A tumor-targeting covalently bound radionuclide probe and a preparation method and application thereof

Abstract

The application belongs to the field of diagnosis and treatment, and discloses a tumor-targeting covalent binding radionuclide probe and a preparation method and application thereof. A compound IR820-2RGD is reacted with SH-DA to obtain a compound IR820-2RGD-DA; the compound IR820-2RGD-DA is reacted with NaI 125 / 131 to obtain a tumor-targeting covalent binding radionuclide probe. The existing internal irradiation strategy has certain limitations, such as complex operation technology, high cost, excessive toxic and side effects on normal organs / tissues, defects in pharmacokinetics, certain tolerance to NSCLC tumors and the like. The application develops a simpler, more mature and long-term diagnosis and treatment method, improves the radiosensitivity of NSCLC tumors, and thus realizes accurate and efficient diagnosis and treatment of NSCLC.

Description

Technical Field

[0001] This invention belongs to the field of tumor diagnosis and treatment, specifically relating to a tumor-targeting covalently bound radionuclide probe, its preparation method, and its application. Background Technology

[0002] Lung cancer is one of the most common malignant solid tumors in humans, with the highest mortality rate among all cancers. It has the highest incidence rate in men and is second only to breast cancer in women. Non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) are the two main histopathological types of lung cancer, with NSCLC accounting for more than 85% of all lung cancers. NSCLC often presents as advanced, unresectable, or metastatic disease. For advanced NSCLC, internal radiation therapy is considered an important treatment strategy. Currently, to improve the effectiveness of radioisotope therapy for radiation-insensitive NSCLC tumors, different treatment strategies have been developed. These are mainly divided into two categories: one is to modify NSCLC proto-oncogenes using specific gene editing technology to increase the radiosensitivity of NSCLC tumors to internal radiation; the other is to combine these strategies with other treatments to reduce the radiosensitivity of NSCLC tumors to internal radiation, thereby improving the treatment efficiency of NSCLC tumors under internal radiation therapy conditions. However, both strategies have certain limitations, such as complex operation techniques, high cost, excessive toxic side effects on normal organs / tissues, pharmacokinetic defects, and certain tolerance in NSCLC tumors. Therefore, there is an urgent need to develop a simpler, more mature, and longer-lasting diagnostic and treatment method to improve the radiosensitivity of NSCLC tumors, thereby achieving accurate and efficient diagnosis and treatment of NSCLC. Summary of the Invention

[0003] This invention discloses a long-term diagnostic and therapeutic strategy for lung cancer that can covalently bind to specific proteins in the tumor microenvironment, and the application of probes using this strategy in multimodal imaging and treatment of non-small cell lung cancer tumors. Therefore, this invention develops a simpler, more mature, and long-term diagnostic and therapeutic method, improving the radiosensitivity of NSCLC tumors, thereby achieving accurate and efficient diagnosis and treatment of NSCLC.

[0004] The present invention adopts the following technical solution.

[0005] A tumor-targeting covalently bound radionuclide probe has the following chemical structure:

[0006] The tumor-targeting covalently bound molecular probe disclosed in this invention can covalently bind to proteins generated under the mediation of the tumor microenvironment, and after radiolabeling, form a tumor-targeting covalently bound radionuclide probe for the diagnosis and treatment of tumors.

[0007] This invention discloses a method for preparing the above-mentioned tumor-targeting covalently bound radionuclide probe, comprising the following steps: reacting compound IR820-2RGD with SH-DA to obtain compound IR820-2RGD-DA; reacting compound IR820-2RGD-DA with radioactive sodium iodide to obtain the tumor-targeting covalently bound radionuclide probe.

[0008] In this invention, compound 3-mercaptopropylamine is reacted with 3,5-dioxanecarboxylic acid to obtain compound SH-DA.

[0009] In this invention, compound GIR820 is reacted with N3-RGD to obtain compound IR820-2RGD.

