A gold monoatomic radiosensitizer with multiple enzyme activities and a preparation method and application thereof

Abstract

The application belongs to the technical field of biological catalytic materials, and particularly relates to a gold monatomic radiosensitizer with multiple enzyme activities and a preparation method and application thereof. The gold monatomic radiosensitizer is a coordination complex formed by a coordination substitution reaction of a gold source containing a tetrachlorogold (III) acid radical ion and 2,4,6-tris (4-pyridyl) -1,3,5-triazine through Au and pyridine nitrogen. The gold monatomic radiosensitizer disclosed in the application is a newly designed biological catalyst with multiple enzyme activities and radioactivation, and can be used as a polymer artificial enzyme for efficient and controllable tumor treatment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological catalytic materials, and particularly relates to a gold monatomic radiosensitizer with multiple enzyme activities and a preparation method and application thereof. BACKGROUND

[0002] Cancer remains one of the leading causes of death worldwide, causing millions of deaths each year, posing a major challenge to global public health. Radiotherapy, which uses high-energy X-rays to achieve local tumor destruction by generating double-stranded DNA breaks and reactive oxygen species (ROS), remains the cornerstone of solid tumor treatment, which is achieved by synergistic effect with chemotherapy or immunotherapy. However, due to factors such as tumor heterogeneity, cancer stem cells and metabolic reprogramming, intrinsic and acquired radioresistance poses a major challenge to the clinical efficacy of radiotherapy for some solid tumors.

[0003] A potential solution is to develop radioactivatable or radiosensitizing agents to absorb and convert X-ray irradiation, such as the clinically approved nitroimidazole and tirapazamine. At the same time, recent basic and clinical research shows that creating radioactivatable or radiosensitizing agents to stimulate adaptive local immune responses and synergize with immunotherapy can maximize tumor killing effects in primary and metastatic tumors. Recently, due to their high atomic number, lanthanides, hafnium, platinum and gold are considered to have the potential to enhance X-ray radiation energy deposition, but their clinical application is limited by chronic toxicity caused by insufficient clearance and long-term retention in vivo. Among them, ultra-small gold nanoclusters (<3 nm) have excellent kidney clearance advantages, which can solve this problem. The application of gold (Au) in cancer treatment, especially in the form of nanoparticles, and its potential application in maximizing the efficacy of radiotherapy clinical protocols by combining radiosensitization and immunomodulation have not been studied. SUMMARY

[0004] To solve the above problems, the present application proposes a brand-new design of a gold monatomic radiosensitizer with multiple enzyme activities and biological catalytic performance and radioactivatable, which can be used as a polymeric artificial enzyme for efficient and controllable tumor treatment.

[0005] Specifically, in a first aspect, the present application provides a gold monatomic radiosensitizer with multiple enzyme activities, which is a coordination complex formed by the coordination substitution reaction of a gold source containing tetrachlorogold (III) acid radical ions with 2,4,6-tris (4-pyridyl)-1,3,5-triazine through the coordination of Au and pyridine nitrogen.

[0006] Further, in the coordination substitution reaction, the pyridine nitrogen ligand (i.e. 2,4,6-tris (4-pyridyl)-1,3,5-triazine) replaces two Cl - in [AuCl4] -to form Au-N coordination.

[0007] Further, the gold monatomic radiosensitizer has Au-N coordination and Au-Cl coordination.

[0008] Further, the gold source containing tetrachloroaurate(III) ion is NaAuCl4 or its hydrate.

[0009] Further, the gold monatomic radiosensitizer has glucose oxidase, lactate oxidase and peroxidase activities, and ROS production performance enhanced by radiation.

[0010] Further, the gold monatomic radiosensitizer has pH-dependent peroxidase activity, and has stronger peroxidase activity under acidic conditions.

[0011] Further, through the multi-enzyme activity, the gold monatomic radiosensitizer can realize cascade efficient generation of ROS.

[0012] In a second aspect, the present application provides a preparation method of the gold monatomic radiosensitizer as described herein, which comprises reacting a gold source containing tetrachloroaurate(III) ion and 2,4,6-tris(4-pyridyl)-1,3,5-triazine in an acidic aqueous solution to prepare the gold monatomic radiosensitizer.

[0013] Further, the molar ratio of the gold source containing tetrachloroaurate(III) ion and 2,4,6-tris(4-pyridyl)-1,3,5-triazine is 1-3:1.

[0014] Further, the gold source containing tetrachloroaurate(III) ion is NaAuCl4 or its hydrate.

[0015] Further, the reaction conditions are stirring the reaction at room temperature for 10-60 min.

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

[0017] Further, 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 tetrachloroaurate(III) ion in water to obtain solution B; mixing solution A and solution B, then reacting under stirring, collecting the reaction product, and obtaining the gold monatomic radiosensitizer after washing and drying.

