An NRP-1 targeted PET molecular probe and a preparation method and application thereof

Abstract

The present application relates to a kind of NRP-1 targeted PET molecular probe and its preparation method and application, belong to chemical technical field.The application provides a kind of molecular probe, the molecular probe is composed of NRP-1 targeted peptide, PEG chain and radioactive label group, the NRP-1 targeted peptide CLKADK of the molecular probe has excellent targeting specificity (the binding constant for NRP-1 protein is 26.24±3.70nM), and, after connecting radionuclide coordination group NOTA, the bioactivity of CK2 is hardly affected (the binding constant for NRP-1 protein is 25.39±1.65nM), therefore, the targeting specificity of the molecular probe for NRP-1 protein is good, sensitivity is high, can effectively distinguish NRP-1 expression negative and positive expression tumor in vitro and in vivo, in early diagnosis and curative effect evaluation of malignant tumor provide guidance in extremely application prospect.

Description

Technical Field

[0001] This invention relates to an NRP-1 targeted PET molecular probe, its preparation method, and its application, belonging to the field of chemical technology. Background Technology

[0002] Neurocilimins (NRPs) are single-transmembrane, non-tyrosine kinase multifunctional surface glycoproteins found in all vertebrates and with a highly conserved structure. Based on their function and ligand binding specificity, NRPs can be divided into two subtypes: neurocilimin-1 (NRP-1) and neurocilimin-2 (NRP-2). NRP-1 is mainly expressed in arterial and capillary endothelial cells, while NRP-2 is mainly expressed in venous and lymphatic endothelial cells. Both NRPs act as neuronal adhesion molecules, playing a regulatory role in nerve cell guidance and axonal growth, and both have been shown to be involved in vascular biology. In addition, both NRPs are multifunctional proteins participating in various biological processes besides angiogenesis and neural development; NRP-1, for example, plays an important role in tumorigenesis and immunity.

[0003] Given that NRP-1 is highly expressed in various tumor tissues and immune cells, and can play an important role as a multifunctional receptor in various solid tumors, hematologic malignancies, and immunological fields, either independently or with the help of other ligand molecules, an increasing number of studies are using NRP-1 as an important molecular marker for tumor diagnosis and anti-tumor drug research. Currently, drugs targeting NRP-1 include monoclonal antibodies, soluble NRP-1 drugs, small molecule peptides, serotonin drugs, and RNA interference technology. Among these, small molecule peptides have gradually become a hot topic in NRP-1 targeted drug research due to their ease of synthesis and optimization, high selectivity, low dosage, excellent tolerability, and limited efficacy. However, existing small molecule peptides targeting NRP-1 suffer from poor targeting specificity, poor biological stability, and low tumor-specific uptake. Therefore, there is an urgent need to develop small molecule peptides targeting NRP-1 with high targeting specificity, good biological stability, and high tumor-specific uptake rates.

[0004] Molecular imaging is the science of using imaging techniques to visualize specific molecules at the tissue, cellular, and subcellular levels, reflecting changes in molecular levels in vivo, and conducting qualitative and quantitative studies on their biological behavior through imaging. As a non-invasive in vivo imaging technique, molecular imaging can detect abnormalities in the levels of disease-causing genes or proteins before tumors undergo anatomical and morphological changes, providing quantitative analysis and real-time assessment of tumor markers in vivo to guide early diagnosis and personalized precision treatment. Therefore, there is an urgent need to develop a small molecule peptide drug with high targeting specificity and sensitivity targeting NRP-1 to guide the early diagnosis and efficacy evaluation of malignant tumors with high NRP-1 expression. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a molecular probe targeting neurocilia protein-1 (NRP-1). The molecular probe comprises an NRP-1 targeting peptide, a PEG chain (azido-tetraethylene glycol-carboxyl group), and a radiolabeled group, and has the following structure: ; Among them, the NRP-1 targeting peptide is CLKADK (CK2), and R is a radionuclide labeling group.

[0006] In one embodiment of the present invention, the radionuclide labeling group is 68 Ga、[ 18 F]AlF、 64 Cu or 89 Zr.