[0010] This invention discloses a method for preparing the above-mentioned tumor-targeting covalently bound radionuclide probe, comprising the following steps: (1) The compound GIR820 was reacted with N3-RGD to obtain the compound IR820-2RGD; (2) The compound 3-mercaptopropylamine was reacted with 3,5-dioxanecarboxylic acid to obtain the compound SH-DA; (3) The compound IR820-2RGD was reacted with SH-DA to obtain the compound IR820-2RGD-DA; (4) The compound IR820-2RGD-DA was reacted with radioactive sodium iodide to obtain a radioactive compound. 125 / 131 I-IR820-2RGD-DA is a tumor-targeting covalently bound radionuclide probe.

[0011] Furthermore, the preparation methods of the above-mentioned tumor-targeting covalently bound molecular probes and tumor-targeting covalently bound radionuclide probes include the following steps: (1) Compound GIR820 and compound N3-RGD were reacted in the presence of an organic solvent, an inorganic copper salt and a reducing agent to obtain compound IR820-2RGD; the organic solvent included N,N-dimethylformamide, the inorganic copper salt included copper sulfate pentahydrate, and the reducing agent included sodium ascorbate; (2) The raw material 3,5-dioxane carboxylic acid is reacted with NHS in the presence of an organic solvent and a condensing agent, and then reacted with 3-mercaptopropylamine in the presence of an organic base and an organic solvent to obtain the compound SH-DA; the condensing agent includes EDC, the organic base includes N,N-diisopropylethylamine, and the organic solvent includes N,N-dimethylformamide, etc. (3) The compound IR820-2RGD was reacted with SH-DA in the presence of an organic base and an organic solvent to obtain the compound IR820-2RGD-DA; the organic solvent included N,N-dimethylformamide and the organic base included triethylamine; (4) The compound IR820-2RGD-DA was reacted with the radioactive iodine compound NaI. 125 / 131 The reaction in an organic solvent and an oxidizing agent yields a radioactive compound. 125 / 131 I-IR820-2RGD-DA is a tumor-targeting covalently bound radionuclide probe; the organic solvent used in the above reaction is N,N-dimethylformamide, and the oxidant is chloramine-T.

[0012] As an example, the preparation method of the above-mentioned tumor-targeting covalently bound radionuclide probe includes the following steps: (1) Compound GIR820 and compound N3-RGD were dissolved in N,N-dimethylformamide, and then a mixture of copper sulfate pentahydrate solution and sodium ascorbate solution was added. The mixture was reacted at room temperature for 2 to 6 hours, and then purified to obtain compound IR820-2RGD. (2) Dissolve 3,5-dioxanecarboxylic acid in N,N-dimethylformamide, add NHS and EDC, react at room temperature for 10-15 hours, then extract and evaporate to dryness; then dissolve the evaporated product in N,N-dimethylformamide, add 3-mercaptopropylamine and N,N-diisopropylethylamine, react at room temperature for 2-5 hours; after the reaction is complete, purify to obtain compound SH-DA; (3) Dissolve compound IR820-2RGD and compound SH-DA in N,N-dimethylformamide, then add triethylamine, react at room temperature for 10-16 hours, and then purify to obtain compound IR820-2RGD-DA; (4) Dissolve compound IR820-2RGD-DA in NaI 125 / 131 In the solution, chloramine-T was then added, and the reaction was carried out at room temperature for 1–5 minutes, followed by purification to obtain the compound. 125 / 131 I-IR820-2RGD-DA is a tumor-targeting covalently bound radionuclide probe.

[0013] In the above technical solution, the equivalent ratio of compound N3-RGD, compound GIR820, copper sulfate pentahydrate solution and sodium ascorbate in step (1) is 1:(0.5~1.5):(0.3~0.8):(0.7~1.3); the equivalent ratio of 3,5-dioxanecarboxylic acid, 3-mercaptopropylamine, NHS, EDC and N,N-diisopropylethylamine in step (2) is 1:(0.5~1.5):(1~1.5):(1~1.5):(1~2); the equivalent ratio of compound IR820-2RGD, SH-DA and triethylamine in step (3) is 1:(0.5~1.5):(1~2).

