme activity biological catalysis performance and can be activated by radiation, and can be used as a polymer artificial enzyme for efficient and controllable tumor treatment.

Description

Technical Field

[0001] This invention belongs to the field of biocatalytic materials technology, specifically relating to a gold single-atom radiosensitizer with multi-enzyme activity, its preparation method, and its application. Background Technology

[0002] Cancer remains one of the leading causes of death worldwide, claiming millions of lives each year and posing a significant challenge to global public health. Radiotherapy, which uses high-energy X-rays to destroy tumors locally by generating double-strand DNA breaks and reactive oxygen species (ROS), remains a cornerstone of solid tumor treatment, often achieved in synergy with chemotherapy or immunotherapy. However, intrinsic and acquired radioresistance, due to factors such as tumor heterogeneity, cancer stem cells, and metabolic reprogramming, poses a significant challenge to the clinical efficacy of radiotherapy for certain solid tumors.

[0003] One potential solution is to develop radioactive or radiosensitizing agents to absorb and convert X-ray irradiation, such as the clinically approved nitroimidazole and telapaamide. Meanwhile, recent basic and clinical studies have shown that creating radioactive or radiosensitizing agents to stimulate adaptive local immune responses and synergize with immunotherapy can maximize tumor-killing effects in both primary and metastatic tumors. Recently, lanthanides, hafnium, platinum, and gold have been considered to have the potential to enhance X-ray radiation energy deposition due to their high atomic numbers, but their clinical application is limited by insufficient in vivo clearance and chronic toxicity caused by long-term retention. Among these, ultrasmall gold nanoclusters (<3 nm) have excellent renal clearance advantages, which could address this issue. The application of gold (Au) in cancer therapy, particularly in the form of nanoparticles, and its potential in combining radiosensitization and immunomodulation to maximize the efficacy of clinical radiotherapy regimens, has not yet been investigated. Summary of the Invention

[0004] To address the aforementioned issues, this invention proposes a novel gold single-atom radiosensitizer with multi-enzyme activity, biocatalytic properties, and radioactive activation, which can be used as a polymeric artificial enzyme for highly efficient and controllable tumor treatment.

[0005] Specifically, in a first aspect, the present invention provides a gold single-atom radiosensitizer with multi-enzyme activity, wherein the gold single-atom radiosensitizer is a coordination complex formed by a gold source containing tetrachloroauro(III) ions and 2,4,6-tris(4-pyridyl)-1,3,5-triazine through a coordination substitution reaction of Au and pyridine nitrogen.

[0006] Furthermore, in the aforementioned coordination substitution reaction, the pyridine nitrogen ligand (i.e., 2,4,6-tris(4-pyridyl)-1,3,5-triazine) substitutes for [AuCl4]. - The two Cl in -This leads to Au-N coordination.

[0007] Furthermore, the gold single-atom radiosensitizer contains Au-N coordination and Au-Cl coordination.

[0008] Furthermore, the gold source containing tetrachloroauro(III) ions is NaAuCl4 or its hydrate.

[0009] Furthermore, the gold single-atom radiosensitizer possesses glucose oxidase, lactate oxidase, and peroxidase activities, as well as radioactively enhanced ROS production performance.

[0010] Furthermore, the gold single-atom radiosensitizer has pH-dependent peroxidase activity, and exhibits stronger peroxidase activity under acidic conditions.

[0011] Furthermore, through the aforementioned multi-enzyme activity, the gold single-atom radiosensitizer can achieve efficient cascade generation of ROS.

[0012] In a second aspect, the present invention provides a method for preparing a gold single-atom radiosensitizer as described herein, comprising reacting a gold source containing tetrachloroauro(III) ions and 2,4,6-tris(4-pyridyl)-1,3,5-triazine in an acidic aqueous solution to obtain the gold single-atom radiosensitizer.

[0013] Furthermore, the molar ratio of the gold source containing tetrachloroauro(III) ions to 2,4,6-tris(4-pyridyl)-1,3,5-triazine is 1 to 3:1.