[0007] In one embodiment of the present invention, when the radionuclide labeling group is 68 At Ga, the molecular probe has the following structure: .

[0008] In one embodiment of the present invention, the labeling precursor of the molecular probe, NOTA-PEG4-CK2, has the following structure: .

[0009] The present invention also provides a method for preparing the above-mentioned molecular probe, the method comprising the following steps: Step 1: Synthesize the NRP-1 targeting peptide CLKADK (CK2); Step 2: PEG-4 and NRP-1 targeting peptide CLKADK (CK2) are condensed and deprotected to obtain compound CK2-PEG4; Step 3: Compound CK2-PEG4 and NOTA-NHS were reacted and deprotected under alkaline conditions to obtain the molecular probe labeled precursor NOTA-PEG4-CK2; Step 4: Radiolabel the molecular probe precursor NOTA-PEG4-CK2 with a radionuclide to obtain the molecular probe; The NRP-1 targeting peptide CLKADK has the following structure: ; The compound CK2-PEG4 has the following structure: .

[0010] In one embodiment of the present invention, in step one, the NRP-1 targeting peptide CLKADK is synthesized using the Fmoc synthesis method; in step two, the Fmoc-protected PEG-4 and the NRP-1 targeting peptide CLKADK are condensed and deprotected to obtain the compound CK2-PEG4. The Fmoc-protected PEG-4 has the following structure: .

[0011] The present invention also provides an NRP-1 targeting peptide, the amino acid sequence of which is shown in SEQ ID NO.1.

[0012] The present invention also provides the application of the above-mentioned molecular probe or the above-mentioned NRP-1 targeting peptide in neurocilia-1 imaging, wherein the application is not for the purpose of disease diagnosis and treatment.

[0013] The present invention also provides an imaging drug that targets neurocilia protein-1, wherein the imaging drug contains the above-mentioned molecular probe or the above-mentioned NRP-1 targeting peptide.

[0014] The present invention also provides the application of the above-mentioned molecular probe or the above-mentioned NRP-1 targeting peptide in the preparation of tumor imaging drugs.

[0015] In one embodiment of the present invention, the imaging drug is used for early diagnosis and efficacy evaluation of tumors.

[0016] In one embodiment of the present invention, the tumor is a malignant tumor that highly expresses NRP-1; the malignant tumor that highly expresses NRP-1 includes at least one of breast cancer, glioma, lung cancer, or pancreatic cancer.

[0017] The present invention also provides an imaging drug targeting tumors, the imaging drug containing the above-mentioned molecular probe or the above-mentioned NRP-1 targeting peptide.

[0018] In one embodiment of the present invention, the imaging drug is used for early diagnosis and efficacy evaluation of tumors.

[0019] In one embodiment of the present invention, the tumor is a malignant tumor that highly expresses NRP-1; the malignant tumor that highly expresses NRP-1 includes at least one of breast cancer, glioma, lung cancer, or pancreatic cancer.

[0020] The technical solution of this invention has the following advantages: This invention provides a molecular probe composed of an NRP-1 targeting peptide, a PEG chain, and a radiolabeled group, which has the following advantages: First, the NRP-1 targeting peptide CLKADK (CK2) of the molecular probe exhibits excellent targeting specificity for the NRP-1 protein (binding constant for NRP-1 protein is 26.24±3.70 nM). Furthermore, after being linked to the radioactive nuclide ligand NOA, the biological activity of CK2 is almost unaffected (binding constant for NRP-1 protein is 25.39±1.65 nM). Therefore, the molecular probe has good targeting specificity and high sensitivity for the NRP-1 protein, and can effectively distinguish between NRP-1-negative and NRP-1-positive tumors in vitro and in vivo. Second, the molecular probe can reach the tumor site quickly (with high tumor-specific uptake rate) and maintain good imaging effect at the tumor site, while having low uptake of non-specific tissues. Third, the molecular probe has a short biological half-life in vivo, and it can be rapidly excreted from non-target tissues through the renal excretion system, thus achieving a good tumor signal-to-noise ratio. Fourth, the molecular probe exhibits good in vitro stability, maintaining good biostability in both PBS and fetal bovine serum (FBS). In summary, the molecular probes described above can provide guidance for the early diagnosis and efficacy assessment of malignant tumors with high NRP-1 expression (e.g., breast cancer) through PET imaging, and have great application potential.

