A cyclic polypeptide molecular probe targeting neuropilin-1, preparation method and application thereof

Abstract

This invention relates to a cyclic polypeptide molecular probe targeting neurocilia protein-1 (NRP-1), its preparation method, and its applications. The cyclic polypeptide molecular probe comprises the structure shown in Formula I or Formula II, where R1 is the structure shown in Formula III or Formula IV; and R2 is a radiolabeled nuclide or a radiolabeled nuclide including a bifunctional chelating group. The technical solution of this invention exhibits excellent targeting specificity and high sensitivity for NRP-1 protein, enabling visualization of NRP-1-highly expressing tumors (such as fibrosarcoma, lung cancer, pancreatic cancer, or breast cancer) and NRP-1-positive lesions (such as metabolic diseases, sclerotic lesions, or autoimmune diseases) on positron emission tomography (PET). It shows great promise in the early diagnosis of NRP-1-highly expressing malignant tumors and in guiding clinical NRP-1 monoclonal antibody treatment regimens.

Description

Technical Field

[0001] This invention belongs to the field of radiochemistry technology, and particularly relates to a cyclic polypeptide molecular probe targeting neurocilia-1, its preparation method, and its application. Background Technology

[0002] Cancer is a disease caused by the uncontrolled growth and proliferation of cells. Common treatments include surgical resection, radiotherapy, chemotherapy, and immunotherapy. The size, depth of invasion, and presence of lymph node or distant metastasis of malignant tumors are strongly correlated with cancer prognosis. Early diagnosis and appropriate treatment can significantly prolong patient survival and improve quality of life. However, because early cancer symptoms are often subtle or nonspecific, and current diagnostic methods (such as endoscopy, CT, and MRI) have limitations, improving the early detection rate of cancer remains a widely discussed topic in the academic community.

[0003] In recent years, molecular imaging, especially positron emission tomography (PET), has made encouraging progress in guiding the early diagnosis and personalized treatment of cancer. This technology can provide complete and high-resolution imaging of malignant tumors in vivo and quantitatively analyze the expression of tumor markers.

[0004] Neuropilin-1 (NRP-1) is a multifunctional transmembrane glycoprotein involved in nervous system development, angiogenesis, and immune regulation. NRP-1 plays a crucial role in tumorigenesis and development, significantly correlated with tumor proliferation, invasion, and poor prognosis. NRP-1 is highly expressed in various tumor cells, including but not limited to lung cancer, fibrosarcoma, pancreatic cancer, and breast cancer. NRP-1 is expressed at low levels in normal tissues but at high levels in some lesions, including metabolic diseases, sclerotic lesions, and autoimmune diseases. Due to its significant pro-cancer effects, research on NRP-1 protein has been a hot topic in recent years. The number of tumor diagnostic and anti-tumor drugs targeting NRP-1 is also increasing, and monoclonal antibodies are already in clinical trials, with potential future clinical applications.

[0005] In molecular imaging, peptides are more suitable for designing molecular probes for PET imaging due to their advantages such as simple synthesis, strong penetration, low immunogenicity, and rapid metabolism. Furthermore, cyclic peptides exhibit better in vitro and in vivo stability compared to linear peptides. While several existing small-molecule peptide drugs targeting NRP-1 can visualize tumors, they suffer from issues such as small differences in uptake between tumor and normal tissues, poor drug stability, and potential biosafety risks. 99 mTc-CK399 mTc-DO3A-Et-RPAR, 68 Ga-NODAGA-K(Cy5)DKPPR, 68 Ga-NOTA-A7R.

[0006] Therefore, developing NRP-1-targeting small molecule cyclic peptide molecular probes with stronger tumor affinity, better biostability, and higher biosafety has important clinical value and social significance. It can be used for early diagnosis of NRP-1-positive cancers and lesions, dynamic monitoring of NRP-1 expression, and provide medication guidance for the clinical application of NRP-1 monoclonal antibodies. Summary of the Invention

[0007] To overcome the shortcomings of existing technologies, this invention provides a cyclic peptide molecular probe for targeting neurocilia-1, its preparation method, and its application. This drug is simple to synthesize, has good in vitro and in vivo stability, and exhibits good tumor imaging effects and biosafety.