[0014] Furthermore, the gold source containing tetrachloroauro(III) ions is NaAuCl4 or its hydrate.

[0015] Furthermore, the reaction conditions are: stirring at room temperature for 10-60 minutes.

[0016] Furthermore, the acidic aqueous solution comprises 0.01M-0.1M hydrochloric acid.

[0017] Further, the preparation method includes dissolving 2,4,6-tris(4-pyridyl)-1,3,5-triazine in acid to form solution A; dissolving a gold source containing tetrachloroauro(III) ions in water to obtain solution B; mixing solution A and solution B, then reacting under stirring, collecting the reaction product, and obtaining the gold single-atom radiosensitizer after washing and drying.

[0018] Furthermore, the acid is 0.01M-0.1M hydrochloric acid.

[0019] Furthermore, the stirring speed is 200-800 rpm.

[0020] Furthermore, the washing process includes washing with 0.01M-0.1M hydrochloric acid and deionized water.

[0021] Furthermore, the drying includes freeze drying.

[0022] In a third aspect, the present invention provides the application of the gold single-atom radiosensitizer as described herein in the preparation of multi-enzyme active biocatalysts with radioenhanced ROS production performance.

[0023] Furthermore, the multi-enzyme activity includes glucose oxidase, lactate oxidase, and peroxidase activity.

[0024] Furthermore, the biocatalyst exhibits pH-dependent peroxidase activity, and even stronger peroxidase activity under acidic conditions.

[0025] Furthermore, through the aforementioned multi-enzyme activity, the biocatalyst can achieve efficient cascade generation of ROS.

[0026] In a fourth aspect, the present invention provides the use of the gold single-atom radiosensitizer as described herein in the preparation of a medicament for radiosensitizing the treatment of tumors.

[0027] Furthermore, the radiosensitization therapy for tumors includes the prevention of tumor metastasis and recurrence.

[0028] Beneficial effects of the present invention

[0029] The gold-coordinated triazine polymer network proposed in this invention (i.e., the gold single-atom radiosensitizer of this invention, referred to as Au-Tpt or Au-Tpt biocatalyst) possesses highly efficient electron transfer, strong electron-hole separation, and an Au2-N2Cl2 catalytic center. It can serve as a multifunctional radiosensitizer co-activated by the tumor microenvironment (TME) and radiotherapy, not only generating excellent reactive oxygen species (ROS) but also synergistically promoting necrosis and radiosensitization, thus becoming a potent immunomodulator for preventing metastasis and recurrence. The research results of this invention reveal that Au-Tpt can effectively inhibit DNA repair, trigger intense necrosis, and increase the sensitivity of cancer cells to X-ray irradiation after the release of damage-associated molecular patterns (DAMPs). This invention suggests that the innovative design of Au-Tpt provides a new pathway to achieving nanomedicines with multi-enzyme activities such as lactate oxidase (LOx), glucose oxidase (GOx), and peroxidase (POD), which can cascade efficiently to produce ROS, induce necrosis, and be radioactivated, thus synergistically promoting radiotherapy and immunotherapy in malignant tumors, particularly in preventing metastasis and recurrence. Attached Figure Description

[0030] Figure 1The diagram shows (a) a schematic diagram of the synthesis of Au-Tpt and (b) a schematic diagram of the cascade catalytic ROS generation of Au-Tpt.

[0031] Figure 2 The following images are shown: (a) Scanning electron microscopy (SEM) image of Au-Tpt; (b) X-ray diffraction analysis of Au-Tpt and Au-Tpb; (c) N 1s XPS spectra of Au-Tpt and Au-Tpb; (d) Au 4f XPS spectra of Au-Tpt and Au-Tpb; (e) X-ray absorption near-edge structure (XANES) spectrum of Au-Tpt; Au k 3 The distribution of the weighted Fourier transform spectrum in R space (f) and the analysis of its fitting results (g); Au L of different samples (h, i, j) 3 Wavelet transform image of edge EXAFS.