[0021] Furthermore, the radionuclide labeling group is 68 Ga; when the radionuclide labeling group is 68 In Ga, the synthesis method of the molecular probe is simple, and it uses nuclides with short radioactive half-lives. 68 When Ga is used for labeling, the labeling conditions are mild, and molecular probes with high radioactivity yield and high radioactivity purity can be obtained without purification, making them easy to use in clinical applications. Attached Figure Description

[0022] Figure 1 High-resolution mass spectrometry analysis of the labeled precursor NOTA-PEG4-CK2.

[0023] Figure 2 High-performance liquid chromatography (HPLC) chromatogram of the labeled precursor NOTA-PEG4-CK2.

[0024] Figure 3 Molecular probes 68 Ga]NOTA-PEG4-CK2 radioactive high-performance liquid chromatography detection image.

[0025] Figure 4 : The binding constant of compound CK2 with NRP-1 was determined.

[0026] Figure 5 : The binding constant of compound Nota-CK2 and NRP-1 was determined.

[0027] Figure 6 : The binding constant of compound CK3 with NRP-1 was determined.

[0028] Figure 7 Molecular probes 68 HPLC analysis of the stability of Ga]NOTA-PEG4-CK2 after incubation in PBS for 0, 0.5, 1, and 2 hours.

[0029] Figure 8 Molecular probes 68 HPLC analysis of the stability of Ga]NOTA-PEG4-CK2 after incubation in fetal bovine serum (FBS) for 0, 0.5, 1, and 2 hours.

[0030] Figure 9 Western blot results of NRP-1 expression in MDA-MB-231 and NCI-H1299 cells.

[0031] Figure 10 Molecular probes 68 Uptake values ​​of Ga]NOTA-PEG4-CK2 in MDA-MB-231 and NCI-H1299 cells.

[0032] Figure 11 Molecular probes 68 MicroPET imaging results of Ga]NOTA-PEG4-CK2 in MDA-MB-231 and NCI-H1299 models.

[0033] Figure 12 Molecular probes 68 The curves of Ga]NOTA-PEG4-CK2 uptake in tumors and muscles in MDA-MB-231 and NCI-H1299 models.

[0034] Figure 13Molecular probes 68 Tumor / muscle target ratio of Ga]NOTA-PEG4-CK2 in MDA-MB-231 and NCI-H1299 models.

[0035] Figure 14 Molecular probes 68 MicroPET imaging results of Ga]NOTA-PEG4-CK2 in an MDA-MB-231 model treated with SB-203580.

[0036] Figure 15 Molecular probes 68 The curve of Ga]NOTA-PEG4-CK2 uptake by tumor and muscle in MDA-MB-231 model treated with SB-203580.

[0037] Figure 16 Molecular probes 68 The tumor / muscle target ratio of Ga]NOTA-PEG4-CK2 in the MDA-MB-231 model treated with SB-203580. Detailed Implementation

[0038] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0039] For any experimental steps or conditions not specified in the following examples, the procedures or conditions described in the literature in this field can be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0040] Example 1: A molecular probe targeting NRP-1 [ 68 Ga]NOTA-PEG4-CK2 This embodiment provides a molecular probe targeting NRP-1. 68 Ga]NOTA-PEG4-CK2, a molecular probe targeting NRP-1 [ 68 [Ga]NOTA-PEG4-CK2 is composed of the NRP-1 targeting peptide CLKADK (CK2) with the amino acid sequence shown in SEQ ID NO.1, a PEG chain, and a radiolabeled group, and has the structure shown below: .

