Tumor-targeted covalent binding type radionuclide probe as well as preparation method and application thereof

Abstract

The invention belongs to the field of diagnosis and treatment, and discloses a tumor targeting covalent binding type radionuclide probe as well as a preparation method and application thereof. The preparation method comprises the following steps: carrying out a reaction on a compound IR820-2RGD and SH-DA to obtain a compound IR820-2RGD-DA; the preparation method comprises the following steps: enabling a compound IR820-2RGD-DA to react with NaI125 / 131, so as to obtain the tumor targeting covalent binding type radionuclide probe. An existing internal irradiation strategy has certain limitations, such as complex operation technology, high cost, overlarge toxic and side effects on normal organs / tissues, pharmacokinetics defects, certain tolerance to NSCLC tumors and the like, a simpler, mature and long-term diagnosis and treatment method is developed, the radiosensitivity of the NSCLC tumors is improved, and the application prospect is wide. Therefore, accurate and efficient diagnosis and treatment of the NSCLC are realized.

Description

Technical Field

[0001] The present invention belongs to the field of tumor diagnosis and treatment, and specifically relates to a tumor-targeted covalently bound radioactive nuclide probe and a preparation method and application thereof. Background Art

[0002] Lung cancer is one of the most common malignant solid tumors in humans. It has the highest mortality rate among all cancers, the highest incidence in men, and the second highest incidence in women after breast cancer. Non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) are the two main histopathological types of lung cancer, of which NSCLC accounts for more than 85% of all lung cancers, and most of them present as advanced, unresectable or metastatic diseases. For advanced non-small cell lung cancer, internal irradiation is considered to be one of the important treatment strategies. At present, in order to improve the effectiveness of radioisotope therapy for NSCLC tumors that are insensitive to radiation, different treatment strategies have been developed. They are mainly divided into two categories: one is to modify NSCLC proto-oncogenes through specific gene editing technology to improve the sensitivity of NSCLC tumors to internal radiation. The other is to combine with other diagnostic and treatment strategies to reduce the insensitivity of NSCLC tumors to internal radiation and improve the diagnostic and treatment efficiency of NSCLC tumors under conditions of internal radiation. However, these two strategies have certain limitations, such as complex operation techniques, high costs, excessive toxic side effects on normal organs / tissues, defects in pharmacokinetics, and certain tolerance to NSCLC tumors. Therefore, there is an urgent need to develop a simpler, mature, and long-lasting diagnostic and therapeutic method to improve the radiosensitivity of NSCLC tumors, thereby achieving accurate and efficient diagnosis and treatment of NSCLC. Summary of the invention

[0003] The present invention discloses a long-term diagnosis and treatment strategy for lung cancer that can be covalently bound to specific proteins in the tumor microenvironment, and the application of the probe in multimodal imaging and treatment of non-small cell lung cancer tumors under this strategy. Therefore, the present invention has developed a simpler, mature, and long-term diagnosis and treatment method to improve the radiosensitivity of NSCLC tumors, thereby achieving accurate and efficient diagnosis and treatment of NSCLC.

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

[0005] A tumor-targeted covalently bound radionuclide probe, the chemical structure of which is as follows:

[0006] The above-mentioned tumor-targeted covalently bound molecular probe disclosed in the present invention can be covalently bound to proteins produced under the guidance of the tumor microenvironment, and after radioactive labeling, a tumor-targeted covalently bound radionuclide probe is formed for the diagnosis and treatment of tumors.