[0032] Figure 3 The following are shown: (a) Comparison of GOx enzyme activities of Au-Tpt and Au-Tpb; (b) Comparison of lactate oxidase activities of Au-Tpt and Au-Tpb; (c) Comparison of POD enzyme activities of Au-Tpt and Au-Tpb; (d) POD enzyme activity of Au-Tpt at different pH values; (e) Comparison of ROS production activities of different concentrations of Au-Tpt and Au-Tpb in the presence of X-ray irradiation; (f) Enhanced ROS production activity of Au-Tpt in the presence of X-ray irradiation. 1 O2 is generated; (g) •O2 is formed during the in-situ Fourier transform infrared spectroscopy display of POD-like processes. - And *OOH intermediate; (h) Au-Tpt comparison of POD enzyme catalytic kinetics with other reported biocatalysts.

[0033] Figure 4 The images show (a) representative JC-1 staining, (b) representative ROS images, (c) representative cytochrome c staining, and (d) representative flow cytometry images of apoptosis in CT26 cells that received different treatments.

[0034] Figure 5 The experimental results show the inhibitory effect of Au-Tpt-enhanced RT on tumor progression: (a) the animal experimental protocol for verifying the therapeutic effect of Au-Tpt-enhanced RT in vivo; (b) changes in tumor volume in mice of different groups; (c) photographs of tumor tissues and changes in tumor volume in individual mice of different groups; (e) changes in tumor weight in mice of different groups; (f) images of in situ tumors in mice of different groups after TUNEL immunostaining and fluorescence images of CRT and HMGB1 expression after immunofluorescence staining. Detailed Implementation

[0035] To obtain the proposed biocatalytically active and radioactively activated gold single-atom radiosensitizer (Au-Tpt) with multi-enzyme activity, a wet chemical self-assembly strategy combining gold salt and 2,4,6-tris(4-pyridyl)-1,3,5-triazine (Tpt) was employed in a typical synthesis process. This resulted in the formation of highly efficient and stable gold single-atom redox centers on Au-Tpt. Figure 1 a). For comparison, a gold single-atom catalyst, termed Au-Tpb or Au-Tpb biocatalyst, was synthesized using 1,3,5-tris(pyridin-4-yl)benzene (Tpb) via the same method. Inspired by the catalytic properties of natural GOx, LOx, and POX, it can be inferred that the developed biocatalytic Au-Tpt may offer the following structural advantages: i) the electron-donating ability of the Tpt ligand; ii) the ability of electron-enriched gold atoms to promote *OOH desorption; and iii) the ability of Au-Tpt to efficiently cascade ROS biocatalysis. Figure 1 b).

[0036] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0037] Example: Synthesis of Au-Tpt

[0038] Tpt (31.2 mg, 0.1 mmol) was dissolved in hydrochloric acid (0.1 M, 25 mL) to form solution A. NaAuCl4·2H2O (61.5 mg, 0.15 mmol) was dissolved in 25 mL of deionized water to form solution B. Solutions A and B were rapidly mixed and vigorously stirred (500 rpm) at room temperature for 0.5 hours. Then, Au-Tpt was centrifuged at 6000 rpm, washed with hydrochloric acid (0.1 M, 25 mL) and deionized water, and lyophilized. Yield: 42.4 mg.

[0039] Comparative Example: Synthesis of Au-Tpb

[0040] Tpb (30.9 mg, 0.1 mmol) was dissolved in hydrochloric acid (0.1 M, 25 mL) to form solution A. NaAuCl4·2H2O (61.5 mg, 0.15 mmol) was dissolved in 25 mL of deionized water to form solution B. Solutions A and B were rapidly mixed and vigorously stirred (500 rpm) at room temperature for 0.5 hours. The resulting Au-Tpb was then centrifuged at 6000 rpm, washed with hydrochloric acid (0.1 M, 25 mL) and deionized water, and lyophilized. Yield: 38.4 mg.