[0041] Example 2: A method for preparing a molecular probe targeting NRP-1 [ 68 The method of Ga]NOTA-PEG4-CK2 This embodiment provides the molecular probe targeting NRP-1 described in Example 1. 68 The preparation method of Ga]NOTA-PEG4-CK2 is as follows: Step 1: Rinse the sand core funnel twice with dichloromethane, then dry it. Add 2-chlorotriphenylmethyl chloride resin (loading amount of 1.106 mmol / g, 271.25 mg) to the dried sand core funnel, and add 10 mL of dichloromethane to soak and swell the 2-chlorotriphenylmethyl chloride resin. After soaking and swelling for 10 min, dry it. Step 2: Add amino acid Fmoc-Lys(Boc)-OH (0.375 mmol) to the sand core funnel obtained in Step 1, and dissolve it with 10 mL of DMF (N,N-dimethylformamide) to obtain a solution; add DIPEA (N,N-diisopropylethylamine) (130 μL, 0.75 mmol) to the solution to adjust the pH to 8, and shake the solution at 25 °C for 3 h. After shaking, remove the solvent by vacuum drying; add 10 mL of a mixed solution of DMF / CH3OH / DIPEA (DMF / CH3OH / DIPEA = 7:2:1, v / v / v) to wash the filter cake, shake for 10 min, and filter by vacuum filtration. Repeat the operation once to remove unreacted amino acids; then use 10 mL of DMF / CH3OH / DIPEA solution to wash the filter cake by vacuum drying. Wash twice with DMF (HPLC type), shake for 2 min, and then filter. Add 10 mL of DMF solution containing 20 vt% piperidine to the sintered glass funnel, shake for 10 min, and then filter. Repeat this process three times to remove the FMOC protecting group on the amino acid. Wash the filter cake five times with 10 mL of DMF (HPLC type) to remove excess piperidine. After washing, dry the solvent and take a sample for Kaiser test. The reagent color should be dark purple, indicating that the FMOC group has been removed and the amino group is exposed, ready to be attached to the next amino acid. Step 3: Based on Step 2, replace Fmoc-Lys(Boc)-OH (0.375mmol) sequentially with Fmoc-Asp(OtBu)-OH (0.375mmol), Fmoc-Ala-OH (0.375mmol), Fmoc-Lys(Boc)-OH (0.375mmol), Fmoc-Leu-OH (0.375mmol), and Fmoc-Cys(Trt)-OH (0.375mmol), and repeat Step 2 to obtain the NRP-1 targeting peptide CLKADK (CK2); The NRP-1 targeting peptide CLKADK (CK2) has the following structure: ; Step 4: Add Fmoc-PEG4-COOH (0.375 mmol) and HBTU (benzotriazole-tetramethylurea hexafluorophosphate) (1 mmol) to the sintered sand funnel obtained in Step 3, and dissolve them in 10 mL of ultra-dry DMF (N,N-dimethylformamide) to obtain a solution; add DIPEA (N,N-diisopropylethylamine) (2 mmol) to the solution to adjust the pH to 8, and shake the solution at 25°C for 3 h. After shaking, remove the solvent by vacuum drying; add 10 mL of DMF solution containing 20 vt% piperidine to the sintered sand funnel, shake for 10 min, and filter. Repeat the operation three times to remove the FMOC protecting groups on the amino acids; add 10 mL of DMF solution containing 1 vt% piperidine to the sintered sand funnel. A TFA (trifluoroacetic acid) CH2Cl2 solution was prepared to obtain a mixture. The mixture was shaken at 25°C for 10 min. After shaking, the filtrate containing compound CK2-PEG4 was filtered out. This operation was repeated until the 2-chlorotriphenylmethyl chloride resin turned wine red and did not fade. The collected filtrate was used to remove the solvent using a rotary evaporator. After precipitation with cold diethyl ether (4°C), the precipitate was transferred to a 50 mL centrifuge tube, centrifuged, and the supernatant was removed. The precipitate was dried to obtain compound CK2-PEG4. The Fmoc-PEG4-COOH has the following structure: ; The compound CK2-PEG4 has the following structure: ; Step 5: Dissolve compounds CK2-PEG4 (0.02 mmol) and NOTA-NHS (0.06 mmol) in 200 μL of DMF (N,N-dimethylformamide) to obtain a solution; add DIPEA (N,N-diisopropylethylamine) (50 µL, 0.28 mmol) to the solution to adjust the pH to 8, and stir at 25 °C and 150 rpm for 3 h under nitrogen protection to obtain a reaction solution; evaporate the reaction solution to dryness using an oil pump, precipitate with cold diethyl ether (4 °C), sonicate at 25 °C for 30 s, transfer to a 50 mL centrifuge tube, centrifuge to remove the supernatant (place at -20 °C for 10 min before centrifugation); dry the precipitate to obtain compound NOTA-PEG4-CK2-1; Step 6: Compound Nota-PEG4-CK2-1 (0.02 mmol) was mixed with 2 mL of TFA (trifluoroacetic acid), 80 µL of acetonitrile, and 80 µL of Tips (triisopropylsilane), and stirred at 25 °C and 150 rpm for 2 h to obtain a reaction solution. The organic solvent was removed from the reaction solution using a rotary evaporator, and the precipitate was precipitated with cold diethyl ether (4 °C). The precipitate was then transferred to a 50 mL centrifuge tube, centrifuged, and the supernatant was removed. After drying the precipitate, the labeled precursor of the molecular probe targeting NRP-1, Nota-PEG4-CK2, was obtained. Step 7: Use 1.4 mL of 0.05 M HCl from... 68 Ge / 68 Elution in an ITG generator 68 Ga ions were mixed with 1.25M NaOAc buffer to adjust the pH to 4.0, resulting in a mixture. This mixture was then directly transferred to a 5mL plastic tube containing 10μg of the NRP-1-targeted molecular probe precursor, NOTA-PEG4-CK2. After mixing, the mixture was incubated in an oil bath at 37°C for 15 minutes to obtain the NRP-1-targeted molecular probe. 68 Ga]NOTA-PEG4-CK2.

