Polypeptide probe for targeted detection of drainage lymph node of metastatic esophageal tumor as well as preparation method and application of polypeptide probe

Abstract

The invention provides a polypeptide probe for targeted detection of metastatic esophageal tumor drainage lymph nodes and a preparation method and application of the polypeptide probe, and belongs to the technical field of biomedicine, and the constructed bimodal polypeptide probe for targeted tumor metastatic lymph nodes comprises NOTA polypeptide, near-infrared two-region fluorescence signal molecules ICG and 18F-FDG. Compared with the prior art, the bimodal polypeptide fluorescent probe prepared by the invention can be enriched in tumor tissues, can efficiently target a target spot of FGF2 in a positive lymph node, prompts an abnormal metabolism increasing area in a tumor by combining PET / CT, has no obvious biotoxicity, and can be used for detecting the tumor metastatic lymph node through bimodal NIR-II / PET / CT imaging. And accurate early diagnosis and accurate navigation in an operation are realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a dual-modal polypeptide probe for targeted detection of metastatic esophageal tumor draining lymph nodes, a preparation method thereof, and an application thereof. Background Art

[0002] Esophageal cancer is a malignant tumor with extremely high incidence and mortality rates in China and the world, seriously affecting the lives and health of the people. Surgical operation is still the only possible curative treatment method for early-stage esophageal cancer patients. In esophageal cancer surgery, lymph node tracing (or lymph node navigation) is a key technology. The lymph node metastasis pathway of esophageal cancer is relatively complex, and it can extend up and down the esophagus to the neck, mediastinum, and abdomen. The lymph node metastasis rate is relatively high and the range is wide. Even after radical resection, 30%-55% of esophageal cancer patients still relapse and die. Because esophageal cancer surgery involves a wide range of areas and requires incisions in multiple anatomical regions (such as the neck, chest, and abdomen), it has high technical requirements, a long operation time, and a relatively high risk of complications. Moreover, the traditional three-field lymph node dissection method may miss some involved lymph nodes or excise too many normal lymph nodes, increasing the risk of postoperative complications. Positive lymph node tracing can help surgeons more accurately identify and excise involved lymph nodes, thereby improving the thoroughness of the operation. Thoroughly cleaning the lymph nodes helps reduce the risk of postoperative recurrence and improve the long-term survival rate of patients. At the same time, lymph node metastasis is an important indicator in the staging of esophageal cancer. Through lymph node tracing, the stage of the tumor can be more accurately determined, and the spread of the tumor can be evaluated. This helps to develop a more precise treatment plan and predict the prognosis of patients.