[0041] Test example:

[0042] The following methods were used to perform structural and functional tests on Au-Tpt and Au-Tpb.

[0043] Structural characterization: Scanning electron microscopy (SEM) was used. X-ray diffraction (XRD) was used to analyze the crystal structure of the catalyst, with Cu Kα radiation in the 2θ range of 5–80°. X-ray photoelectron spectroscopy (XPS) was performed on a K-Alpha™+ X-ray photoelectron spectroscopy system equipped with a hemispherical 180° double-focusing analyzer and a 128-channel detector. Fourier transform infrared (FT-IR) spectroscopy and in-situ attenuated total reflectance surface-enhanced infrared absorption spectroscopy (ATRSEIRAS) measurements were performed on a Nicolet-560 spectrophotometer. Electron paramagnetic resonance (EPR) measurements were performed using a Bruker EPR EMX Plus.

[0044] The dynamic parameters of the enzyme: the Michels-Menton constant is calculated based on the Michels-Menton saturation curve. For each H2O2, the Beer-Lambert law (equation (1)) is used (ε is 39,000 M). -1 cm -1 The initial reaction rate (V0) was calculated from the absorbance change (where c represents the concentration of oxTMB and lcm represents the length of the solution in the optical path). The reaction rate was then compared with the corresponding H2O2 and fitted using a Michaelis-Menten curve. In addition, a linear double reciprocal plot (Lineweaver-Burk plot, Equation (2)) was used to determine the maximum reaction rate (Vmax) and the Michaelis constant (Km). Furthermore, the number of turnovers (TON) was calculated according to Equation (3).

[0045] (1)

[0046] (2) (3)

[0047] [S] is the concentration of H2O2, and [E0] is the molar concentration of the metal in the nanozyme.

[0048] Glucose oxidase (GOx)-like activity: GOx is an enzyme that catalyzes the oxidation of glucose to gluconic acid and H₂O₂. H₂O₂ can oxidize iodide ions (I₂O₂). - ) generates triiodide ions (I - 3), then use I -3. Quantification of H₂O₂ by absorption characteristics in the UV-Vis band. First, 50 μL of catalyst solution (4 mg / mL) was mixed with 880 μL of PBS and 70 μL of glucose solution (360 mg / mL) at 42 °C for 30 minutes to form solution A. Potassium iodide solution (0.4 mol / L) and potassium hydrogen phthalate solution (0.1 mol / L) were mixed in a 1:1 ratio to form solution B. To detect H₂O₂, 100 μL of solution A and 100 μL of solution B were added to each well of a 96-well plate, and analysis was performed using a UV-Vis spectrophotometer. The relative absorbance at 350 nm was used to quantify the glucose degradation rate.

[0049] Peroxidase (POD) activity: POD-like activity was determined by colorimetric method. 5 μL of catalyst (10 mg / mL) was added. -1 ), 25 μL TMB (10 mg mL) -1 25 μL of H2O2 (0.1 M) and 25 μL of sodium acetate-acetic acid (NaOAc / HOAc) buffer [100 mM (pH 4.5)] were added to 2 mL of the solution. The catalytic oxidation of TMB (oxTMB) was investigated by measuring the absorption change of the oxidized form of TMB at λmax = 652 nm (Ɛ = 39,000 M-1 cm-1). Km and V max It uses the Michaelis-Menten equation V=V max The result is calculated from the double reciprocal of the Lineweaver-Burk plot of ×[S] / (Km+[S]), where TON=V. max / [E], where [S] is the concentration of H2O2 and [E] is the molar concentration of the metal in the catalyst.

[0050] EPR Measurement: Detection of Catalyst Formation by 2,2,6,6-Tetramethylpiperidine Oxide (TEMP) 1 O2 capacity. Add 10 μL of catalyst (10 mg / mL) to 500 μL buffer. -1 Add 10 μL of H2O2 (10M) and then add 20 μL of TEMP. The formation of 5,5-dimethyl-1-pyrrolidone N-oxide (DMPO) was detected by analysis. 1 O2 capacity. Add 10 μL of catalyst (10 mg / mL) to 500 μL DMSO. -1 Add 10 μL of H2O2 (10M) and then add 10 μL of DMPO.