[0008] To achieve the above objectives, the present invention proposes the following technical solution:

[0009] In a first aspect, the present invention proposes a cyclic polypeptide molecular probe targeting neurocilia protein-1, wherein the cyclic polypeptide molecular probe comprises the structure shown in Formula I or Formula II as follows:

[0010]

[0011]

[0012] Wherein, R1 is the structure shown in Formula III or Formula IV; R2 is a radiolabeled nuclide or a radiolabeled nuclide including a bifunctional chelating group;

[0013] Equation III or Equation IV is shown below:

[0014]

[0015] In Equation III or Equation IV, n is an integer between 0 and 10.

[0016] Preferably, the bifunctional chelating group is one of the structures shown in Formula V to Formula XII:

[0017]

[0018]

[0019]

[0020] Preferably, the radiolabeled nuclide is 68Ga、 18 F, 64 Cu、 89 Zr or 177 One or more of Lu.

[0021] Secondly, the present invention provides a method for preparing the above-mentioned cyclic polypeptide molecular probe, the method comprising the following steps:

[0022] Step S1: Mix the cyclic polypeptide molecule with the bifunctional chelating agent to obtain the bifunctional chelating agent-cyclic polypeptide molecule;

[0023] Step S2: The bifunctional chelating agent-cyclic polypeptide molecule is mixed with the radiolabeled nuclide to obtain the radionuclide-bifunctional chelating agent-cyclic polypeptide molecule.

[0024] Preferably, the cyclic polypeptide molecule is CEND1.

[0025] The structure of the cyclic polypeptide molecule is shown in Formula XIII below:

[0026]

[0027] Preferably, the specific operation of step S1 is as follows:

[0028] The cyclic polypeptide molecule is dissolved in a suitable solvent, the pH is adjusted to 7.5-9.5, a bifunctional chelating agent of 2-20 times the molecular weight of the cyclic polypeptide is added, the mixture is mixed and reacted at room temperature for 2-24 hours. The reaction mixture is separated and purified by HPLC, the product peak is collected, and the collected product peak liquid is lyophilized to obtain a white powder, which is the bifunctional chelating agent-cyclic polypeptide molecule.

[0029] The suitable solvent is one of water for injection, DMSO, or DMF.

[0030] It should be noted that the alkaline reagent used to adjust the pH in the above process is triethanolamine (TEA) or N,N'-diisopropylethylamine (DIEA).

[0031] Preferably, the HPLC is a semi-preparative HPLC method, and the chromatographic conditions include: mobile phase: phase A is an aqueous solution of 0.1% v / v trifluoroacetic acid, and phase B is an organic phase of acetonitrile containing 0.1% v / v trifluoroacetic acid; elution conditions: minute 0: phase A 100% v / v, phase B 0% v / v; minute 3: phase A 95% v / v, phase B 5% v / v; minute 10: phase A 5% v / v, phase B 95% v / v; minute 13: phase A 5% v / v, phase B 95% v / v; minute 14: phase A 100% v / v, phase B 0% v / v; elution stops at minute 15.

[0032] Preferably, the specific operation of step S2 is as follows:

[0033] The bifunctional chelating agent-cyclic polypeptide molecule is dissolved in a suitable solvent, the pH is adjusted to 4.0-4.5, and then 5 MCI-2 Ci of radionuclide is added. The mixture is heated in a water bath at 80-120℃ for 10-30 min to prepare the radionuclide-bifunctional chelating agent-cyclic polypeptide molecule.

[0034] The suitable solvent is one of water for injection, DMSO, or DMF.