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

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

[0020] Further, the washing comprises washing with 0.01M-0.1M hydrochloric acid and deionized water.

[0021] Further, the drying comprises freeze-drying.

[0022] In a third aspect, the present application provides use of the gold monatomic radiosensitizer as described herein in the preparation of a multi-enzyme activity biological catalyst with ROS-producing performance enhanced by radiation.

[0023] Further, the multi-enzyme activity comprises glucose oxidase, lactate oxidase and peroxidase activity.

[0024] Further, the biological catalyst has pH-dependent peroxidase activity, and has stronger peroxidase activity under acidic conditions.

[0025] Further, through the multi-enzyme activity, the biological catalyst can achieve cascade efficient generation of ROS.

[0026] In a fourth aspect, the present application provides use of the gold monatomic radiosensitizer as described herein in the preparation of a drug for radiosensitization treatment of tumors.

[0027] Further, the radiosensitization treatment of tumors comprises preventing metastasis and recurrence of tumors.

[0028] Advantages of the present application

[0029] The gold coordination triazine-based polymer network (i.e. the gold monatomic radiosensitizer of the present application, referred to as Au-Tpt or Au-Tpt biological catalyst) has efficient electron transfer, strong electron-hole separation and Au2-N2Cl2 catalytic center, and can be used as a multifunctional radiosensitizer activated by tumor microenvironment (TME) and radiotherapy. It not only can produce excellent reactive oxygen species (ROS), but also can synergize with necrosis and radiosensitization, and become a powerful immunomodulator for preventing metastasis and recurrence. The research results of the present application show that Au-Tpt can effectively inhibit DNA repair, trigger strong necrosis, and increase the sensitivity of cancer cells to X-ray irradiation after releasing damage-associated molecular patterns (DAMPs). The present application believes that the innovative design of Au-Tpt provides a new way for realizing multi-enzyme activity such as lactate oxidase (LOx), glucose oxidase (GOx) and peroxidase (POD), and can cascade efficient ROS production, induce necrosis and radioactivation of nanodrugs, so as to synergize radiotherapy and immunotherapy in malignant tumors, especially in preventing metastasis and recurrence. BRIEF DESCRIPTION OF DRAWINGS

[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 Distribution of weighted Fourier transform spectrum in R space (f) and 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 bio-catalytic performance with multi-enzyme activities and the radioactivatable gold monoatomic radiosensitizer (Au-Tpt) proposed in the present application, in a typical synthesis process, the wet-chemical self-assembly strategy by combining gold salt and 2,4,6-tris(4-pyridyl)-1,3,5-triazine (Tpt) was used to construct Au-Tpt, which enables the formation of efficient and stable gold monoatomic redox centers on Au-Tpt Figure 1 a). For comparison, a gold monoatomic catalyst was synthesized by the same method using 1,3,5-tris(pyridin-4-yl)benzene (Tpb), referred to as Au-Tpb or Au-Tpb bio-catalyst. Inspired by the catalytic properties of native GOx, LOx and POX, it can be inferred that the developed bio-catalytic Au-Tpt can provide the following structural advantages: i) the electron donor ability of Tpt ligand; ii) the ability of electron-rich gold atom to promote the desorption of *OOH; and iii) the ability of Au-Tpt to efficiently cascade ROS bio-catalysis Figure 1 b).

[0036] The present application will be further described in conjunction with specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the 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. Solution A and solution B were quickly mixed and stirred vigorously (500 rpm) at room temperature for 0.5 hours. Then, Au-Tpt was centrifuged at a speed of 6000 rpm, washed with hydrochloric acid (0.1 M, 25 mL) and deionized water, and freeze-dried. 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. Solution A and solution B were quickly mixed and stirred vigorously (500 rpm) at room temperature for 0.5 hours. Then, the generated Au-Tpb was centrifuged at a speed of 6000 rpm, washed with hydrochloric acid (0.1 M, 25 mL) and deionized water, and freeze-dried. Yield: 38.4 mg.

[0041] Test examples:

[0042] Au-Tpt and Au-Tpb were structurally and functionally tested using the following methods.

[0043] Structural characterization: Scanning electron microscopy (SEM) was used. X-ray diffraction (XRD) was used to analyze the crystal structure of the catalysts, Cu Ka radiation in the range of 5-80°. X-ray photoelectron spectroscopy (XPS) spectra were measured on a K-Alpha™+ X-ray photoelectron spectrometer 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 reflection surface-enhanced infrared absorption spectroscopy (ATR SEIRAS) measurements were performed on a Nicolet-560 spectrophotometer. Electron paramagnetic resonance (EPR) measurements were performed by a Bruker EPR EMX Plus.

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

[0045] (1)

[0046] (2)

[0047] (3)

[0048] [S] is the concentration of H2O2 and [E0] is the molar concentration of metals in the nanoszyme.