[0051] Cytotoxicity assay: CT26 cells were cultured overnight in 96-well plates and then treated with Au-Tpb or Au-Tpt at concentrations ranging from 0 to 60 μg / ml. After 4 hours of treatment, the cells were irradiated with 160 kV and 20 mA X-rays at doses of 0 or 6 Gy. After 48 hours of culture, the viability of CT26 cells was determined using a CCK-8 assay kit.

[0052] Apoptosis detection: CT26 cells were cultured overnight in six-well plates and irradiated with PBS / Au-Tpb / Au-Tpt at a concentration of 40 μg / ml for 4 hours, followed by X-ray irradiation at 6 Gy. After 48 hours, cells were stained using the Annexin V-FITC / PI apoptosis kit and analyzed using FACS FCM and Flowjo software.

[0053] Intracellular ROS detection: ROS production was assessed using a reactive oxygen species (ROS) detection kit. After different treatments, CT26 cells were exposed to DCFH-DA (10 μM) for 30 min and then observed using an Olympus IX83 in vivo microscope.

[0054] JC-1 staining: CT26 cells were pre-incubated with PBS / Au-Tpb / Au-Tpt and irradiated as instructed, then cultured for 48 hours. Subsequently, the cells were stained with JC-1 using a mitochondrial membrane potential assay kit and imaged using a laser scanning confocal microscope.

[0055] Cytochrome C release assay: Cytochrome C release was assessed using immunocytochemistry with cytochrome C / CYCS antibodies. In short, 1×10⁻⁶ antibodies were used... 6 CT26 cells were seeded onto a glass slide and incubated with PBS / Au-Tpb (40 μg / ml) / Au-Tpt (40 μg / ml) for 4 hours, followed by 6 Gy irradiation. After a 48-hour incubation period, antibodies were added according to the manufacturer's instructions. The mounted slides were thoroughly examined using a focused laser scanning microscope (CLSM).

[0056] Antitumor efficacy of Au-Tpt material: To establish the CT26 mouse model, 5×10 5 One CT26 cell was injected into the right ventral region of BALB / c mice. When the tumor volume reached 80-100 mm... 3Mice were randomly divided into six groups: control group, RT group, Au-Tpb group, Au-Tpt group, Au-Tpb+RT group, and Au-Tpt+RT group. Each mouse received an intratumoral injection of 100 μg / ml (100 μl) of Au-Tpb or Au-Tpt. Four hours after injection, mice were anesthetized with 2% (v / v) isoflurane and the tumors were irradiated with 6 Gy X-rays. Tumor volume and body weight were monitored every two days. Tumor volume (mm) 3 Use the formula (length × width) 2 Calculated by multiplying the value by 0.5. When the tumor volume in the control group mice reached 2 cm... 3 All mice were euthanized. Tumors were removed, and sections were then subjected to immunofluorescence staining.

[0057] Multicolor immunofluorescence staining: Tumor sections were prepared and stained using multiplex immunofluorescence staining. After blocking with 20% goat serum, sections were incubated with single primary antibodies, including H MGB1, CRT, and TUNEL. For terminal deoxynucleotidyl transferase-mediated dUTP nick-end marker (TUNEL) immunofluorescence detection, all steps were performed according to the manufacturer's instructions. Cell nuclei were stained with DAPI prior to mounting. All images were acquired using Nikon Eclipse C1 and analyzed using CaseViewer software.

[0058] Statistical analysis. Quantitative data are expressed as mean ± SD. When comparing two or more groups, unpaired Student's t-tests or one-way ANOVA were performed using GraphPadPrism software to calculate statistical differences. Survival curves were analyzed using the log-rank test.

[0059] The test results are as follows.