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

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

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

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

[0011] Furthermore, the preparation method of the above-mentioned tumor-targeting covalently bound molecular probe and tumor-targeting covalently bound radionuclide probe comprises the following steps: (1) reacting the compound GIR820 with the compound N3-RGD in the presence of an organic solvent, an inorganic copper salt, and a reducing agent to obtain the compound IR820-2RGD; the organic solvent includes N,N-dimethylformamide, the inorganic copper salt includes copper sulfate pentahydrate, and the reducing agent includes sodium ascorbate; (2) reacting the raw material 3,5-dioxocyclohexanecarboxylic acid with NHS in the presence of an organic solvent and a condensing agent, and then reacting with 3-mercaptopropylamine in the presence of an organic base and an organic solvent to obtain a 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) reacting the compound IR820-2RGD with SH-DA in the presence of an organic base and an organic solvent to obtain a compound IR820-2RGD-DA; the organic solvent includes N,N-dimethylformamide, and the organic base includes triethylamine; (4) Compound IR820-2RGD-DA and radioactive iodine compound NaI 125 / 131 React with an oxidant in an organic solvent to obtain a radioactive compound 125 / 131 I-IR820-2RGD-DA is a tumor-targeted covalently bound radionuclide probe; the organic solvents used in the above reactions are all N,N-dimethylformamide, and the oxidant is chloramine-T.

[0012] As an example, the method for preparing the above tumor-targeting covalently bound radionuclide probe comprises the following steps: (1) Compound GIR820 and compound N3-RGD are dissolved in N,N-dimethylformamide, and then a mixture of copper sulfate pentahydrate solution and sodium ascorbate solution is added, reacted at room temperature for 2 to 6 hours, and then purified to obtain compound IR820-2RGD; (2) dissolving 3,5-dioxocyclohexanecarboxylic acid in N,N-dimethylformamide, adding NHS and EDC thereto, reacting at room temperature for 10 to 15 hours, then extracting and drying by spin drying; then dissolving the spin dried product in N,N-dimethylformamide, adding 3-mercaptopropylamine and N,N-diisopropylethylamine thereto, reacting at room temperature for 2 to 5 hours; after the reaction is completed, purifying to obtain compound SH-DA; (3) Compound IR820-2RGD and compound SH-DA were dissolved in N,N-dimethylformamide, followed by the addition of triethylamine, and reacted at room temperature for 10 to 16 hours, followed by purification to obtain compound IR820-2RGD-DA; (4) Dissolve the compound IR820-2RGD-DA in NaI 125 / 131 The solution was then added with chloramine-T, reacted at room temperature for 1 to 5 minutes, and then purified to obtain the compound 125 / 131 I-IR820-2RGD-DA is a tumor-targeted covalently bound radionuclide probe.

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

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

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

[0016] In the present invention, the chemical structures of compounds N3-RGD and GIR820 are as follows:

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

[0018]

[0019] The present invention discloses the use of the above-mentioned tumor-targeted covalently bound radionuclide probe in the preparation of imaging agents, preferably, the use of the above-mentioned tumor-targeted covalently bound radionuclide probe in non-small cell lung cancer imaging agents such as tumor long window period multimodal imaging agents; or the use of the above-mentioned tumor-targeted 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 use of the above-mentioned tumor-targeted covalently bound radionuclide probe in radiotherapy of non-small cell lung cancer; or the use of the above-mentioned tumor-targeted covalently bound radionuclide probe in the preparation of radiotherapy agents for non-small cell lung cancer.

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

[0021] The invention discloses a tumor diagnosis and treatment reagent, the active component of which comprises a tumor-targeted covalently bound radioactive nuclide probe.

[0022] The method for long-window multimodal imaging using the above-mentioned tumor-targeted covalently bound radionuclide probe comprises the following steps: injecting the tumor-targeted covalently bound radionuclide probe solution into the body, such as by conventional intravenous injection, to achieve multimodal imaging at different time points.

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

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

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

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

[0027] 3. The tumor-targeted covalently bound radionuclide probe of the present invention has a significant therapeutic effect on non-small cell lung cancer tumors, realizing the integration of radioactive diagnosis and treatment of non-small cell lung cancer tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of the synthesis of tumor-targeted covalently bound radionuclide probes.

[0029] Figure 2 Schematic diagram of the synthesis of the control probe.