[0042] The labeled precursor NOTA-PEG4-CK2 was analyzed using high-resolution mass spectrometry and high-performance liquid chromatography. The results are shown in the figure. Figure 1 and Figure 2 Using the Gabi Nova radiometric detector to [ 68 The reaction solution labeled with Ga]NOTA-PEG4-CK2 was subjected to radioactive HPLC detection. The results are shown in the figure. Figure 3 Calculated by dividing the peak area of ​​radioactive products by the total peak area. 68 The radiochemical purity (RCP) of Ga]NOTA-PEG4-CK2 was calculated to be higher than 99%.

[0043] After attenuation correction, the results were calculated using radioactive HPLC detection. 68 The final radiochemical yield (RCY) of Ga]NOTA-PEG4-CK2 was calculated to be 98%, obtained by calculating the molar activity (Am) = product radioactivity / M (molar mass of the precursor). 68 The molar activity of Ga]NOTA-PEG4-CK2 was calculated to be 21.92 ± 2.06 GBq / μmol.

[0044] Comparative Example 1: An NRP-1-targeting cyclic heptapeptide CLKADKAKC (CK3) This comparative example provides an NRP-1-targeted cyclic heptapeptide CLKADKAKC (CK3), the amino acid sequence of which is shown in SEQ ID NO.2, and has the following structure: .

[0045] Comparative Example 2: A method for preparing NRP-1-targeting cyclic heptapeptide CLKADKAKC (CK3) This comparative example provides the preparation method of NRP-1-targeting cyclic heptapeptide CLKADKAKC (CK3) of Comparative Example 1, which can be found in the literature "Feng GK, et al. SPECT and near-infrared fluorescence imaging of breast cancer with a neuropilin-1-targeting peptide. J Control Release. 2014;192:236-42."

[0046] Experimental Example 1: Experiment on the binding constant of NRP-1 protein by a molecular probe targeting NRP-1 This experimental example provides an experiment on the binding constant of a molecular probe targeting NRP-1 to the NRP-1 protein. The specific procedure is as follows: Using the MO-L018 RED-tris-NTA protein labeling kit (purchased from NanoTemper Technologies), and a Monolith NT.115 microthermophoresis apparatus, the binding constants of the NRP-1 targeting peptide CLKADK (CK2) prepared in Example 2, compound Nota-CK2, and the NRP-1 targeting cyclic heptapeptide CLKADKAKC (CK3) prepared in Comparative Example 2, with NRP-1 protein (purchased from Sinocare), were determined. The results are as follows: Figures 4-6 As shown.