[0014] Preferably, in the above technical solution, the equivalent ratio of compound N3-RGD, compound GIR820, copper sulfate pentahydrate solution, and sodium ascorbate in step (1) is 1:(0.8-1.2):(0.3-0.6):(0.9-1.2); the equivalent ratio of 3,5-dioxanecarboxylic acid, 3-mercaptopropylamine, NHS, EDC, and N,N-diisopropylethylamine in step (2) is 1:(0.9-1.2):(1.1-1.4):(1.2-1.5):(1.3-1.5); and the equivalent ratio of compound IR820-2RGD, SH-DA, and triethylamine in step (3) is 1:(0.8-1.2):(1.2-1.7).

[0015] As an example, the equivalent ratio of compound N3-RGD, compound GIR820, copper sulfate pentahydrate solution, and sodium ascorbate in step (1) is 1:1:0.5:1; the equivalent ratio of 3,5-dioxanecarboxylic acid, 3-mercaptopropylamine, NHS, EDC, and N,N-diisopropylethylamine in step (2) is 1:1:1.2:1.3:1.5; and the equivalent ratio of compound IR820-2RGD, SH-DA, and triethylamine in step (3) is 1:1:1.5.

[0016] In this invention, the chemical structural formulas of compounds N3-RGD and GIR820 are as follows:

[0017] In this invention, the chemical structural formulas of compounds IR820-2RGD, SH-DA, and IR820-2RGD-DA are as follows:

[0018]

[0019] This invention discloses the application of the aforementioned tumor-targeting covalently bound radionuclide probe in the preparation of imaging reagents. Preferably, the application of the aforementioned tumor-targeting covalently bound radionuclide probe in imaging reagents for non-small cell lung cancer, such as tumor long-window period multimodal imaging reagents; or the application of the aforementioned tumor-targeting covalently bound radionuclide probe in the preparation of drugs, preferably, the drugs include anti-tumor drugs, specifically, the drugs include drugs for treating solid tumors, such as drugs for treating lung cancer; or the application of the aforementioned tumor-targeting covalently bound radionuclide probe in radiotherapy for non-small cell lung cancer; or the application of the aforementioned tumor-targeting covalently bound radionuclide probe in the preparation of radiotherapy agents for non-small cell lung cancer.

[0020] This invention discloses the application of the above-mentioned tumor-targeting covalently bound radionuclide probe in the preparation of diagnostic and therapeutic reagents, wherein the diagnosis and treatment include diagnosis and treatment; preferably, this invention discloses the application of the above-mentioned tumor-targeting covalently bound radionuclide probe in the preparation of tumor diagnostic and therapeutic reagents, specifically, the tumor includes solid tumors, such as lung cancer.

[0021] This invention discloses a tumor diagnostic reagent, the active ingredient of which includes a tumor-targeting covalently bound radionuclide probe.

[0022] The method for long-window multimodal imaging using the above-mentioned tumor-targeting covalently bound radionuclide probes includes the following steps: injecting the tumor-targeting covalently bound radionuclide probe solution into the body, for example, via conventional intravenous injection, to achieve multimodal imaging at different time points.

[0023] Furthermore, the method for tumor long-window multimodal imaging using the above-mentioned tumor-targeting covalently bound radionuclide probes includes the following steps: injecting the tumor-targeting covalently bound radionuclide probe solution into the patient's body, for example, via conventional tail vein injection, and observing multimodal images at different time points under anesthesia.

[0024] The method of using the above-mentioned tumor-targeting covalently bound radionuclide probe for treatment includes the following steps: injecting the above-mentioned tumor-targeting covalently bound radionuclide probe solution into the patient's body, for example, by conventional intravenous injection, to achieve tumor treatment; taking tumor-bearing mice as an example, a single injection of 3.7 MBq / 100 μL is injected into non-small cell lung cancer tumor-bearing mice via tail vein injection, and the tumor volume, changes in the weight of tumor-bearing mice, and safety assessment of normal tissues after treatment are recorded for 21 consecutive days to determine the efficacy and biosafety of the probe for the diagnosis and treatment of non-small cell lung cancer tumors.

[0025] Due to the application of the above technical solutions, the advantages of this invention are: 1. This invention first designs and synthesizes a tumor-targeting covalently bound probe and a corresponding radionuclide probe. The probe can directly undergo a covalent binding reaction with protein sulfinic acid under the mediation of H2O2 in cells, prolonging the retention time of the probe in non-small cell lung cancer tumors and realizing long-window fluorescence imaging of non-small cell lung cancer tumors.