[0030] Figure 3 The present invention relates to the in vitro characterization of the covalently bound probes to the sulfonated proteins produced by H2O2 in the tumor microenvironment and the corresponding radionuclide probes; including: (a) the UV absorption change and fluorescence spectrum of the covalently bound probes to the sulfonated proteins produced by H2O2 in the tumor microenvironment; (b) the nano-characterization of the covalently bound probes to the sulfonated proteins produced by H2O2 in the tumor microenvironment; (cd) the radiostability of the tumor-targeted covalently bound radionuclide probes covalently bound to the sulfonated proteins produced by H2O2 in the tumor microenvironment in PBS and FBS solutions, respectively.

[0031] Figure 4 This is an in vitro binding study of a covalently bound probe that can bind to sulfonated proteins produced under the mediation of H2O2 in the tumor microenvironment; wherein: (ab) agarose gel electrophoresis and near-infrared second-zone fluorescence images of the covalently bound probe that can bind to sulfonated proteins produced under the mediation of H2O2 in the tumor microenvironment after incubation with bovine serum albumin and A549 cell protein in vitro, respectively.

[0032] Figure 5The present invention is a cell-related study on probes that covalently bind to sulfonated proteins produced under the mediation of H2O2 in the tumor microenvironment; wherein: (ad) fluorescence imaging and quantitative results of probe IR820-2RGD-DA that covalently binds to sulfonated proteins produced under the mediation of H2O2 in the tumor microenvironment and control probe IR820-2RGD-COOH (10 μM) at different time points in non-small cell lung cancer A549 cells and normal lung epithelial BEAS-2B cells; (eg) distribution, fluorescence quantification and potential of probe IR820-2RGD-DA (10 μM) that covalently binds to sulfonated proteins produced under the mediation of H2O2 in the tumor microenvironment in different organelles of non-small cell lung cancer A549 cells; (hi) fluorescence quantification and potential of probe IR820-2RGD-DA and control probe IR820-2RGD-COOH (10 μM) fluorescence imaging and quantitative results of cell retention at different time points in non-small cell lung cancer A549; (g) The experimental probe IR820-2RGD-DA and the control probe IR820-2RGD-COOH (10 μM) labeling of subsulfonic acid protein in non-small cell lung cancer A549 under the mediation of a certain concentration of H2O2 (50 μM); (kl) Fluorescence imaging and quantitative results of the experimental probe IR820-2RGD-DA and the control probe IR820-2RGD-COOH (10 μM) labeling of subsulfonic acid protein in non-small cell lung cancer A549 under the mediation of different concentrations of H2O2 (0-50 μM).

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

[0034] Figure 7The present invention is a study of in vivo and in vitro treatment of tumor-targeted covalently bound probes and corresponding radionuclide probes; wherein: (a-b) cell survival rates of experimental and control probes in A549 and BEAS-2B cells before and after radiolabeling; (c) live / dead staining images of A549 cells with probes in different treatment groups (scale bar = 100 μm); (d) changes in tumor size of nude mice bearing A549 tumors treated with probes with different treatments; (e) changes in body weight of nude mice bearing A549 tumors treated with probes with different treatments; (f) survival curves of nude mice bearing A549 tumors treated with probes with different treatments; (g) changes in appearance of nude mice bearing A549 tumors treated with probes with different treatments after treatment; (h) changes in appearance of nude mice bearing A549 tumors treated with probes with different treatments after treatment; (i) tissue sections of nude mice bearing A549 tumors treated with probes with different treatments after treatment (scale bar = 100 The statistical significance level was *p<0.05, **p<0.01 (n=5).