[0047] Depend on Figures 4-6 It can be seen that both the NRP-1 targeting peptide CLKADK (CK2) and the compound NOTA-CK2 have excellent binding ability to NRP-1 protein, with binding constants of 26.24±3.70 nM and 25.39±1.65 nM, respectively. Although the NRP-1 targeting cyclic heptapeptide CLKADKAKC (CK3) also conforms to the C-terminal rule like the NRP-1 targeting peptide CLKADK (CK2), its binding constant is 867.27±1.77 nM. These results indicate that the NRP-1 targeting peptide CLKADK (CK2) and the compound NOTA-CK2 have higher targeting specificity and binding ability to NRP-1 protein.

[0048] Experimental Example 2: In vitro stability experiment of molecular probes targeting NRP-1 This experimental example provides an in vitro stability experiment of a molecular probe targeting NRP-1, the specific procedure of which is as follows: Experiment 1: The molecular probe targeting NRP-1 prepared in Example 2 was used... 68 GaNOTA-PEG4-CK2 was mixed with PBS buffer (pH 7.4, 0.01M) at a volume ratio of 1:9 to obtain a mixture. The mixture was incubated at 37°C for 0, 0.5, 1, and 2 hours. After incubation, the incubation solution was analyzed by radioactive HPLC using a Gabi Nova radiometric detector. The results are shown below. Figure 7 .

[0049] Experiment 2: The molecular probe targeting NRP-1 prepared in Example 2 was used... 68 GaNOTA-PEG4-CK2 was mixed with fetal bovine serum (FBS, purchased from BI) at a volume ratio of 1:9 to obtain a mixture. The mixture was incubated at 37°C for 0, 0.5, 1, and 2 hours. After incubation, 20 μL of the incubation solution was taken, and an equal volume of acetonitrile was added. The mixture was centrifuged at 12000g for 5 minutes to separate the serum from the protein. The supernatant was then analyzed by radioactive HPLC using a Gabi Nova detector. The results are shown in the figure. Figure 8 .

[0050] Depend on Figures 7-8 It can be seen that molecular probes [ 68 After incubation in PBS and fetal bovine serum for 2 hours, the main peak ratio of the HPLC chromatogram of Ga]NOTA-PEG4-CK2 remained greater than 95%, indicating that the molecular probe [ 68 Ga]NOTA-PEG4-CK2 exhibits good stability.

[0051] Experimental Example 3: Cellular Uptake Experiment of Molecular Probe Targeting NRP-1 The expression of NRP-1 protein in triple-negative breast cancer cells MDA-MB-231 and lung cancer cells NCI-H1299 was determined by Western blot. The results are shown in the figure below. Figure 9 .from Figure 9 As can be seen, MDA-MB-231 cells and NCI-H1299 cells are NRP-1 positive and NRP-1 negative expression cell lines, respectively, which can be used as targeted research models for molecular probes targeting NRP-1.

[0052] This experiment used MDA-MB-231 cells and NCI-H1299 cells to demonstrate the cellular uptake of a molecular probe targeting NRP-1. The specific procedure is as follows: 1×10 6 After placing MDA-MB-231 cells (NRP-1 positive cells, purchased from the Shanghai Institute of Cell Bank, Chinese Academy of Sciences) and NCI-H1299 cells (NRP-1 negative cells, purchased from the Shanghai Institute of Cell Bank, Chinese Academy of Sciences) into radioimmunoassay tubes, the molecular probe targeting NRP-1 prepared in Example 2 was added to the radioimmunoassay tubes. 68 GaNOTA-PEG4-CK2 (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, 0.01M) was added to the radioimmunoassay tube, and the tube was centrifuged at 4000 r / min 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 cellular uptake %AD result is expressed as the ratio of intracellular CPM to total dose CPM. The detection results are shown in the figure. Figure 10 .