[0060] Scanning electron microscopy (SEM) images show that Au-Tpt exhibits a well-preserved nanorod structure. Figure 2 a). X-ray diffraction analysis confirmed that all biocatalysts showed clear and similar diffraction peaks, and no diffraction peaks associated with crystalline gold species were detected in Au-Tpb and Au-Tpt, which may be attributed to the isolated dispersion of gold atoms. Figure 2 b).

[0061] X-ray photoelectron spectroscopy (XPS) is used to elucidate the coordination environment and chemical state of gold sites in biocatalysts. The N 1s spectrum of Au-Tpt shows a positive central level shift compared to Au-Tpb. Figure 2 c). The high-resolution spectrum of Au 4f can be decomposed into two doublets, with the dominant peak signal labeled as Au. + and Au 3+ Meanwhile, Au in Au-Tpt+ / Au 3+ The strength ratio is 1.11, which is much greater than that of Au-Tpb (0.34). Figure 2 d). Au in Au-Tpt + The increase in indicates that an excess of electrons is transferred from the triazine molecule of Tpt to Au. This electron transfer may help overcome the multi-electron reaction of the oxygen intermediate at the gold site, thus ensuring a rapid redox reaction.

[0062] Subsequently, X-ray absorption near-edge structure (XANES) spectroscopy and extended X-ray absorption fine structure (EXAFS) spectroscopy were used to elucidate the atomic coordination environment of the gold center in biocatalytic Au-Tpt. Analysis of the gold L3 edge XANES spectrum showed that the white line intensity of Au-Tpt was between that of gold foil and AuCl3, and the simulated oxidation state of gold in Au-Tpt was approximately +2.39 (…). Figure 2 e). Furthermore, this invention further evaluated the local atomic structure of gold using Fourier transform (FT) and wavelet transform (WT) of the extended X-ray absorption fine structure (EXAFS) spectrum. Au-Tpt showed a characteristic peak near 1.93 Å ( Figure 2 f), indicating the presence of Au-Cl / N. No typical Au-Au peaks were observed at longer distances (greater than 2.5 Å), suggesting that the gold atoms are isolated and dispersed. To obtain quantitative structural parameters of gold in Au-Tpt, this invention performed a least-R space fitting ( Figure 2 g) and provided Au L for different samples. 3 Wavelet transform image of edge EXAFS ( Figure 2 The results showed that the coordination number of Au was approximately 4 (1.7 Au-N and 1.8 Au-Cl).

[0063] Based on the above fitting results, it can be proven that the Au atom is anchored by four coordinating atoms: N and Cl. These results strongly demonstrate that Au exists as a single atom in Au-Tpt.

[0064] Enzyme-like ROS biocatalytic activity: After characterizing the morphology and electronic structure of the synthesized Au-Tpt, especially the gold site where it coordinates with the Tpt ligand, this invention systematically verified its enzyme-mimicking biocatalytic ability. Compared with the control biocatalyst Au-Tpb, the biocatalyst Au-Tpt exhibits superior cascade catalytic function in glucose consumption, lactate oxidation, and ROS generation; simultaneously, compared with the control biocatalyst Au-Tpb, the biocatalyst Au-Tpt can generate a large amount of ROS under X-ray irradiation, thereby enhancing radiation sensitivity, reducing radiation resistance, and improving overall antitumor efficacy.

[0065] GOx and LOx can catalyze the oxidation of glucose and lactate, respectively, to produce H2O2. The glucose consumption activity was studied by determining the concentration of H2O2 generated by GOx using the potassium iodide method. The results showed that Au-Tpt exhibits excellent performance similar to GOx. Figure 3 a). Lactic acid consumption activity was studied by measuring LOx using a lactate assay kit, and the results showed that Au-Tpt has high LOx efficiency ( Figure 3 b). The POD enzyme activity of Au-Tpt was evaluated using 3,3′,5,5′-tetramethylbenzidine (TMB) and H2O2 assay. The results showed that Au-Tpt had a faster ability to generate ROS compared to Au-Tpb. Figure 3 c). Furthermore, the POD enzyme activity of Au-Tpt at different pH values ​​was evaluated, and the results showed that it exhibited stronger POD enzyme activity under acidic conditions. Figure 3 d).