[0035] Preferably, in step S1, the retention time of the product peak is in the range of 8 to 10 minutes;

[0036] Preferably, in step S2, the weakly acidic reagent used to adjust the pH is a NaAc buffer solution with a concentration of 0.5–2 mol / L;

[0037] Thirdly, this invention proposes the application of the aforementioned cyclic polypeptide molecular probe in the preparation of PET / CT tumor imaging diagnostic reagents.

[0038] Preferably, the tumor is a tumor that highly expresses NRP-1, including but not limited to lung cancer, breast cancer, glioma, fibrosarcoma, and pancreatic cancer. Fourthly, this invention proposes the application of the above-mentioned cyclic polypeptide molecular probe in the preparation of diagnostic reagents for NRP-1 positive lesions.

[0039] Preferably, the NRP-1 positive lesions include, but are not limited to, metabolic diseases, sclerotic lesions, autoimmune diseases, and precancerous lesions.

[0040] When the bifunctional chelating agent is DOTA, the cyclic polypeptide molecular probe precursor has the structure of formula IXV:

[0041]

[0042] When the radiolabeled group is 68 At Ga, the cyclic polypeptide molecular probe has the following structure:

[0043]

[0044] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0045] In the technical solution described in this invention, a cyclic peptide molecular probe precursor targeting NRP-1 is obtained by labeling the cyclic peptide CEND1 with a bifunctional chelating agent DOTA, NOA, or a derivative thereof. A radioactive labeling group is then chelated onto this precursor to obtain a cyclic polypeptide molecular probe targeting NRP-1. This cyclic polypeptide molecular probe enables PET imaging diagnosis of tumors or lesions with high NRP-1 expression in vivo through the specific binding of the cyclic peptide CEND1 to the NRP-1 protein.

[0046] Furthermore, the technical solution described in this invention modifies the structure of the NRP-1-targeting cyclic peptide CEND1, reserving an amino group capable of condensing with a bifunctional chelating group. The radiolabeled group can chelate onto the drug precursor via the bifunctional chelating group, enabling PET imaging diagnosis of NRP-1-positive tumors and lesions and monitoring of NRP-1 expression, with high specificity and sensitivity. Compared to existing peptide molecular probes, the NRP-1-targeting cyclic peptide molecular probe involved in this invention has advantages such as stronger tumor affinity, better biological stability, and higher biological safety, enabling non-invasive, early, and dynamic diagnosis of NRP-1-positive tumors and lesions. Moreover, PET / CT imaging can screen patients with high NRP-1 expression, guiding personalized treatment, such as the use of NRP-1 monoclonal antibodies.

[0047] Furthermore, the technical solution described in this invention has excellent targeting specificity and high sensitivity for NRP-1 protein, and can visualize NRP-1 highly expressing tumors (such as fibrosarcoma, lung cancer, pancreatic cancer, or breast cancer) and NRP-1 positive lesions (such as metabolic diseases, sclerotic lesions, or autoimmune diseases) on positron emission tomography (PET). It has great application prospects in the early diagnosis of NRP-1 highly expressing malignant tumors and in guiding clinical NRP-1 monoclonal antibody drug regimens.

[0048] Finally, the technical solution described in this invention transforms the conventional drug CEND1 into a nuclear medicine molecular imaging probe, which can diagnose tumors and lesions with high NRP-1 expression at an early stage, guide patients in choosing the most suitable treatment plan, and has positive significance for further research on the NRP-1 target and the development of new drugs. Attached Figure Description

[0049] To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the embodiments will be briefly described below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on the drawings without creative effort.