[0053] from Figure 10 It can be seen that MDA-MB-231 cells respond well to molecular probes [ 68 The uptake of Ga]NOTA-PEG4-CK2 was significantly higher in Ga]NOTA-PEG4-CK2 than in NCI-H1299 cells, indicating that the molecular probe [ 68 Ga]NOTA-PEG4-CK2 exhibits good targeting specificity.

[0054] Experiment Example 4: Mouse PET Imaging Experiment Using NRP-1 as a Molecular Probe This experimental example provides a mouse PET imaging experiment using NRP-1 as a molecular probe. The specific procedure is as follows: Experiment 1: MDA-MB-231 cells (NRP-1 positive cells, purchased from the Shanghai Institute of Cell Bank, Chinese Academy of Sciences) and NCI-H1299 cells (NRP-1 negative cells, purchased from the Shanghai Institute of Cell Bank, Chinese Academy of Sciences) were respectively divided into 5×10⁻⁶ cells and 10⁻⁶ cells. 6 The recommended dose was administered subcutaneously to the right forelimb axilla of female BALB / c mice (5 weeks old, purchased from Changzhou Cavens Laboratory Animal Co., Ltd.); tumor diameter was monitored every other day, and the tumor was classified as 200.0 ± 25.0 mm when the tumor volume reached this value. 3 (Formula for calculating tumor volume: 1 / 2 × major diameter × minor diameter) 2 Mice were anesthetized with oxygen containing 2 wt% isoflurane at a flow rate of 2 L / min; after fixing the limbs and tail of the mice, 150 μCi of the molecular probe targeting NRP-1 prepared in Example 2 was dissolved in 100 µL of physiological saline.68 Ga]NOTA-PEG4-CK2 was injected via the tail vein; immediately after the probe injection, a 60-minute dynamic PET scan was performed, and the PET imaging results are shown below. Figure 11 After scanning, the OSEM3D / MAP algorithm was used to segment the 60-minute PET imaging results into 12 frames, one frame every 5 minutes, to achieve real-time analysis of in vivo imaging in mice. The region of interest (ROI) technique in ASIPRO software was used to delineate the accumulation of probes in tumor and muscle tissue. The analysis results are shown below. Figure 12 The uptake values ​​of the molecular probe in various tissues in vivo are expressed as %ID / mL (percentage of the injected dose per milliliter). The uptake of the probe in tumors and muscles was compared, and the signal-to-noise ratio of the probe in animal models was studied. The analysis results are shown in […]. Figure 13 .

[0055] Experiment 2: MDA-MB-231 cells (NRP-1 positive cells, purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences) were processed at a rate of 5 × 10⁻⁶ cells / year. 6 The recommended dose was administered subcutaneously to the right forelimb axilla of female BALB / c mice (5 weeks old, purchased from Changzhou Cavens Laboratory Animal Co., Ltd.); tumor diameter was monitored every other day, and the tumor was classified as 150.0 ± 25.0 mm when the tumor volume reached 150.0 ± 25.0 mm. 3 (Formula for calculating tumor volume: 1 / 2 × major diameter × minor diameter) 2 When the tumor was treated with the chemotherapy drug SB-203580 (purchased from Sigma-Aldrich), the treatment regimen was as follows: the dosage was 2 mg / kg, administered via tail vein injection, once every two days, for a total of 5 treatments. The tumor size was monitored during the treatment. After the treatment, the mice were anesthetized with oxygen containing 2 wt% isoflurane at a flow rate of 2 L / min. After fixing the limbs and tail of the mice, 150 μCi of the molecular probe targeting NRP-1 prepared in Example 2 was dissolved in 100 µL of physiological saline. 68 [Ga]NOTA-PEG4-CK2 was administered via tail vein injection; immediately after injection, a 60-minute dynamic PET scan was performed on the mice. The scan results are as follows: Figure 14 As shown; ROI analysis was performed on the accumulation of probes during tumor treatment, such as... Figure 15 As shown; the tumor / muscle uptake ratio is as follows: Figure 16 As shown.