[0003] Lymph node metastasis of malignant tumors has always been a focus in tumor research. At present, there are certain challenges in differentiating benign and malignant lymph nodes based on morphological features. Although some features can provide clues, no single morphological feature can completely and reliably distinguish between benign and malignant lymph nodes. Therefore, it is usually necessary to combine multiple diagnostic methods to improve accuracy. In recent years, lymph node tracer technology has developed to a certain extent. The main guiding methods are divided into image guidance and tracer guidance. Image guidance mainly uses PET / CT as the main technology. Preoperative CT or PET / CT scans can help plan lymph node dissection strategies and be guided by real-time imaging devices during surgery. In particular, PET / CT can provide functional information and help detect tiny metastatic foci. This method is non-invasive and provides immediate feedback, but it requires technical experience. However, the current mainstream PET imaging agent, 18F-FDG, has low specificity for normal lymph nodes and is easily confused with inflammatory hyperplasia with the same increased metabolism, resulting in false positive results. Generally speaking, due to tumor heterogeneity and the limitations of the technology itself, non-invasive imaging techniques may have insufficient sensitivity and specificity, may produce false negative and false positive results, and have limitations in spatial resolution, making it impossible to evaluate the lesions of tiny lymph nodes in detail. Early tracers used dyes, but they had the defect of insufficient visibility. There are also radioactive tracers. Although the visibility has been improved, the radioactivity they contain needs to be treated with caution for both doctors and patients. In recent years, fluorescent tracers have been introduced. When combined with corresponding professional imaging devices, they can perform lymph node imaging safely and efficiently, and are easy to operate and can be used with endoscopes. Among them, the representative ones are Cyanine 7 (Cy7) and Indocyanine Green (ICG). Cy7 is a commonly used near-infrared fluorescent dye. The excitation wavelength is usually between 740 - 760 nm, and the emission wavelength is usually between 760 - 780 nm. Cy7 has a relatively high fluorescence quantum yield, which can reach about 0.28 in some solvents. ICG has a maximum absorption peak of about 800 nm in aqueous solution, and the emission wavelength is about 830 nm. Its excitation and emission characteristics in the near-infrared region make it suitable for deep tissue imaging. Cy7 and ICG are mainly metabolized by the liver and excreted through bile, and have relatively good biosafety. They are generally soluble in organic solvents (such as DMSO, ethanol) and certain buffers (such as PBS), and the excitation and emission wavelengths are in the near-infrared region, with low background noise and high tissue penetration depth, which are very suitable for fluorescence imaging of live animals. For example, they can be used in angiogenesis imaging research to help understand angiogenesis and hemodynamics; they can be conjugated with biological macromolecules (such as proteins, antibodies, nucleic acids, etc.) to detect and locate tumors in the body. Therefore, there is an urgent need to explore a more perfect lymph node tracer method to bring further benefits to the individualized treatment of esophageal cancer patients.

[0004] In recent years, peptide drugs have received extensive attention in the fields of oncology, metabolic diseases, and infectious diseases due to their good biocompatibility, high specificity, low toxicity, and easy modification characteristics. Peptide drugs can bind to target proteins, receptors, or enzymes with high selectivity. Compared with traditional small molecule drugs, peptide drugs usually have higher targeting and lower side effects, and some structures with short in vivo half-lives are more suitable as diagnostic reagents for imaging. Among them, polypeptide tumor imaging probes have become an important direction in the field of tumor diagnosis. By binding peptide sequences with fluorescent dyes or radioactive markers, researchers have been able to develop highly sensitive probes to achieve rapid identification of specific tumor markers. Common modification methods include connecting with near-infrared fluorescent dyes such as ICG, enabling the probe to penetrate deep into tissues and reduce background fluorescence interference. Peptide-ICG probes have shown good imaging effects in preclinical studies and can be used for intraoperative navigation and molecular imaging diagnosis to improve the recognition accuracy of tumor tissues. Such probes are expected to be combined with immune checkpoint inhibitors or other targeted therapies to help doctors more effectively determine the location and metastasis of tumors and improve the thoroughness of surgical resection. In addition, with the progress of polypeptide synthesis technology and the upgrading of imaging equipment, the development of peptide drugs and imaging probes is gradually entering the clinical stage, providing new solutions for tumor diagnosis and treatment. Summary of the Invention

[0005] In view of the above problems, the purpose of the present invention is to provide a dual-modal polypeptide probe for targeted detection of metastatic esophageal tumor draining lymph nodes, its preparation method, and application. The present invention uses a polypeptide to connect a near-infrared region fluorescent signal molecule ICG and constructs a dual-modal polypeptide fluorescent probe by connecting 18F-FDG through a NOTA polypeptide. It can be enriched in tumor tissues, can efficiently target the FGFR2 target in positive lymph nodes, and combined with PET / CT to indicate regions with abnormally increased metabolism in tumors. This probe can not only be used as a diagnostic agent for tumor imaging but also as a diagnostic agent for detecting tumor lymph node metastasis.

[0006] The specific solutions adopted by the present invention are as follows:

[0007] In the first aspect, the present invention provides a polypeptide probe for targeted detection of metastatic esophageal tumor draining lymph nodes, including: a NOTA polypeptide; the structural formula of the NOTA polypeptide is NOTA-CLQLQAEERC.