[0066] Subsequently, this invention conducted an in-depth study of the kinetics of POD-type processes. The catalytic Michelson constant (K...) m ), maximum reaction rate (V) max Basic parameters such as Kt and turnover times (TON, representing the maximum substrate conversion per catalytic atom) were determined using the Michaelis-Menten analysis method. Compared to Au-Tpb, Au-Tpt exhibits a lower Kt. m (K) m =0.042mM), higher V max (1.24 µM s) -1 ) and TON (17.86×10 -3 s -1 This indicates that Au-Tpt has a higher affinity for the substrate and more efficient catalytic kinetics. Importantly, compared with recently reported biocatalysts, Au-Tpt exhibits the highest TON value (…). Figure 3 h). Electron paramagnetic resonance (EPR) spectroscopy determined the type of ROS generated by Au-Tpt in the presence of H2O2, verifying the presence of •O2. - and 1 O2 is the main ROS product, and the enhanced ROS production of Au-Tpt in the presence of X-ray radiation, as revealed by biocatalysis, was also observed. Figure 3 e, f). In-situ Fourier transform infrared spectroscopy has also been used to detect structural changes in ROS intermediates during POD-like processes, indicating the formation of •O2. - and *OOH intermediate products ( Figure 3 g).

[0067] In vitro detection of Au-Tpt-enhanced radiofrequency ablation (RT): double-strand breaks (DDSBs) are widely recognized as the most lethal type of damage induced by ionizing radiation and are a major indicator of RT efficacy. JC-1 staining revealed that, compared to RT alone, Au-Tpt+RT resulted in a significant decrease in mitochondrial membrane potential (ΔΨm), manifested by a shift in fluorescence emission from red to green. Figure 4 a). Multiple studies have shown that ROS is associated with changes in ψm. When exposed to RT, ROS generated by Au-Tpt can directly or indirectly induce tumor cell apoptosis, thereby increasing radiosensitivity. ROS levels were assessed using the ROS probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). Results showed that fluorescence imaging ( Figure 4 b) indicates that cells treated with Au-Tpt and RT produced the highest levels of ROS. Furthermore, mitochondrial depolarization, in turn, led to the translocation of cytochrome c to the cell membrane (…). Figure 4 c), and activated the intrinsic pathway of apoptosis. Similarly, the Au-Tpt and RT groups showed the strongest antitumor activity and the highest apoptosis rate ( Figure 4 d). This indicates a positive correlation between superoxide ions and mitochondrial events in Au-Tpt+RT-induced apoptosis in CT26 cells. These results clearly suggest that Au-Tpt holds promise as a potent radiosensitizer.

[0068] Inhibitory effect of Au-Tpt-enhanced RT on tumor progression: Due to the excellent performance of Au-Tpt-based combination therapy in vitro, this invention subsequently investigated its efficacy in animal tumor models. To verify the therapeutic effect of Au-Tpt-enhanced RT in vivo, this invention established a CT26 tumor mouse model (… Figure 5 a). On day 7 after CT26 cell inoculation, BALB / c mice were randomly divided into 6 groups (n=5), including a control group (PBS), a RT group, an Au-Tpb group, an Au-Tpt group, an Au-Tpb+RT group, and an Au-Tpt+RT group (Au-Tpb dose = 10 mg / kg, Au-Tpt dose = 10 mg / kg). Mice were intravenously injected with PBS / Au-Tpb / Au-Tpt on day 0, and then received X-ray irradiation (6 Gy) 4 hours after injection. Tumor images showed that Au-Tpt had a superior inhibitory effect on tumor growth compared to Au-Tpb (…). Figure 5While RT alone can partially delay tumor growth, its inhibitory effect is stronger when used in combination with Au-Tpt. Notably, the growth rate of CT26 tumors was slowed after Au-Tpt combined with X-ray irradiation. Immunofluorescence staining showed a significant increase in TdT-mediated dUTP nick-end markers (TUNEL) after Au-Tpt+RT treatment, indicating successful induction of tumor cell apoptosis. Figure 5 f). Meanwhile, compared to the control group, Au-Tpt-based RT treatment significantly increased the expression of calreticulin (CRT) and high-mobility group box 1 (HMGB1). Figure 5 f). These in vivo experimental results successfully demonstrate that the Au-Tpt-based combined strategy employed in the CT26 cancer model can enhance the anti-tumor efficacy of X-ray irradiation.