[0050] Figure 1 A cyclic peptide radiopharmaceutical targeting NRP-168 Schematic diagram of Ga-DOTA-CEND1;

[0051] Figure 2 Prepared as in Example 1 68 HPLC results of Ga-DOTA-CEND1;

[0052] Figure 3 Prepared as in Example 1 68 HPLC results of the stability of Ga-DOTA-CEND1 after incubation in PBS, NaCl, and fetal bovine serum (FBS) for 0, 0.5, 1, and 2 hours;

[0053] Figure 4 Prepared as in Example 1 68 HPLC results of the in vivo stability of Ga-DOTA-CEND1 at 0.5, 1, and 2 hours;

[0054] Figure 5 Prepared as in Example 1 68 The allocation coefficient results for Ga-DOTA-CEND1;

[0055] Figure 6 For injection 68 Pharmacokinetic curves of Ga-DOTA-CEND1;

[0056] Figure 7 for 68 Uptake values ​​of Ga-DOTA-CEND1 in A549 cells, HT-1080 cells, and Siha cells;

[0057] Figure 8 The results of Western blot detection of NRP-1 expression;

[0058] Figure 9 For injection 68 PET / CT images of BALB / c-Nude mice bearing A549 tumors (NRP-1 positive) at 30, 60 and 90 min after Ga-DOTA-CEND1;

[0059] Figure 10 For injection 68 PET / CT images of BALB / c-Nude mice bearing HT-1080 tumors (NRP-1 positive) at 30, 60 and 90 min after Ga-DOTA-CEND1;

[0060] Figure 11 For injection 68PET / CT images of BALB / c-Nude mice bearing CFPAC-1 tumors (NRP-1 positive) at 30, 60 and 90 min after Ga-DOTA-CEND1;

[0061] Figure 12 For injection 68 PET / CT images of BALB / c-Nude mice bearing MCF-7 tumors (NRP-1 positive) at 30, 60 and 90 min after Ga-DOTA-CEND1;

[0062] Figure 13 For injection 68 PET / CT images of Siha tumor-bearing (NRP-1 negative) BALB / c-Nude mice at 30, 60 and 90 min after Ga-DOTA-CEND1. Detailed Implementation

[0063] The embodiments of this application will now be described in detail with reference to the accompanying drawings.

[0064] The following specific examples illustrate the implementation of this application. Those skilled in the art can easily understand other advantages and effects of this application from the content disclosed in this specification. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of them. This application can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of this application. It should be noted that, in the absence of conflict, the following embodiments and features in the embodiments can be combined with each other. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0065] It should be noted that various aspects of embodiments within the scope of the appended claims are described below. It will be apparent that the aspects described herein can be embodied in a wide variety of forms, and any particular structure and / or function described herein is merely illustrative. Based on this application, those skilled in the art will understand that one aspect described herein can be implemented independently of any other aspect, and two or more of these aspects can be combined in various ways. For example, any number and aspects set forth herein can be used to implement the device and / or practice the method. Additionally, this device and / or method can be implemented using structures and / or functionalities other than one or more of the aspects set forth herein.

[0066] Example 1: 68 Ga-DOTA-CEND1 crafting and tagging

[0067] (1) A cyclic peptide radiopharmaceutical for NRP-1 targeting, the bifunctional chelator (DOTA)-CEND1 is DOTA-NHS, which has the following IXV structure:

[0068]

[0069] (2) 68 Preparation method of Ga-DOTA-CEND1 cyclic peptide molecular probe:

[0070] Step 1: Weigh 1.0–100 mg of CEND1 and 2–20 equivalents of the bifunctional chelating agent and dissolve them in water for injection. The bifunctional chelating agent is DOTA-NHS. Add an appropriate amount of triethanolamine (TEA) or N,N'-diisopropylethylamine (DIEA) to adjust the pH to weakly alkaline (7.5–9.5). Mix well and react overnight at room temperature. After the reaction is complete, dilute the reaction solution with water for injection and separate and purify it by semi-preparative HPLC. Collect the product peak, concentrate by rotary evaporation, and freeze dry it into a white powder, which is the radiopharmaceutical precursor DOTA-CEND1, i.e., the molecule with the structure shown in Formula IXV.