[0026] 2. The tumor-targeting covalently bound radionuclide probe of the present invention causes damage to tumor cells, such as non-small cell lung cancer cells, after undergoing a covalent binding reaction in cells, leading to severe apoptosis of non-small cell lung cancer cells, thereby inhibiting tumor growth.

[0027] 3. The tumor-targeting covalently bound radionuclide probe of this invention has a significant therapeutic effect on non-small cell lung cancer tumors, realizing the integration of radiotherapy and diagnosis for non-small cell lung cancer tumors. Attached Figure Description

[0028] Figure 1 This is a schematic diagram illustrating the synthesis of a tumor-targeting covalently bound radionuclide probe.

[0029] Figure 2 This is a schematic diagram of the synthesis of the control probe.

[0030] Figure 3 The in vitro characterization of probes covalently bound to hyposulfonated proteins generated under H2O2-mediated conditions in the tumor microenvironment and the corresponding radionuclide probes included: (a) UV absorption changes and fluorescence spectra of probes covalently bound to hyposulfonated proteins generated under H2O2-mediated conditions in the tumor microenvironment; (b) nanoscale characterization of probes covalently bound to hyposulfonated proteins generated under H2O2-mediated conditions in the tumor microenvironment; and (cd) radiostability of tumor-targeting covalently bound radionuclide probes covalently bound to hyposulfonated proteins generated under H2O2-mediated conditions in PBS and FBS solutions.

[0031] Figure 4 To study the in vitro binding of probes covalently bound to hyposulfonated proteins generated under H2O2-mediated conditions in the tumor microenvironment; wherein: (ab) the agarose gel electrophoresis and near-infrared fluorescence images of probes covalently bound to hyposulfonated proteins generated under H2O2-mediated conditions in the tumor microenvironment after co-incubation with bovine serum albumin and A549 cell protein, respectively.

[0032] Figure 5This study investigates the relationship between the experimental probe IR820-2RGD-DA and the control probe IR820-2RGD-COOH (10 μM) covalently bound to hyposulfonated proteins generated under H2O2-mediated conditions in the tumor microenvironment. Specifically: (ad) fluorescence imaging and quantification results of the experimental probe IR820-2RGD-DA and the control probe IR820-2RGD-COOH (10 μM) covalently bound to hyposulfonated proteins generated under H2O2-mediated conditions in the non-small cell lung cancer A549 cells and normal lung epithelial BEAS-2B cells at different time points; (eg) distribution, fluorescence quantification, and potential of the experimental probe IR820-2RGD-DA (10 μM) covalently bound to hyposulfonated proteins generated under H2O2-mediated conditions in the non-small cell lung cancer A549 cells; (hi) comparison of the experimental probe IR820-2RGD-DA and the control probe IR820-2RGD-COOH (10 μM) in different organelles of the non-small cell lung cancer A549 cells. (j) Fluorescence imaging and quantification results of cell retention at different time points in non-small cell lung cancer A549 using experimental probe IR820-2RGD-DA and control probe IR820-2RGD-COOH (10 μM) mediated by a certain concentration of H2O2 (50 μM); (kl) Fluorescence imaging and quantification results of cell retention at different time points in non-small cell lung cancer A549 using experimental probe IR820-2RGD-DA and control probe IR820-2RGD-COOH (10 μM) mediated by different concentrations of H2O2 (0-50 μM).

[0033] Figure 6 This study investigates the in vivo multimodal imaging of tumor-targeting covalently bound probes and corresponding radionuclide probes. The results include: (a) in vivo fluorescence imaging of A549 tumor-bearing nude mice with different treatment groups; (b) changes in biodistribution of A549 tumor-bearing nude mice with different treatment groups 24 hours after in vivo imaging; (c) results of intraoperative tumor resection in A549 tumor-bearing nude mice before and after tail vein administration of the experimental probe group; (d) in vivo SPECT / CT imaging of A549 tumor-bearing nude mice treated with different radionuclide probes; (e) changes in tumor uptake in A549 tumor-bearing nude mice treated with different radionuclide probes from 0 to 48 hours; (f) changes in the tumor / muscle uptake ratio in A549 tumor-bearing nude mice treated with different radionuclide probes from 0 to 48 hours; (g) in vivo biodistribution of A549 tumor-bearing nude mice treated with the experimental radionuclide probe from 0 to 36 hours; and (h) in vivo biodistribution of A549 tumor-bearing nude mice treated with the control radionuclide probe from 0 to 36 hours. The statistical significance level is *p<0.05, **p<0.01 (n=3).