[0056] from Figure 11 It can be clearly seen from the image that the molecular probe [ 68 Ga]NOTA-PEG4-CK2 uptake in MDA-MB-231 tumors was significantly higher than in NCI-H1299 tumors. Figure 12 ROI analysis showed that the molecular probe [68 The highest tumor uptake of Ga]NOTA-PEG4-CK2 was observed 10 minutes after injection, with uptake values ​​of 4.16±0.67%ID / mL in MDA-MB-231 tumors and 1.03±0.27%ID / mL in NCI-H1299 tumors, indicating that the molecular probe [ 68 Ga]NOTA-PEG4-CK2 exhibits good NRP-1 targeting. Meanwhile... Figure 13 The tumor / muscle target ratio shows that molecular probes [ 68 Ga]NOTA-PEG4-CK2 showed a good signal-to-noise ratio in MDA-MB-231 tumors, with a maximum target-to-species ratio of 5.22±0.1.8 60 min after injection, which was significantly different from that in NCI-H1299 tumors.

[0057] from Figure 14 It can be clearly seen from the image that the molecular probe [ 68 Ga]NOTA-PEG4-CK2 showed almost no scintillation in MDA-MB-231 tumors after SB-203580 treatment. Figure 15 ROI analysis showed that the molecular probe [ 68 [Ga]NOTA-PEG4-CK2 showed comparable tumor uptake to muscle uptake within 60 minutes of injection, indicating that the molecular probe [ 68 Ga]NOTA-PEG4-CK2 uptake was lower in MDA-MB-231 cells after SB-203580 treatment. Meanwhile, Figure 16 The tumor / muscle target ratio also confirms the effectiveness of molecular probes. 68 Ga]NOTA-PEG4-CK2 showed no significant uptake in MDA-MB-231 tumors treated with SB-203580. The results indicate that the molecular probe [ 68 Ga]NOTA-PEG4-CK2 can be used to evaluate the therapeutic effects of breast cancer tumors.

[0058] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A molecular probe targeting neurocilia protein-1, characterized in that, The molecular probe has the following structure: ； Wherein, R is a radionuclide labeling group.

2. The molecular probe as described in claim 1, characterized in that, The radionuclide labeling group is 68 Ga、[ 18 F]AlF、 64 Cu or 89 Zr.

3. The molecular probe as described in claim 2, characterized in that, When the radionuclide labeling group is 68 At Ga, the molecular probe has the following structure: 。 4. The molecular probe according to any one of claims 1 to 3, characterized in that, The labeled precursor of the molecular probe has the following structure: 。 5. A method for preparing the molecular probe according to any one of claims 1 to 4, characterized in that, The method includes the following steps: Step 1: Synthesize the NRP-1 targeting peptide CLKADK; Step 2: PEG-4 and NRP-1 targeting peptide CLKADK are condensed and deprotected to obtain compound CK2-PEG4; Step 3: Compound CK2-PEG4 and NOTA-NHS were reacted and deprotected under alkaline conditions to obtain the molecular probe labeled precursor NOTA-PEG4-CK2; Step 4: Radiolabel the molecular probe precursor NOTA-PEG4-CK2 with a radionuclide to obtain the molecular probe; The NRP-1 targeting peptide CLKADK has the following structure: ； The compound CK2-PEG4 has the following structure: 。 6. An NRP-1 targeting peptide, characterized in that, The amino acid sequence of the NRP-1 targeting peptide is shown in SEQ ID NO.

1.

7. The application of the molecular probe according to any one of claims 1 to 4 in neurociliacin-1 imaging, characterized in that, The application is for non-disease diagnosis and treatment purposes.

8. An imaging drug targeting neurocilia protein-1, characterized in that, The imaging drug contains the molecular probe according to any one of claims 1 to 4.

9. The use of the molecular probe according to any one of claims 1 to 4 in the preparation of tumor imaging drugs, characterized in that, The tumor is a malignant tumor that highly expresses NRP-1; the malignant tumor that highly expresses NRP-1 includes at least one of breast cancer, glioma, lung cancer, or pancreatic cancer.

Images