[0071] Step 2: Use 5 mL of 0.01 M HCl from... 68 Ge / 68 Elution in an ITG generator 68 Ga ions were mixed with a 1.5M NaOAc buffer solution to adjust the pH to approximately 4.0, resulting in a mixture. This mixture was then directly transferred to a 2mL plastic tube containing 20–50 μg of the NRP-1-targeted radiopharmaceutical precursor DOTA-CEND1. After mixing, the mixture was heated at 100°C for 10–15 minutes to prepare... 68 Ga-DOTA-CEND1, see structure below Figure 1 (i.e., the structure shown in formula XV).

[0072] (3) 68 Method for determining the labeling rate of Ga-DOTA-CEND1:

[0073] The labeling rate was determined using a high-performance liquid chromatography (Radio-HPLC) method: gradient analysis was employed, the analytical column was a Kinetex 5μm EVO C18 (250.0mm×4.6mm); the UV detection wavelength was 214nm; the flow rate was 1.0mL / min; the injection volume was 20μL, and the mobile phase program is shown in Table 1 below.

[0074] Table 1.

[0075]

[0076] Labeling rate results are shown in Figure 2 Under this marking condition, 68 The Ga-DOTA-CEND1 drug had a radiochemical purity greater than 95%, meeting the experimental purity requirements. To reduce the impact of free gallium on tumor diagnosis, purification was performed using a Sep-Pak Plus Light C18 solid-phase extraction column. After purification with the Sep-Pak Plus Light C18 column, the drug's radiochemical purity approached 100%. To ensure drug consistency, all subsequent experiments used Sep-Pak Plus Light C18 column purification.

[0077] Example 2: 68 In vivo and in vitro stability of Ga-DOTA-CEND1

[0078] The preparation from Example 1 68 Ga-DOTA-CEND1 drug was added to 200 μL of NaCl, PBS, and FBS solutions (approximately 100 μCi), respectively, and incubated in a metal bath at 37°C for different times. The radiochemical purity was then determined (Radio-HPLC). Results are shown below. Figure 3 In normal mice, the tail vein was injected with... 68 After 0.5, 1, and 2 hours of Ga-DOTA-CEND1 (100 μCi), mouse urine was collected and its radiochemical purity was determined (Radio-HPLC). The results are shown below. Figure 4 This indicates that the probe has good in vitro and in vivo stability.

[0079] Example 3: 68 Determination of the distribution coefficient of Ga-DOTA-CEND1

[0080] The preparation from Example 1 68 The Ga-DOTA-CEND1 (2-4 μCi) probe was added to 500 μL of DDW and n-octanol, respectively, and thoroughly mixed by vortexing. The mixture was then centrifuged at 12000 rpm for 5 min. 200 μL of each layer was taken and added to separate radioimmunoassay tubes. The γ value was measured and calculated using the Log P formula: Log P = Log(aqueous phase γ value / n-octanol phase γ value). The results are shown below. Figure 5 The LogP value is -2.33, indicating that the probe is hydrophilic.

[0081] Example 4: 68 Pharmacokinetic analysis of Ga-DOTA-CEND1

[0082] Five healthy six-week-old mice were injected with 0.2 mCi via the tail vein. 68Pharmacokinetic studies were conducted on Ga-DOTA-CEND1. Blood samples were collected via tail vein dissection at 1, 3, 10, 15, 30, 60, 90, and 120 minutes post-dose. After gamma counting, blood weight was collected, and after radiation decay correction, the percentage of radioactive count per gram of blood relative to the total injected radioactive count (%ID / g) was calculated and expressed. The blood half-life was calculated using GraphPad Prism 8.0 software. The results are shown below. Figure 6 The results showed: 68 Ga-DOTA-CEND1 drug in blood T 1 / 2(α) It was 1.063 min, T 1 / 2(β) The time was 16.98 min, indicating that the probe was cleared from the blood relatively quickly.