[0034] Figure 7This study investigates the in vivo and in vitro therapeutic effects of tumor-targeting covalently bound probes and corresponding radionuclide probes. The results include: (ab) cell viability in A549 and BEAS-2B cells before and after radiolabeling of the experimental and control probes; (c) live / dead staining images of A549 cells with probes from different treatment groups (scale bar = 100 μm); (d) tumor size changes in A549 tumor-bearing nude mice treated with different probe treatments; (e) weight changes in A549 tumor-bearing nude mice treated with different probe treatments; (f) survival curves of A549 tumor-bearing nude mice treated with different probe treatments; (g) morphological changes in A549 tumor-bearing nude mice treated with different probe treatments after treatment; (h) tumor morphological changes in A549 tumor-bearing nude mice treated with different probe treatments after treatment; and (i) tissue sections of A549 tumor-bearing nude mice treated with different probe treatments after the treatment process (scale bar = 100 μm). The results (μm) showed statistical significance at the levels of *p<0.05 and **p<0.01 (n=5).

[0035] Figure 8 This is a schematic diagram of the tumor-targeting covalently bound radionuclide probe of the present invention; wherein: (a) H2O2-mediated cross-linking promotes covalent binding between the radionuclide probe and the hyposulfonated protein; (b) a schematic diagram of the potential advantages of the hyposulfonated covalently bound radionuclide probe for targeted radiotherapy compared with traditional radionuclide probes. Detailed Implementation

[0036] To overcome the shortcomings of existing molecular probe technologies, such as low uptake and short retention at tumor sites, this invention provides a tumor-targeting covalently bound radionuclide probe for long-term diagnosis and treatment of NSCLC. In vitro studies have demonstrated that the designed probe can specifically bind to proteins mediated by the tumor microenvironment, thereby enabling dynamic monitoring of protein hyposulfonation. Furthermore, excitingly, this probe can also be used to assist in guiding precise tumor resection during surgery, and in vivo multimodal imaging has shown that the probe has a longer tumor retention time. Moreover, after radiolabeling with iodine-131, the probe can more effectively inhibit the growth of NSCLC tumors. In summary, the tumor-targeting covalently bound radionuclide probe developed in this invention shows great promise for long-term diagnosis and treatment of NSCLC.

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and data analysis was performed using conventional statistical methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available or obtained using existing methods. For example, compounds N3-RGD and GIR820 are existing compounds. Unless otherwise specified, in the compounds of this invention, if anion coordination is present, it is an iodide ion. The tumor-bearing female nude mice were modeled using conventional subcutaneous tumor methods, and the process complied with the animal experiment ethics requirements of Soochow University.

[0038] Thirty female BALB / c nude mice, 6-7 weeks old and weighing 17-21 g, were subjected to standard conditions (temperature 25 ± 2°C). o Mice were housed at C (relative humidity 60 ± 10%) with a 12-hour light / dark cycle. Human non-small cell lung cancer A549 tumor fragments were transplanted into the left hind limb of each female BALB / c nude mouse using xenografting technology. The subcutaneous tumors reached approximately 300 mm in size. 3 (Approximately 7-10 days) After that, subsequent animal experiments can be conducted.