[0035] Figure 8 Schematic diagram of the tumor-targeting covalently bound radionuclide probe of the present invention; wherein: (a) H2O2-mediated cross-linking promotes the covalent binding between the radionuclide probe and the subsulfonated protein; (b) schematic diagram of the potential advantages of the subsulfonated covalently bound radioactive probe in targeted radiotherapy compared with traditional radioactive probes. DETAILED DESCRIPTION

[0036] In order to overcome the shortcomings of low uptake and short retention in tumor sites in existing molecular probe technologies, the present invention provides a tumor-targeted covalently bound radionuclide probe for long-term diagnosis and treatment of NSCLC. In vitro studies have shown that the designed probe can specifically bind to proteins mediated by the tumor microenvironment, thereby achieving dynamic monitoring of protein subsulfonation. In addition, it is exciting that the probe can also be used to assist in guiding the precise resection of tumors during surgery, and the in vivo multimodal imaging of the probe also proves that the probe has a longer tumor retention time. Furthermore, after radiolabeling with iodine-131, the probe can more effectively inhibit the growth of NSCLC tumors. In summary, the tumor-targeted covalently bound radionuclide probe developed by the present invention has a good application prospect in the long-term diagnosis and treatment of NSCLC.

[0037] The experimental methods used in the following examples are conventional methods unless otherwise specified, and conventional statistical methods are used for data analysis. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources or according to existing methods. For example, compounds N3-RGD and GIR820 are existing compounds. If there is an anion coordination in the compounds of the present invention, it is an iodide ion. Tumor-bearing female nude mice were modeled by conventional subcutaneous tumor methods, and they met the animal experiment ethics requirements of Soochow University.

[0038] Thirty female BALB / c nude mice, 6-7 weeks old, weighing 17-21 g, were cultured under standard conditions (temperature 25 ± 2 o C, relative humidity 60 ± 10%), light / dark cycle 12 h. Human non-small cell lung cancer A549 tumor was transplanted into the left lower limb of each female BALB / c nude mouse by tumor xenograft technique. When the subcutaneous tumor size was about 300 mm 3 (about 7 to 10 days), subsequent related animal experiments can be carried out.

[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 added to a round-bottom flask containing N,N-dimethylformamide / water solution (4:1, 5 mL) and reacted under nitrogen protection at room temperature 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 C1N 24 O 16 The calculated value ([M] + ) = 1977.9467; the observed value is 1978.0569; (2) 3,5-Dioxadimethoxycyclohexanecarboxylic acid (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 added simultaneously to a round-bottom flask containing N,N-dimethylformamide solution (DMF, 30 mL) and reacted at room temperature under nitrogen protection for 12 h. After the reaction was completed, the reaction solution was purified by dichloromethane / water mixed solution extraction method and spin-dried, and 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 continued at room temperature under nitrogen protection for 4 h to obtain a crude product. Finally, a yellow oily compound SH-DA (401 mg, 1.09 mmol, 76.3 %) was obtained by preparative high performance liquid chromatography. LC-MS (m / z): C9H 13 Calculated value of NO3S ([M] + ) = 215.06; the actual observation 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 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 C1N 24 O 16 The calculated value ([M] + ) = 1977.9467; the observed value is 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, preparative HPLC was used to obtain compound IR820-2RGD-DA (6.7 mg, 0.003 mmol, 52.0 %). Maldi-TOF (m / z): C 111 H 138 N 25 O 19 S + The calculated value ([M] + )=2157.0316; The actual observation is 2158.1648.

[0040] Comparison example The control probe NOTA-BY was prepared as Figure 2 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 HPLC. Maldi-TOF (m / z): C 105 H 131 N 24 O 18 S + Calculated value ([M+2H] + )=2049.9788; the actual observation 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: Commercial NaI was added to the 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 / 131solution (37 MBq, 100 μL, Xinke Biotechnology) and PBS solution (60 μL) were stirred at room temperature for 4 min, and then the radioactive probe was purified using 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 was used for subsequent in vitro and in vivo studies.