[0008] As a further optimization of the above polypeptide probe, the polypeptide probe further includes: ICG connected by a polypeptide, and its structural formula is ICG-NHS-CLQLQAEERC. Further, the polypeptide probe further includes: a imaging agent. The imaging agent is 18 F connected through a NOTA polypeptide.

[0009] Second aspect, the present invention provides a method for preparing the above-mentioned polypeptide probe, comprising the following steps: (1) First, synthesize a single-chain polypeptide, form a disulfide bond through air oxidation, and then obtain NOTA-polypeptide through purification treatment; (2) Dissolve the product obtained in step (1) in DMF, add ICG-NHS active ester, and react to form a polypeptide-ICG composite structure; (3) Use 18 F to synthesize 18 F-SFB, and incubate with the product obtained in step (2) in PBS at pH 8.5 for a conjugation reaction; (4) After separation, purification, analysis and identification, obtain 18 F-ICG-NOTA-CLQLQAEERC polypeptide probe.

[0010] Third aspect, the present invention provides the application of the above-mentioned polypeptide probe in the preparation of drugs for treating esophageal cancer or diagnostic reagents for esophageal cancer.

[0011] Fourth aspect, the present invention provides the application of the above-mentioned polypeptide probe in the preparation of diagnostic reagents for differentiating tumor-metastatic lymph nodes from inflammatory hyperplastic lymph nodes.

[0012] Fifth aspect, the present invention provides the application of the above-mentioned polypeptide probe in the preparation of tumor imaging diagnostic agents or diagnostic agents for detecting tumor lymphatic metastasis.

[0013] Beneficial effects: The present invention constructs a novel fluorescence / PET probe, verifies the binding ability of the probe using the patient's tumor sample in vitro, constructs immune-deficient mouse esophageal cancer CDX and PDX tumor lymphatic metastasis models, uses the fluorescence / PET probe to monitor the occurrence, development and metastasis of tumors in mice, and uses two near-infrared fluorescence probes to perform imaging at different time points to dynamically monitor the occurrence, development and metastasis process of tumors, and understand the growth rate and metastasis pattern of tumors. The combination of fluorescent molecules, polypeptides and 18F can make up for the deficiencies of a single imaging mode and improve the accuracy and reliability of detection. Description of the Drawings

[0014] Figure 1 For 18 Synthesis route diagram of

[0015] Figure 2 For 18 Mass spectrometry detection diagram and HPLC detection result diagram of

[0016] Figure 3 For 18 Cytotoxicity test results of

[0017] Figure 4 For 18 Cell uptake experiment and in vitro targeting experiment results of F-ICG-CLQLQAEERC.

[0018] Figure 5 For 18 In vivo distribution and pharmacokinetic experiment results of F-ICG-CLQLQAEERC.

[0019] Figure 6 For 18 Subcutaneous tumor PET / CT imaging of F-ICG-CLQLQAEERC.

[0020] Figure 7 For 18 PET / CT imaging of lymph node metastases of F-ICG-CLQLQAEERC.

[0021] Figure 8 For 18 Near-infrared second-region imaging of lymph node metastases of F-ICG-CLQLQAEERC. Detailed implementation manners

[0022] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments and drawings of the present invention. In the following embodiments, unless otherwise specified, the reagents used are all commercially available, and the methods used are all conventional technical means.

[0023] The present invention synthesizes single-chain polypeptides by the following steps:

[0024] 1. Treatment of solvents: DMF and methanol are soaked in molecular sieves with G3 pores overnight before use to remove impurities and water.

[0025] 2. Sufficient swelling of the resin: Weigh 2.0 g of blank Wang resin into a clean and dry reaction tube, add 15 mL of DMF, and activate at room temperature for about 30 min.