[0039] Example 1: The synthesis steps of IR820-2RGD-DA are as follows Figure 1 As shown, the details are as follows: (1) Compound GIR820 (20 mg, 0.057 mmol), compound N3-RGD (36 mg, 0.057 mmol), copper sulfate pentahydrate (7.07 mg, 0.028 mmol), and sodium L-ascorbate (11.19 mg, 0.057 mmol) were simultaneously added to a round-bottom flask containing N,N-dimethylformamide / water solution (4:1, 5 mL), and reacted at room temperature under nitrogen protection for 2 hours. After the reaction was completed, pure compound IR820-2RGD (18 mg, 0.009 mmol, 31.91%) was obtained by preparative high-performance liquid chromatography. Maldi-TOF (m / z): C 102 H 126 ClN 24 O 16 The calculated value ([M]) + =1977.9467; Actual observation was 1978.0569; (2) 3,5-dioxane carboxyl (200 mg, 1.43 mmol), N-hydroxysuccinimide (NHS, 197.14 mg, 1.71 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 327.43 mg, 1.71 mmol) were simultaneously added to a round-bottom flask containing N,N-dimethylformamide solution (DMF, 30 mL). The reaction was carried out at room temperature under nitrogen protection for 12 h. After the reaction was completed, the reaction solution was purified by dichloromethane / water extraction and evaporated to dryness. Then, 3-mercaptopropylamine (351.43 mg, 1.43 mmol), N,N-dimethylformamide solution (DMF, 30 mL) and diisopropylethylamine (DIPEA, 372.55 μL, 2.14 mmol) were added. The reaction was carried out at room temperature under nitrogen protection for 4 h to obtain the crude product. Finally, the yellow oily compound SH-DA (401 mg, 1.09 mmol, 76.3%) was obtained by preparative high-performance liquid chromatography (HPLC). LC-MS (m / z): C9H 13 Calculated value of NO3S ([M]) + =215.06; Actual measured value is 215.31; (3) Compound IR820 (20 mg, 0.057 mmol), compound N3-RGD (36 mg, 0.057 mmol), copper sulfate pentahydrate (7.07 mg, 0.028 mmol), and sodium L-ascorbate (11.19 mg, 0.057 mmol) were simultaneously added to a round-bottom flask containing N,N-dimethylformamide / water solution (4:1, 5 mL). The reaction was carried out at room temperature under nitrogen protection for 2 hours. After the reaction was completed, pure compound IR820-2RGD (18 mg, 0.009 mmol, 31.91%) was obtained by preparative high-performance liquid chromatography. Maldi-TOF (m / z): C 102 H 126 ClN 24 O 16 The calculated value ([M]) + =1977.9467; Actual observation was 1978.0569; (4) Compound IR820-2RGD (10 mg, 0.005 mmol), SH-DA (4.9 mg, 0.012 mmol), N,N-dimethylformamide solution (DMF, 5 mL), and triethylamine (TEA, 2.1 μL, 0.012 mmol) adjusted to pH 8 were reacted at room temperature under nitrogen protection for 12 h to obtain the product. Finally, compound IR820-2RGD-DA (6.7 mg, 0.003 mmol, 52.0%) was obtained by preparative high-performance liquid chromatography. Maldi-TOF (m / z): C 111 H 138 N 25 O 19 S + The calculated value ([M]) + =2157.0316;; Actual measured value is 2158.1648.

[0040] Comparison Example Preparation of the control probe NOA-BY as follows Figure 2 The compound IR820-2RGD (13 mg), mercaptoethanethiol (8.47 mg, 0.021 mmol), N,N-dimethylformamide solution (DMF, 5 mL), and triethylamine (TEA, 3.68 μL, 0.021 mmol) adjusted to pH 8-9 were reacted at room temperature under nitrogen protection for 4 h to obtain the product. Finally, IR820-2RGD-COOH (10.2 mg, 0.005 mmol, 56.67%) was purified by preparative high-performance liquid chromatography. Maldi-TOF (m / z): C 105 H 131 N 24 O 18 S + The calculated value ([M+2H)) + =2049.9788; the actual measured value is 2049.6463.

[0041] Example 2 125 / 131 The radiosynthesis steps of I-IR820-2RGD-DA are as follows: Figure 1 As shown, the details are as follows: Commercially available NaI was added to a mixture of IR820-2RGD-DA (50 μg, 0.023 μmol, 1 eq) and chloramine-T (6.32 μg, 0.027 μmol, 1.2 eq). 125 / 131The radioactive probe was mixed with 37 MBq (100 μL, Xinke Biotechnology) and PBS (60 μL) at room temperature for 4 min, and then purified using a Sep-Pak C18 solid-phase extraction column, deionized water, and DMSO solution. 125 / 131 I-IR820-2RGD-DA. Prepared radioactive experimental probe. 125 / 131 I-IR820-2RGD-DA will be used for subsequent in vitro and in vivo studies.