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

[0043] Example 3 In vitro characterization of probes The probe IR820-2RGD-DA (5 μM) was dissolved in 5% DMSO aqueous solution, and then the UV absorption spectrum and fluorescence spectrum were measured. Figure 3 a and Figure 3 As shown in b, the maximum UV absorption peak and emission peak of the probe IR820-2RGD-DA are at 820 nm and 860 nm, respectively. After ultrasound, the probe IR820-2RGD-DA will self-assemble into nanoparticles. At the same time, after the probe is radiolabeled, it has 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-targeted covalently bound probe IR820-2RGD-DA The in vitro binding experiment of the probe IR820-2RGD-DA was verified by a small animal near-infrared zone II imager. Figure 4 As shown in Figures 4a and 4b, under the mediation of H2O2 in vitro, only the experimental probe IR820-2RGD-DA can covalently bind to albumin in vitro and sulfonylated proteins in A549 cells, while the control probe IR820-2RGD-COOH does not have the corresponding binding ability; in addition, it is also confirmed that the binding ability of the probe IR820-2RGD-DA to protein sulfonic acid is positively correlated with the in vitro mediated H2O2 concentration.

[0045] Example 5 Related cell studies of tumor-targeted covalently bound probe IR820-2RGD-DA A549 and BEAS-2B cells were plated in confocal dishes at 3×10 4The cells were cultured at a density of 100 cells / 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 the culture dish. After incubation at different time points (0, 2, 4, 8, 12, 24, and 48 hours), the cells were washed three times with PBS. Fresh culture medium was then added and culture continued. Confocal laser scanning microscopy (CLSM) was used to observe the cellular uptake of the probes. Afterwards, after the optimal cellular 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 incubated at 37°C for 30 min. After washing three times with PBS, new culture medium was added and CLSM imaging was performed to determine the distribution of the experimental probes in the cells. The research results are shown in the following figure. Figure 5 As shown in ad and 5e-g, the cell uptake experiment and cell co-localization experiment of the probe verified that compared with the shorter uptake platform period (4-8 h) of the control probe, the experimental probe was taken up by non-small cell lung cancer A549 cells after a longer platform period (4-24 h), and during this period, the experimental probe was mainly endocytosed by the mitochondria in the tumor cells. This shows that the probe IR820-2RGD-DA has a good retention effect on A549 cells and is mainly present in the mitochondria of the cells. Finally, the covalent binding of the probe to the subsulfonated protein in A549 cells was studied. After the optimal cell 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, and then incubated at 37°C for different times (0-48 h). The covalent binding with the intracellular subsulfonated protein was observed using a laser confocal microscope and a near-infrared second-zone fluorescence imaging system. As shown Figure 5 As shown in hl, the results of the probe's cell covalent binding study showed that compared with the control probe's short intracellular retention and inability to covalently bind to the intracellular sulfenic acid protein, the experimental probe can covalently bind to the intracellular sulfenic acid protein under 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-targeted 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 131I-IR820-2RGD-COOH in multimodal imaging of non-small cell lung cancer tumor-bearing nude mice, such as Figure 6 As shown in a-6h, after the probes of different treatment groups were administered through the tail vein of tumor-bearing nude mice, the experimental probes and radioactive experimental probes had longer multimodal signal retention results in the tumor compared with the shorter multimodal signal retention results of the probes in other control groups; afterwards, as Figure 6 As shown in Figure c, the assisted surgery fluorescence navigation system was used to verify that the probe can assist in the surgical resection of the tumor during surgery. This shows that the probe of the present invention has a good retention effect in the tumor and can be used for complete resection of the tumor during surgery.