[0083] Example 5: 68 Cellular uptake assay of Ga-DOTA-CEND1

[0084] 1×10 6 After placing lung cancer cells A549, fibrosarcoma cells HT-1080, and cervical cancer cells Siha into radioimmunoassay tubes, the molecular probe targeting NRP1 prepared in Example 1 was added to the radioimmunoassay tubes. 68 Ga-DOTA-CEND1 (1 μCi / tube) was incubated in a 37°C water bath for 0.5 h, 1 h, 1.5 h, and 2 h, respectively. After incubation, 500 μL of cold (4°C) PBS buffer (pH = 7.4, 0.01 M) was added to the radioimmunoassay tube, and the tube was centrifuged at 4000 rpm for 5 min. After centrifugation, the above operation was repeated once, and the CPM value of the sample was detected using a gamma counter. The %AD uptake result was expressed as the ratio of intracellular CPM to total dose CPM. The results are shown in the figure. Figure 7 A549 cells and HT-1080 cells on 68 Ga-DOTA-CEND1 uptake was significantly higher in Ga-DOTA-CEND1 cells than in Siha cells, indicating that the molecular probe... 68 Ga-DOTA-CEND1 exhibits good target specificity.

[0085] Example 6: Western Blot Experiment of NRP-1 Expression

[0086] The expression of NRP1 protein in lung cancer cells A549, fibrosarcoma HT-1080, pancreatic cancer CFPAC-1, breast cancer MDB-MA-231, and cervical cancer Siha was determined by Western blotting. The results are shown in [Figure number missing]. Figure 8 .from Figure 8As can be seen, A549, HT-1080, CFPAC-1, and MDB-MA-231 cells highly express NRP-1 protein, while Siha cells hardly express NRP-1 protein. A549, HT-1080, CFPAC-1, and MDB-MA-231 can be used as positive models for targeting studies of molecular probes targeting NRP-1, while Siha can be used as a negative model.

[0087] Example 7: 68 Biological evaluation of Ga-DOTA-CEND1

[0088] The following describes the NRP-1-targeted probe prepared according to the method of Example 1 of the present invention. 68 The PET / CT imaging performance of Ga-DOTA-CEND1 is described as follows:

[0089] (1) Preparation of mouse tumor model

[0090] A549 cells (NRP 1 positive cells), HT-1080 cells (NRP 1 positive cells), CFPAC-1 cells (NRP 1 positive cells), MCF-7 cells (NRP 1 positive cells), and Siha cells (NRP 1 negative cells) were digested with 0.25 wt% trypsin / 0.02 wt% EDTA, washed with sterile PBS, and resuspended in sterile physiological saline to prepare a cell suspension of 4 × 10⁴ / μL. The three cell types were then divided into three groups of cells at 5 × 10⁴ cells per cell line. 6 A dose of 100 μL per mouse was injected subcutaneously into the right forelimb axilla of 4–5 week old female BALB / c-Nude mice. The mice were housed in an SPF-grade animal facility, and tumor diameter was monitored every other day. After approximately 2–3 weeks, when the average tumor diameter reached 0.8–1.0 cm, the mice were used for experiments.

[0091] (2) 68 MicroPET / CT imaging of Ga-DOTA-CEND1 in a lung cancer model

[0092] (1) The tumor-bearing mice (n=3) were anesthetized and placed prone on the PET / CT bed. 0.2 mL of a probe of about 150 μCi was injected via the tail vein. Static PET and CT scans were performed for 10 min and 5 min respectively 30, 60 and 90 min after the probe injection.

[0093] PET imaging results of four NRP-1 positive models, A549, HT-1080, CFPAC-1, and MCF-7, are shown below. Figure 9 , Figure 10 , Figure 11 , Figure 12 PET imaging results of the NRP-1 negative model Siha are shown below. Figure 13 .