[0042] Similarly, the radioactive labeling of the control probe was performed using the same labeling conditions.

[0043] Example 3: In vitro characterization of the probe The probe IR820-2RGD-DA (5 μM) was dissolved in a 5% DMSO aqueous solution, and then the UV absorption and fluorescence spectra were measured. The results are as follows: Figure 3 a and Figure 3 As shown in b, the maximum UV absorption and emission peaks of the probe IR820-2RGD-DA are at 820 nm and 860 nm, respectively. After sonication, the probe IR820-2RGD-DA self-assembles into nanoparticles. Furthermore, after radiolabeling, the probe exhibits good in vitro radiostability in both PBS and FBS solutions. Figure 3 c and Figure 3 d).

[0044] Example 4: In vitro covalent binding study of tumor-targeting covalently binding probe IR820-2RGD-DA The in vitro binding assay of probe IR820-2RGD-DA was verified using a small animal near-infrared II imager, such as... Figure 4 As shown in a and 4b, under the mediation of H2O2 in vitro, only the experimental probe IR820-2RGD-DA could covalently bind to albumin and hyposulfonated protein in A549 cells, while the control probe IR820-2RGD-COOH did not have the corresponding binding ability. In addition, it was also confirmed that the binding ability of probe IR820-2RGD-DA to protein hyposulfonate was positively correlated with the concentration of H2O2 mediated in vitro.

[0045] Example 5: Cellular Study of Tumor-Targeting Covalently Binding Probe IR820-2RGD-DA A549 and BEAS-2B cells were seeded in a confocal dish at a density of 3 × 10⁻⁶. 4Cells were cultured at a density of 10 μM / mL for 24 hours. After discarding the culture medium, complete culture medium containing the experimental probe IR820-2RGD-DA and the control probe IR820-2RGD-COOH (10 μM) was added to culture dishes. Cells were washed three times with PBS after incubation at different time points (0, 2, 4, 8, 12, 24, and 48 hours). Fresh culture medium was then added, and the cells were cultured further. Cellular uptake of the probes was observed using confocal laser scanning microscopy (CLSM). Subsequently, after the optimal cell uptake time of the experimental probes, 10 μM Mito-Tracker green, Lyso-Tracker green, and ER-trailer green were added to different groups of A549 cells, and the cells were incubated at 37°C for 30 min. After washing three times with PBS, fresh culture medium was added, and CLSM imaging was performed to determine the intracellular distribution of the experimental probes. The results are as follows: Figure 5 As shown in ad and 5e-g, cellular uptake and colocalization experiments of the probe verified that, compared to the shorter uptake plateau period (4-8 h) of the control probe, the experimental probe had a longer uptake plateau period (4-24 h) in non-small cell lung cancer A549 cells, and during this period, the probe was mainly endocytosed by mitochondria within tumor cells. This indicates that the probe IR820-2RGD-DA has a good retention effect in A549 cells and is mainly present in the mitochondria. Finally, the covalent binding of the probe to hyposulfonated proteins in A549 cells was investigated. After the optimal uptake time of the experimental probe, IR820-2RGD-DA and the control probe IR820-2RGD-COOH were added to A549 cells containing the same or different concentrations of H2O2, respectively. After incubation at 37°C for different times (0-48 h), the covalent binding with intracellular hyposulfonated proteins was observed using laser confocal microscopy and near-infrared II fluorescence imaging system. Figure 5 As shown in Figure hl, the results of the cell covalent binding study of the probe showed that, compared with the control probe's short intracellular retention and inability to covalently bind to intracellular hyposulfonate protein, the experimental probe could covalently bind to intracellular hyposulfonate protein at a certain concentration of H2O2 (0-50 μM), thereby prolonging the probe's intracellular retention time.