[0047] Example 7 Tumor-targeted covalently bound radionuclide probe 131 Antitumor study of I-IR820-2RGD-DA in vivo and in vitro Probes of 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, to allow them to completely adhere to the 96-well plate and incubate at 37°C for 24 hours. After incubation, the culture medium was replaced with fresh culture medium containing MTT (10 μL of 5 mg / mL solution). After another 2 hours of incubation, the culture medium was removed and 100 μL of DMSO was added to each well. The plate was shaken vigorously at room temperature for 10 minutes, and the absorbance at 490 nm was measured using a multifunctional enzyme meter. Figure 7 As shown in ab, the cytotoxicity test results of the probe show that with the radioactive experimental probe 131 Increasing radioactive doses of I-IR820-2RGD-DA, a radioactive experimental probe 131 I-IR820-2RGD-DA affects non-small cell lung cancer A549 cells ( Figure 7 a) gradually increased in toxicity and had an adverse effect on normal lung epithelial BEAS-2B cells ( Figure 7 b) has less cytotoxicity; in addition, Figure 7 c shows that the live-death cell experiment proved that compared with the survival status of tumor cells in other control groups, the low-dose radioactive experimental probe 131 I-IR820-2RGD-DA has a significant inhibitory effect on non-small cell lung cancer A549 cells.

[0048] The therapeutic efficacy of different radioactive probes on tumor-bearing nude mice was studied. 15 tumor-bearing nude mice were divided into three groups: radioactive experimental group, control group and PBS group (n=5). The treatment cycle was 21 days. Figure 7As shown in Figure ci, after a single tail vein administration (3.7 MBq) of tumor-bearing nude mice for a 21-day treatment cycle, the therapeutic effect of the radioactive experimental probe group was significantly better than that of the other two control probe groups, and the normal tissue sections after the experimental group probe treatment showed that the radioactive experimental probe had high biosafety. This result to a certain extent shows that the tumor-targeted covalently bound radionuclide probe of the present invention 131 I-IR820-2RGD-DA has certain clinical application potential Based on all the above research results, the tumor-targeted covalently bound radionuclide probe of the present invention 125 / 131 The covalent binding reaction between I-IR820-2RGD-DA and intracellular sulfenic acid protein not only realizes long-window multimodal tumor imaging, but also promotes the inhibition of radiation-insensitive tumors by radiotherapy, thereby achieving the purpose of tumor treatment. Figure 8 , (a) H2O2-mediated cross-linking promotes the covalent binding between radionuclide probes and sulfonylated proteins, (b) Schematic diagram of the potential advantages of sulfonylated covalently bound radioactive probes for targeted radiotherapy compared to traditional radioactive probes.

Claims

1. A tumor-targeted covalently bound radionuclide probe, the chemical structure of which is as follows: 。 2. The method for preparing the tumor-targeting covalently bound radionuclide probe according to claim 1, comprising the following steps: The compound IR820-2RGD is reacted with SH-DA to obtain the compound IR820-2RGD-DA; The compound IR820-2RGD-DA is reacted with a radioactive iodine compound to obtain a tumor-targeted covalently bound radionuclide probe.

3. The method for preparing the tumor-targeting covalently bound radionuclide probe according to claim 2, characterized in that: The compound 3-mercaptopropylamine is reacted with 3,5-dioxacyclohexanecarboxylic acid to obtain the compound SH-DA; the compound GIR820 is reacted with N3-RGD to obtain the compound IR820-2RGD.

4. The method for preparing the tumor-targeting covalently bound radionuclide probe according to claim 2, characterized in that: The compound IR820-2RGD-DA is reacted with a radioactive iodine compound in the presence of an organic solvent and an oxidant to prepare a tumor-targeted covalently bound radionuclide probe.

5. Use of the tumor-targeting covalently bound radionuclide probe according to claim 1 in the preparation of imaging agents.

6. Use of the tumor-targeting covalently bound radionuclide probe according to claim 1 in the preparation of drugs.

7. Use of the tumor-targeted covalently bound radionuclide probe according to claim 1 in the preparation of a reagent for detecting the level of sulfenic acid in tumor tissue.

8. Use of the tumor-targeting covalently bound radionuclide probe according to claim 1 in the preparation of diagnostic and therapeutic reagents.

9. The use according to claim 8, characterized in that: Diagnostic and therapeutic agents include tumor diagnostic and therapeutic agents.

10. A diagnostic and / or therapeutic agent, the active ingredient of which comprises the tumor-targeting covalently bound radionuclide probe according to claim 1.

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