[0094] like Figure 13 As shown, the results illustrate 68 The Ga-DOTA-CEND1 probe was readily taken up by tumors in four positive tumor models after injection, and the uptake was significantly higher than in the negative model Siha. This indicates that the probe can bind well to tumor NRP-1 and can be used for molecular imaging monitoring of tumor NRP-1 expression.

[0095] The above embodiments are merely illustrative of the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made based on the essence of the content of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A cyclic polypeptide molecular probe targeting neurocilia protein-1, characterized in that, The cyclic polypeptide molecular probe comprises the structure shown in Formula I or Formula II: Wherein, R1 is the structure shown in Formula III or Formula IV; R2 is a radiolabeled nuclide or a radiolabeled nuclide including a bifunctional chelating group; Equation III or Equation IV is shown below: In Equation III or Equation IV, n is an integer between 0 and 10.

2. The cyclic polypeptide molecular probe according to claim 1, characterized in that, The bifunctional chelating group is one of the structures shown in Formula V to Formula XII:

3. The cyclic polypeptide molecular probe according to claim 1, characterized in that, The radiolabeled nuclide is 68 Ga、 18 F, 64 Cu、 89 Zr or 177 One or more of Lu.

4. A method for preparing the cyclic polypeptide molecular probe according to any one of claims 1 to 3, characterized in that, The preparation method includes the following steps: Step S1: Mix the cyclic polypeptide molecule with the bifunctional chelating agent to obtain the bifunctional chelating agent-cyclic polypeptide molecule; Step S2: The bifunctional chelating agent-cyclic polypeptide molecule is mixed with the radiolabeled nuclide to obtain the radionuclide-bifunctional chelating agent-cyclic polypeptide molecule.

5. The preparation method according to claim 4, characterized in that, The cyclic polypeptide molecule is CEND1.

6. The preparation method according to claim 4 or 5, characterized in that, The specific operation of step S1 is as follows: The cyclic polypeptide molecule is dissolved in a suitable solvent, the pH is adjusted to 7.5-9.5, a bifunctional chelating agent of 2-20 times the molecular weight of the cyclic polypeptide is added, the mixture is mixed and reacted at room temperature for 2-24 hours. The reaction mixture is separated and purified by HPLC, the product peak is collected, and the collected product peak liquid is lyophilized to obtain a white powder, which is the bifunctional chelating agent-cyclic polypeptide molecule. The suitable solvent is one of water for injection, DMSO, or DMF.

7. The preparation method according to claim 6, characterized in that, The HPLC method described is a semi-preparative HPLC method. The chromatographic conditions include: mobile phase: phase A is an aqueous solution of 0.1% v / v trifluoroacetic acid, and phase B is an organic phase of acetonitrile containing 0.1% v / v trifluoroacetic acid; elution conditions: minute 0: phase A 100% v / v, phase B 0% v / v; minute 3: phase A 95% v / v, phase B 5% v / v; minute 10: phase A 5% v / v, phase B 95% v / v; minute 13: phase A 5% v / v, phase B 95% v / v; minute 14: phase A 100% v / v, phase B 0% v / v; elution is stopped at minute 15.

8. The preparation method according to claim 4 or 5, characterized in that, The specific operation of step S2 is as follows: The bifunctional chelating agent-cyclic polypeptide molecule is dissolved in a suitable solvent, the pH is adjusted to 4.0-4.5, and then 5 MCI-2 Ci of radionuclide is added. The mixture is heated in a water bath at 80-120℃ for 10-30 min to prepare the radionuclide-bifunctional chelating agent-cyclic polypeptide molecule. The suitable solvent is one of water for injection, DMSO, or DMF.

9. The use of the cyclic polypeptide molecular probe as described in any one of claims 1 to 3 in the preparation of PET / CT tumor imaging diagnostic reagents.

10. The use of the cyclic polypeptide molecular probe according to any one of claims 1 to 3 in the preparation of diagnostic reagents for NRP-1 positive lesions.

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