[0069] In summary, this invention reports a novel biocatalytically and radioactively activated gold single-atom polymer network that can serve as a polymeric artificial enzyme for highly efficient and controllable tumor immunotherapy. The research of this invention demonstrates that the gold-coordinated triazine polymer network (Au-Tpt) possesses highly efficient electron transport, strong electron-hole separation, and an Au2-N2Cl2 catalytic center. It can act as a multifunctional radiosensitizer synergistically activating TME and RT, not only reversing immunosuppressive TME but also becoming a potent immunomodulator for preventing metastasis and recurrence through superior ROS generation, synergistic necrosis, and radiosensitization.

[0070] It should be noted that while the preferred embodiments of the present invention are given in the specification and accompanying drawings, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. These embodiments are not intended to impose additional limitations on the content of the present invention; their purpose is to provide a more thorough and comprehensive understanding of the disclosure of the present invention. Furthermore, the above-described technical features can be combined with each other to form various embodiments not listed above, all of which are considered to be within the scope of the present invention specification. Moreover, those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A gold monoatomic radiosensitizer having multiple enzymatic activities, characterized in that, The gold monatomic radiosensitizer is a coordination complex formed by coordination substitution reaction of a gold source containing tetrachlorogold(III) acid radical ion and 2,4,6-tris(4-pyridyl)-1,3,5-triazine through Au and pyridine nitrogen.

2. The gold monooatom radiosensitizer according to claim 1, wherein The gold source containing tetrachlorogold(III) acid radical ion is NaAuCl4 or its hydrate.

3. A method of preparing the gold monoatom radiation sensitizer according to claim 1 or 2, characterized in that, The preparation method comprises reacting the gold source containing tetrachlorogold(III) acid radical ion and 2,4,6-tris(4-pyridyl)-1,3,5-triazine in an acidic aqueous solution to obtain the gold monatomic radiosensitizer.

4. The production method according to claim 3, characterized by, The molar ratio of the gold source containing tetrachlorogold(III) acid radical ion and 2,4,6-tris(4-pyridyl)-1,3,5-triazine is 1-3:

1.

5. The preparation method according to claim 3, characterized in that, The reaction condition is stirring the reaction at room temperature for 10-60 min.

6. The preparation method according to claim 3, characterized in that, The acidic aqueous solution comprises 0.01M-0.1M hydrochloric acid.

7. The preparation method according to claim 3, characterized in that, The preparation method comprises dissolving 2,4,6-tris(4-pyridyl)-1,3,5-triazine in acid to form solution A; dissolving the gold source containing tetrachlorogold(III) acid radical ion in water to obtain solution B; mixing solution A and solution B, then reacting under stirring, collecting the reaction product, and washing and drying to obtain the gold monatomic radiosensitizer.

8. Use of the gold monatomic radiosensitizer according to claim 1 or 2 in the preparation of a multi-enzyme active biological catalyst with ROS production performance enhanced by radiation.

9. Use according to claim 8, characterized in that, The multi-enzyme activity comprises glucose oxidase, lactate oxidase and peroxidase activity.

10. Use of the gold monatomic radiosensitizer according to claim 1 or 2 in the preparation of a drug for radiosensitization treatment of tumors.

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