[0026] 3. Attachment of the first amino acid: At room temperature, filter off the solvent from the previous step through a sintered glass funnel, add 1 mmol of the first amino acid at the C-terminus in 5-fold molar excess, 5-fold molar excess of DMAP, 5-fold molar excess of DIC, and use DMF as the solvent to react at room temperature for 3 h. After the reaction is completed, wash with DMF 4 - 6 times, 5 - 6 mL each time. Then add an appropriate amount of pyridine and acetic anhydride with a volume ratio of 1:1 and react for 30 min. After the reaction is completed, wash with DMF 4 - 6 times, 5 - 6 mL each time. (Function: Block the active sites on the unreacted empty resin.)

[0027] 4. Removal of the Fmoc protecting group: Filter off the solvent from the previous step by suction filtration. Add 10 mL of 20% piperidine in DMF solution to the resin. Stir with N₂ for 10 min, then filter out the solution. Add another 10 mL of 20% piperidine in DMF solution, blow and stir with N₂ for 5 min and then filter off the solution. Repeat this operation twice. Wash with DMF 4 times and with methanol 2 times, 5 - 6 mL each time.

[0028] 5. Ninhydrin test for the removal effect: Take out a small amount of resin, wash it three times with methanol. Add one drop each of ninhydrin, KCN, and phenol solution, and heat at 105 °C - 110 °C for 5 min. A dark blue color indicates a positive reaction, meaning the removal is complete and the next step of the reaction can be carried out; if it is colorless, it means the protecting group has not been completely removed, and the above deprotection operation needs to be repeated.

[0029] 6. Coupling of the second amino acid and removal of the Fmoc protecting group: Weigh 3 - fold molar excess of the second C - terminal amino acid, 3 - fold molar excess of HBTU, and 3 - fold molar excess of HOBT into a reaction tube. After adding an appropriate amount of DMF solution to completely dissolve them, add 10 - fold molar excess of (pure) DIEA. React at room temperature for 40 min, wash with DMF 4 - 6 times, 5 - 6 mL each time. Take a small amount of resin and test it with ninhydrin detection reagent. If it is colorless, then add 10 mL of 20% piperidine in DMF solution to remove Fmoc, do this twice, for 10 min and 5 min respectively. Then wash with DMF 4 times and with methanol 2 times, 5 - 6 mL each time. Take a small amount of resin and test it with ninhydrin detection reagent. If the test shows blue, the next step of the reaction can be carried out.

[0030] 7. And so on, repeat the steps in 2.6 until the synthesis reaches the last N - terminal amino acid, remove the Fmoc protecting group, and then dry it by suction.

[0031] 8. Cleavage of the resin and separation and detection of the pure product: Finally, cut with trifluoroacetic acid cleavage solution (95% TFA: 2% TIS: 2% EDT: 1% H 2 O) for 2 h. Filter the reaction solution by suction to obtain a trifluoroacetic acid solution of the polypeptide. Try to blow dry the cleavage solution with nitrogen, then precipitate with ether, centrifuge, and then wash with ether 3 - 5 times to obtain a white solid. Dissolve it in pure water, desalt and purify it by HPLC, and freeze - dry to precipitate crystals. Take a small amount for MS analysis.

[0032] Example 1 Synthesis 18 F - ICG - NOTA - CLQLQAEERC and carry out corresponding quality inspections

[0033] First, a single-chain polypeptide was synthesized and obtained, which was oxidized by the air oxidation method to form disulfide bonds in the polypeptide chain, thereby obtaining a polypeptide containing disulfide bonds. This polypeptide was purified to obtain the target product NOTA-KcLQLQAEERc (where "c" represents the disulfide bond structure formed by cysteine). Then, the purified polypeptide was dissolved in a dimethylformamide (DMF) solution to ensure good solubility of the polypeptide. Subsequently, ICG-NHS active ester was added to the solution to react with the amino group in the polypeptide, forming a polypeptide-ICG linked composite structure. This reaction was carried out at room temperature to ensure full utilization of the activity of ICG-NHS. Then, we synthesized 18F-SFB (N-succinimidyl-4-18Ffluorobenzoate) by 18F labeling. This molecule contains an active ester group and can react with the free amino group on the NOTA-polypeptide to form a stable amide bond. Incubation was carried out in PBS at pH 8.5 to promote the ligation reaction. The synthetic route is as Figure 1 shown. After the reaction was completed, the product was separated and purified by high performance liquid chromatography (HPLC), and the purified target polypeptide probe product was identified by mass spectrometry (Mass) analysis, thereby obtaining 18 the 18F-ICG-NOTA-CLQLQAEERC polypeptide probe.