[0046] Example 6: In vivo multimodal imaging of tumor-targeting covalently bound probes and corresponding radionuclide probes. Twelve tumor-bearing nude mice were used to study the experimental probe IR820-2RGD-DA, the control probe IR820-2RGD-COOH, and the radioactive experimental probe. 131 I-IR820-2RGD-DA and radioactive control probe 131Multimodal tumor imaging of I-IR820-2RGD-COOH in nude mice with non-small cell lung cancer, such as Figure 6 As shown in a-6h, after administration of probes via the tail vein of tumor-bearing nude mice, the experimental probes and the radioactive experimental probes exhibited longer multimodal signal retention within the tumor compared to the shorter multimodal signal retention times observed in other control groups. Subsequently, as... Figure 6 As shown in Figure c, the use of an auxiliary surgical fluorescence navigation system further verified that the probe can assist in the surgical resection of tumors during surgery. This demonstrates that the probe of the present invention has a good retention effect within the tumor and can be used for complete resection of the tumor during surgery.

[0047] Example 7: Tumor-targeting covalently bound radionuclide probe 131 In vitro and in vivo antitumor studies of I-IR820-2RGD-DA Probes at different concentrations (0, 5, 10, 20, 40, 50 μM) or different radioactive doses (0, 0.037, 0.074, 0.111, 0.185, 0.37 MBq) were added to non-small cell lung cancer A549 cells and normal lung epithelial BEAS-2B cells, respectively, allowing them to adhere completely to 96-well plates and incubate at 37°C for 24 hours. After incubation, the medium was replaced with fresh medium containing MTT (10 μL of 5 mg / mL solution). After another 2 hours of incubation, the medium was removed, and 100 μL of DMSO was added to each well. The plates were shaken vigorously for 10 minutes at room temperature, and the absorbance at 490 nm was measured using a multifunctional enzyme analyzer. Figure 7 As shown in ab, the cytotoxicity test results of the probe indicate that, with the radioactive experimental probe... 131 The increased radioactive dose of I-IR820-2RGD-DA, and the radioactive experimental probe 131 I-IR820-2RGD-DA on non-small cell lung cancer A549 cells ( Figure 7 a) gradually increases in toxicity and is particularly harmful to normal lung epithelial BEAS-2B cells ( Figure 7 b) has lower cytotoxicity; in addition, such as Figure 7 As shown in Figure c, the live-dead cell experiment demonstrated the survival status of tumor cells compared to other control groups, with the low-dose radioactive probe... 131 I-IR820-2RGD-DA has a significant inhibitory effect on non-small cell lung cancer A549 cells.

[0048] This study investigated the therapeutic efficacy of different radioactive probes on tumor-bearing nude mice. Fifteen tumor-bearing nude mice were randomly divided into three groups: a radioactive experimental group, a control group, and a PBS group (n=5). The treatment period was 21 days. Figure 7As shown in Figure 1, after a 21-day treatment cycle with a single tail vein administration (3.7 MBq) in tumor-bearing nude mice, the treatment effect of the radioactive experimental probe group was significantly better than that of the other two control probe groups. Furthermore, normal tissue sections after treatment in the experimental group showed that the radioactive experimental probe exhibited high biocompatibility. This result, to some extent, demonstrates the tumor-targeting covalently bound radionuclide probe of this invention. 131 I-IR820-2RGD-DA has certain potential for clinical application. Based on all the above research results, the tumor-targeting covalently bound radionuclide probe of the present invention... 125 / 131 The covalent binding reaction between I-IR820-2RGD-DA and intracellular hyposulfonic acid protein not only enables long-window multimodal tumor imaging, but this binding reaction also promotes the inhibition of radiation-insensitive tumors by radiotherapy, thereby achieving the goal of tumor treatment. (See also...) Figure 8 (a) H2O2-mediated cross-linking promotes covalent binding between the radionuclide probe and the hyposulfonated protein. (b) Schematic diagram of the potential advantages of hyposulfonated covalently bound radioactive probes for targeted radiotherapy compared to conventional radioactive probes.

Claims

1. The application of a tumor-targeting covalently bound radionuclide probe in the preparation of tumor diagnostic and therapeutic reagents, characterized in that, The chemical structure of the tumor-targeting covalently bound radionuclide probe is as follows: ； The tumor is lung cancer.

Images