[0049] Glucose oxidase (GOx)-like activity: GOx is an enzyme that catalyzes the oxidation of glucose to gluconic acid and H2O2. H2O2 can oxidize iodine ions (I - ) to triiodine ions (I - 3), which is then used to measure the activity of the enzyme. -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.

[0050] 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.

[0051] 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.

[0052] 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, cells were exposed to X-ray at 160 KV and 20 mA with a dose of 0 or 6 Gy. After 48 hours of incubation, the viability of CT26 cells was determined using a CCK-8 kit.

[0053] Apoptosis detection: CT26 cells were cultured overnight in six-well plates and exposed to 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 an Annexin V-FITC / PI apoptosis kit and analyzed using FACS FCM and Flowjo software.

[0054] Intracellular ROS detection: ROS production was evaluated using an active oxygen detection kit. After different treatments, CT26 cells were exposed to DCFH-DA (10 μM) for 30 minutes and then observed using an Olympus IX83 live microscope.

[0055] JC-1 staining: After pre-incubation of CT26 cells with PBS / Au-Tpb / Au-Tpt and irradiation as indicated, cells were incubated for 48 hours. Subsequently, cells were stained with a mitochondrial membrane potential detection kit and imaged using a laser scanning confocal microscope.

[0056] Cytochrome c release assay: Release of cytochrome c was evaluated using an immunocytochemical cytochrome c / CYCS antibody. Briefly, 1 x 10 6 CT26 cells were seeded onto chambered glass slides and then 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, the antibody was added according to the manufacturer's protocol. The mounted slides were thoroughly examined using a confocal laser scanning microscope (CLSM).

[0057] Antitumor efficacy of Au-Tpt material: To establish a CT26 mouse model, 5 x 10 5 CT26 cells were injected into the right flank 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.

[0058] 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.

[0059] 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.

[0060] The test results are as follows.

[0061] 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).

[0062] 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 intensity ratio is 1.11, much larger than that of Au-Tpb (0.34) Figure 2 d). The increase of Au in Au-Tpt indicates that there is an excess of electron transfer from the triazine molecules of Tpt to Au. This electron transfer may help to overcome the multi-electron reaction of oxygen intermediates on the gold site, thus ensuring a fast redox reaction. +

[0063] Subsequently, the atomic coordination environment of the gold center in the biocatalytic Au-Tpt was elucidated using X-ray absorption near-edge structure (XANES) spectroscopy and extended X-ray absorption fine structure (EXAFS) spectroscopy. 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 about +2.39 Figure 2 e). In addition, the Fourier transform (FT) and wavelet transform (WT) of the extended X-ray absorption fine structure (EXAFS) spectrum were further evaluated to assess the local atomic structure of gold. Au-Tpt showed a characteristic peak at 1.93 Å Figure 2 f), indicating the presence of Au-Cl / N. No typical Au-Au peak was observed at longer distances (greater than 2.5 Å), indicating that the gold atoms were isolated and dispersed. To obtain the quantitative structural parameters of gold in Au-Tpt, R-space fitting Figure 2 g) was performed and wavelet transform images of Au L 3 edge EXAFS of different samples were provided Figure 3 h-j). The results showed that the coordination number of Au was about 4 (1.7 Au-N and 1.8 Au-Cl)

[0064] According to the above fitting results, it can be proved that the Au atom is anchored by four coordination atoms of N atoms and Cl atoms. The above results strongly prove that the Au element exists in the form of a single atom in Au-Tpt.

[0065] Enzyme-mimicking ROS biocatalytic activity: After characterizing the morphology and electronic structure of the synthesized Au-Tpt, especially the gold site coordinated with the Tpt ligand, the biocatalytic ability of the enzyme-mimicking ROS biocatalyst Au-Tpt was systematically verified. Compared with the control biocatalyst Au-Tpb, the biocatalyst Au-Tpt showed excellent cascade catalytic function in glucose consumption, lactic acid oxidation and ROS production; at the same time, compared with the control biocatalyst Au-Tpb, the biocatalyst Au-Tpt could produce a large amount of ROS under X-ray radiation, thereby enhancing radiation sensitivity, reducing radiation resistance and improving overall antitumor efficacy.

[0066] ​GOx and LOx can catalyze glucose oxidation and lactate oxidation to produce H2O2, respectively. The glucose consumption activity was investigated by determining the concentration of H2O2 produced by GOx using the potassium iodide method, and the results showed that Au-Tpt has excellent performance similar to GOx Figure 3 a). The lactate consumption activity was investigated by determining LOx using a lactate detection kit, and the results showed that Au-Tpt has high LOx performance Figure 3 b). The POD enzyme activity of Au-Tpt was evaluated using 3,3',5,5'-tetramethylbenzidine (TMB) and H2O2 detection method, and the results showed that Au-Tpt has the ability to generate ROS quickly compared with Au-Tpb Figure 3 c). In addition, the POD enzyme activity of Au-Tpt at different pH was also evaluated, and the results showed that it has stronger POD enzyme activity under acidic conditions Figure 3 d).