[0034] Example 2 Mass spectrometry detection and HPLC detection of purity

[0035] Mass spectrometry detection: An appropriate amount of the HPLC purified product was taken, dissolved in an appropriate amount of 50:50 (V / V) water-acetonitrile solution, and 0.1% formic acid was added to facilitate ionization. A liquid chromatography-mass spectrometry (LC-MS) system was used, the electrospray ionization source (ESI) was selected and set to the positive ion mode. The spray voltage was adjusted to +4.0 kV and the capillary temperature was 320 °C. Data was collected within the target molecular weight range, and the molecular ion peak on the mass spectrometry graph was analyzed. The theoretical mass of the target molecule was compared with the actually detected mass to ensure that the molecular weights were consistent, and the 18 purity and structure of 18F-ICG-NOTA-CLQLQAEERC were confirmed.

[0036] HPLC detection of purity: An appropriate amount of the polypeptide-ICG probe product was taken, dissolved in an appropriate solvent (such as water or 0.1% trifluoroacetic acid (TFA) aqueous solution), and passed through a 0.22 μm filter to remove insoluble substances. A reverse phase high performance liquid chromatography (RP-HPLC) system was used, equipped with a C18 column (such as 5 μm, 4.6 × 250 mm). The column temperature was set at 30 °C and the flow rate was 1.0 mL / min. Gradient elution was used, solvent A was 0.1% TFA aqueous solution, and solvent B was 0.1% TFA acetonitrile solution. Starting from 5% B, it was linearly increased to 95% B within 30 minutes to ensure18 Good separation of F-ICG-NOTA-CLQLQAEERC during the entire elution process. Set the wavelength at 780 - 800 nm to detect the characteristic peak of the ICG fluorescently labeled part, and at the same time, the detection at 220 nm can be combined to monitor the polypeptide part. Collect the chromatogram of the sample and analyze the peak area of the target peak. The purity calculation method is the ratio of the target peak area to the total area to determine 18 the purity of F-ICG-NOTA-CLQLQAEERC.

[0037] The results of mass spectrometry detection and HPLC detection are as Figure 2 shown. Mass spectrometry analysis and HPLC analysis show that the main component in the sample is 18 F-ICG-CLQLQAEERC, accounting for 96.0359% of the total components, and there is no other organic matter contamination in the sample.

[0038] Example 3 Cytotoxicity Experiment

[0039] Treat esophageal cancer cells KYSE140 with different concentrations of 18 F-ICG-NOTA-CLQLQAEERC for 24 hours, and use the CCK-8 reagent to detect cell viability. Set up three replicate wells for each group and measure the absorbance value at a wavelength of 450 nm. The results are as Figure 3 shown. According to the cell survival rates of the probe and ICG with and without laser irradiation respectively, the cell survival rate of the probe + laser group decreased to less than 50% at a concentration of 2 mM, and the cell survival rate of the ICG + laser group decreased to less than 50% at a concentration of 10 mM, indicating that the probe and ICG have no significant effect on cell activity within the detection concentration range, suggesting that the probe has good biocompatibility and no significant enhancement of the cell killing effect by laser irradiation.