[0067] Subsequently, the kinetics of the POD-like process were further investigated. The basic parameters such as catalytic Michaelis constant (K m ), maximum reaction rate (V max ) and turnover number (TON, representing the maximum substrate conversion per catalytic atom) were determined by Michaelis-Menten analysis. Compared with Au-Tpb, Au-Tpt showed lower K m (K m = 0.042 mM), higher V max (1.24 µM s -1 ) and TON (17.86 × 10 -3 s -1 ), indicating that it has higher affinity for the substrate and more efficient catalytic kinetics. Importantly, the TON value of Au-Tpt is the highest compared with the recently reported biocatalysts Figure 3 h). Electron paramagnetic resonance (EPR) spectroscopy determined the type of ROS produced by Au-Tpt in the presence of H2O2, verifying that •O2 - and 1 O2 are the main ROS products, and revealing the enhanced ROS production of biocatalytic Au-Tpt in the presence of X-ray radiation Figure 3 e, f). In situ Fourier transform infrared spectroscopy was also used to detect the structural changes of ROS intermediates in the POD-like process, indicating the formation of •O2 - and *OOH intermediates Figure 4 g).

[0068] 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 5 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.

[0069] 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 5b-e). Although RT alone can partially delay tumor growth, it has a stronger effect on inhibiting tumor growth when combined with Au-Tpt. Notably, the growth rate of CT26 tumors is the slowest after Au-Tpt combined with X-ray irradiation treatment. Immunofluorescence staining shows that TdT-mediated dUTP nick end labeling (TUNEL) is significantly increased after Au-Tpt+RT treatment, indicating successful induction of tumor cell apoptosis Figure 5 f). Meanwhile, the expression of calreticulin (CRT) and high mobility group box 1 (HMGB1) is significantly increased after Au-Tpt-based RT treatment compared with the control group ​ f). These in vivo experimental results successfully demonstrate that the Au-Tpt-based combined strategy adopted in the CT26 cancer model of the present application can enhance the anti-tumor effect of X-ray irradiation.

[0070] In summary, the present application reports a newly designed biocatalytic and radioactivated gold monatomic polymer network that can be used as a polymer artificial enzyme for efficient and controllable tumor immunotherapy. The research of the present application shows that the gold-coordinated triazine-based polymer network (Au-Tpt) has efficient electron transfer, strong electron-hole separation and Au2-N2Cl2 catalytic center, which can be used as a multifunctional radiosensitizer activated by TME and RT synergy, not only reversing the immunosuppressive TME, but also becoming a powerful immunomodulator for preventing metastasis and recurrence through excellent ROS generation, synergistic necrosis and radiosensitization.

[0071] It should be noted that the present application is described in the specification and drawings of the present application, but the present application can be implemented in many different forms and is not limited to the embodiments described in the specification. These embodiments are not additional limitations on the content of the present application, and the purpose of providing these embodiments is to make the disclosure of the present application more thorough and comprehensive. Furthermore, each of the above technical features continues to be combined, forming various embodiments not listed above, which are considered to be within the scope of the present application. Furthermore, those skilled in the art can improve or modify the above description, and all such improvements and modifications are within the scope of the appended claims of the present application.

Claims

1. A gold single-atom radiosensitizer with multi-enzyme activity, characterized in that, 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.

2. The gold single-atom radiosensitizer according to claim 1, characterized in that, The gold source containing tetrachloroauro(III) ions is NaAuCl4 or its hydrate.

3. A method for preparing a gold single-atom radiosensitizer according to claim 1 or 2, characterized in that, The gold single-atom radiosensitizer is prepared by reacting a gold source containing tetrachlorogold(III) ions and 2,4,6-tris(4-pyridyl)-1,3,5-triazine in an acidic aqueous solution.

4. The preparation method according to claim 3, characterized in that, The molar ratio of the gold source containing tetrachloroauro(III) ions to 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 conditions are: stirring at room temperature for 10-60 minutes.

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

7. The preparation method according to claim 3, characterized in that, 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, reacting under stirring, collecting the reaction product, and obtaining the gold single-atom radiosensitizer after washing and drying.

8. The application of the gold single-atom radiosensitizer according to claim 1 or 2 in the preparation of multi-enzyme active biocatalysts with radioenhanced ROS production performance.

9. The application according to claim 8, characterized in that, The multi-enzyme activities include glucose oxidase, lactate oxidase, and peroxidase activities.

10. The use of the gold single-atom radiosensitizer according to claim 1 or 2 in the preparation of a medicament for radiosensitizing the treatment of tumors.

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