[0040] Example 4 Cell Uptake Efficiency Experiment and In Vitro Targeting Experiment

[0041] Add different concentrations of 18 F-ICG-NOTA-CLQLQAEERC to three culture systems: HUVEC cells, KYSE-140 cells, and co-culture of HUVEC cells and KYSE140 cells. After incubation for a certain time, collect the cells and wash them with PBS, and then detect the intracellular fluorescence intensity with a flow cytometer. The results are as Figure 4 shown. Fluorescence can be seen in both HUVEC cells and KYSE-140 cells, indicating that the cells have a certain uptake ability for 18 F-ICG-NOTA-CLQLQAEERC; the fluorescence intensity of HUVEC cells is weak, the intracellular fluorescence intensity of KYSE cells is good, and the fluorescence intensity of KYSE-140 cells in the co-culture system is higher, indicating 18F-ICG-NOTA-CLQLQAEERC has good targeting ability to KYSE-140 cells.

[0042] Example 5 In Vivo Distribution and Pharmacokinetics Experiments

[0043] Dissolve 18 F-ICG-NOTA-CLQLQAEERC was administered to tumor-bearing mice in the brain via tail vein injection. Main organs and blood samples were collected at different time points after administration, and fluorescence imaging was used to analyze the tissue distribution and metabolism of the probe. The results are as Figure 5 shown. After injecting 18 F-ICG-NOTA-CLQLQAEERC, the fluorescence intensity of the whole body and each important organ of the mice in the imaging decreased with time, indicating that the probe was gradually cleared from the blood and various organ tissues and was basically completely cleared at 12 hours. There were no significant differences in the indicators of liver and kidney functions, showing its good tumor targeting and pharmacokinetic characteristics.

[0044] Example 6 In Vivo Subcutaneous Esophageal Cancer Tumor Targeting Experiment

[0045] KYSE140 cells were cultured and amplified. Approximately 10 6 KYSE140 cells were added to PBS and blown evenly to form a cell suspension. After mixing with Matrigel at a ratio of 1:1, the mixture was inoculated subcutaneously on the back of 6-week-old nude mice to construct a CDX model. When the tumor volume reached a certain size, 18 F-ICG-NOTA-CLQLQAEERC was injected via tail vein. Imaging was performed using a small animal imager and PET / CT at 0 min, 30 min, and 60 min. The results are as Figure 6 shown. In the PET / CT images, except for normal hypermetabolic sites, the tumor site showed stronger signals compared with the healthy side, and the signals of other sites were weaker, verifying the targeting of the probe to subcutaneous tumors and its good PET / CT imaging efficacy.

[0046] Example 7 In Vivo Orthotopic Tumor Targeting Experiment

[0047] KYSE140 cells were cultured and amplified. Approximately 10 6 KYSE140 cells were added to PBS and blown evenly to form a cell suspension. After mixing with Matrigel at a ratio of 1:1, the mixture was inoculated subcutaneously on the back of 6-week-old nude mice to construct a CDX model. When the tumor volume reached a certain size, approximately 10 5 KYSE140 cells were added to PBS and blown evenly to form a cell suspension, which was then injected into the lower limb footpad on the tumor side to form lymph node metastases. One week later, 18After using F-ICG-NOTA-CLQLQAEERC, imaging was performed using PET / CT at 0 min, 30 min, and 60 min, and the PET signal intensity at the tumor site was recorded. After completion, the popliteal fossa, caudal root, and inguinal lymph nodes were obtained by dissection, paraffin-embedded, and then subjected to HE staining. If tumor cells were confirmed in the lymph node metastasis foci and corresponded to the imaging results, it indicated that the probe could effectively target esophageal cancer lymph node metastasis foci. The results were as Figure 7 shown. In the PET / CT images, except for normal hypermetabolic sites, high-intensity signals appeared in the tumor and metastatic lymph node regions compared to the healthy side, and the signals in other parts were weak, indicating that the probe could effectively target tumors and lymphatic metastases and had good PET / CT imaging performance.

[0048] Example 8 In Vivo Lymph Node Metastasis Targeting Experiment

[0049] KYSE140 cells were cultured and amplified, and approximately 10 5 KYSE140 cells were inoculated by injection into the footpads of the lower limbs of 6-week-old nude mice to form lymph node metastases. One week later, 18 the F-ICG-NOTA-CLQLQAEERC probe was injected into one footpad, and ICG was injected into the other footpad. Imaging was performed using a NIR-II small animal imager from 0 min to 1 min to 5 min. After completion, the popliteal fossa, caudal root, and inguinal lymph nodes were obtained by dissection, paraffin-embedded, and then subjected to HE staining. If tumor cells were confirmed in the lymph node metastasis foci and corresponded to the imaging results, it indicated that the NIR-II fluorescence probe could effectively target esophageal cancer lymph node metastasis foci. The results were as Figure 8 shown. After injecting the probe, strong fluorescence signals were visible in the footpads of the mice, and the fluorescence signal intensities of the lymph nodes in both lower limbs increased with time. After 5 minutes, significant fluorescence signals were monitored in the popliteal lymph nodes and caudal root lymph nodes, indicating that the probe could effectively target lymphatic metastases and had good NIR-II fluorescence imaging performance.

[0050] A mouse model with KYSE140 cell metastases in the popliteal lymph nodes, caudal root lymph nodes, or inguinal lymph nodes was constructed by injecting through the footpads. After intravenous injection of 18 F-ICG-NOTA-CLQLQAEERC, real-time imaging was performed to observe and record the fluorescence signals at the lymph node sites. Strong fluorescence was shown in the metastatic lymph nodes, verifying the targeting ability of the probe, especially its recognition ability in the metastatic foci.

[0051] In summary, the dual-modal probe of the present invention can regulate tumor cells in the tumor microenvironment through NIR-II / PET / CT imaging, detect the tumor microenvironment, achieve precise early diagnosis, and can treat postoperative recurrence models. At the same time, the dual-modal probe meets the requirements of multi-modal imaging, and has high targeting and specificity for tumors and positive lymph nodes.

[0052] The above-described embodiments are only preferred embodiments given to fully illustrate the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. A polypeptide probe for targeted detection of lymph nodes draining metastatic esophageal tumors, characterized in that: include: NOTA polypeptide; The structural formula of the NOTA polypeptide is NOTA-CLQLQAEERC.

2. The polypeptide probe according to claim 1, characterized in that Also includes: The structural formula of ICG linked to the polypeptide is ICG-NHS-CLQLQAEERC.

3. The polypeptide probe according to claim 2, characterized in that Also includes: Imaging agent.

4. The polypeptide probe according to claim 3, characterized in that The imaging agent is 18F linked via a NOTA polypeptide.

5. The method for preparing a polypeptide probe according to claim 2, characterized in that: The following steps are involved: (1) First, a single-chain polypeptide is synthesized, and disulfide bonds are formed by air oxidation, followed by purification to obtain NOTA-peptide; (2) dissolving the product obtained in step (1) in DMF, adding ICG-NHS active ester, and reacting to form a polypeptide-ICG composite structure; (3) Synthesis using 18F labeling 18 F-SFB, incubated with the product obtained in step (2) in PBS at pH 8.5 for ligation reaction; (4) After separation, purification, analysis and identification, 18 F-ICG-NOTA-CLQLQAEERC peptide probe.

6. Use of the polypeptide probe according to any one of claims 1 to 4 in the preparation of a drug for treating esophageal cancer or a reagent for diagnosing esophageal cancer.

7. Use of the polypeptide probe according to any one of claims 1 to 4 in the preparation of a diagnostic reagent for distinguishing lymph nodes with tumor metastasis from lymph nodes with inflammatory hyperplasia.

8. Use of the polypeptide probe according to any one of claims 1 to 4 in the preparation of a tumor imaging diagnostic agent or a diagnostic agent for detecting tumor lymphatic metastasis.

Images