An NRP-1 tumor-targeting fluorescence imaging probe and a preparation method and application thereof

Abstract

The present application relates to a kind of NRP-1 tumor targeting fluorescence imaging probe and preparation method and application, belong to chemical technical field.The application provides a kind of molecular probe, the molecular probe is composed of fluorescent molecule G0 and first targeting molecule N3-PEG4-ALKADK, or, by fluorescent molecule G0, first targeting molecule N3-PEG4-ALKADK and second targeting molecule 2-azido-2-deoxy-D-glucose.The introduction of targeting molecule not only improves the hydrophilicity of the molecular probe, also improves the optical property of the molecular probe, and, the molecular probe has very strong targeting specificity to NRP-1 high expression tumor, therefore, the molecular probe has very strong application prospect in NRP-1 high expression tumor (for example, breast cancer, brain glioma, lung cancer or pancreatic cancer) detection and subsequent tumor resection visualization guidance.

Description

Technical Field

[0001] This invention relates to an NRP-1 tumor-targeting fluorescent imaging probe, its preparation method, and its application, belonging to the field of chemical technology. Background Technology

[0002] Cancer, also known as malignant tumor, is a disease caused by the abnormal control of cell growth and proliferation mechanisms. In addition to uncontrolled growth, cancer cells can invade surrounding normal tissues and even metastasize to other parts of the body via the circulatory or lymphatic systems. Common cancer treatments include surgery, radiotherapy, chemotherapy, and immunotherapy. Surgical removal of the tumor remains one of the most important cancer treatments to date. However, a significant proportion of patients still face the risk of recurrence after surgical tumor removal, largely due to the failure to completely identify and remove tumor margins and micrometastases. Therefore, a high-resolution imaging technique is needed to provide complete visualization of tumor tissue, enabling doctors to visually identify malignant lesions and successfully complete tumor resection surgery.

[0003] Near-infrared (NIR) imaging technology offers advantages such as real-time imaging, high spatial resolution, and no radioactive hazards, making fluorescence-guided surgery possible. Furthermore, compared to conventional fluorescence imaging, the NIR region (700–900 nm) exhibits lower background signal and stronger tissue penetration, making it more suitable for tissue imaging. Mayeul Collot's team designed and synthesized a tumor-targeting NIR fluorescent molecule (i.e., fluorescent molecule G0) for fluorescence imaging. This molecule is an improvement upon the fluorescent dye ICG, possessing superior optical properties and three modifiable ends, providing a basis for functional expansion and enabling its application in tumor imaging technology (see: Chemicalcommunications 2017, 53, 6117-6120).

[0004] However, the fluorescent molecule G0 belongs to the anthocyanin class of dyes, which are hydrophobic and tend to aggregate in water, significantly reducing their light utilization. Furthermore, the targeting effect of a single dye on tumors is limited, restricting its development in tumor imaging. Therefore, there is an urgent need to improve the hydrophilicity and tumor-targeting specificity of tumor-targeting NIR fluorescent molecules that can be used for fluorescence imaging, providing visualization guidance for tumor detection and subsequent tumor resection. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a molecular probe targeting neurocilia protein-1, the molecular probe comprising a first targeting molecule and a fluorescent molecule; the fluorescent molecule is an anthocyanin dye containing two modifiable alkynyl ends; the first targeting molecule comprises the NRP-1 targeting peptide ALKADK as shown in SEQ ID NO.1 and a PEG chain (azido-tetraethylene glycol-carboxyl), and the first targeting molecule is modified on one or both modifiable alkynyl ends of the fluorescent molecule GO;

[0006] Alternatively, the molecular probe may consist of a first targeting molecule, a second targeting molecule, and a fluorescent molecule; the fluorescent molecule may be an anthocyanin dye containing two modifiable alkynyl ends; the first targeting molecule may consist of the NRP-1 targeting peptide ALKADK as shown in SEQ ID NO. 1 and a PEG chain (azido-tetraethylene glycol-carboxyl); the second targeting molecule may be 2-azido-2-deoxy-D-glucose; the first and second targeting molecules may be respectively modified at the two modifiable alkynyl ends of the fluorescent molecule GO;

[0007] When the first targeting molecule is modified onto a modifiable alkyne terminus of the fluorescent molecule, the molecular probe has the following structure:

[0008]

[0009] When the first targeting molecule is modified at the two modifiable alkyne ends of the fluorescent molecule, the molecular probe has the following structure:

[0010]

[0011] When the first and second targeting molecules are respectively modified onto the two modifiable alkyne ends of the fluorescent molecule, the molecular probe has the following structure:

[0012]

[0013] The present invention also provides a method for preparing the above-mentioned molecular probe, wherein when the first targeting molecule is modified on one or two modifiable alkyne ends of a fluorescent molecule, the method comprises the following steps:

[0014] Step 1: Synthesize the fluorescent molecule G0 and the first targeting molecule N3-PEG4-ALKADK;

[0015] Step 2: The first targeting molecule N3-PEG4-ALKADK and the fluorescent molecule G0 are condensed in the presence of a catalyst to obtain a molecular probe;

[0016] When the first and second targeting molecules are respectively modified onto the two modifiable alkyne ends of the fluorescent molecule, the method includes the following steps:

[0017] Step 1: Synthesize the fluorescent molecule G0 and the first targeting molecule N3-PEG4-ALKADK;

[0018] Step 2: The first targeting molecule N3-PEG4-ALKADK and the fluorescent molecule G0 are condensed under the action of a catalyst to obtain intermediate QS-1;

[0019] Step 3: The intermediate QS-1 is condensed with the second target molecule 2-azido-2-deoxy-D-glucose under the action of a catalyst to obtain the molecular probe;

[0020] The fluorescent molecule G0 has the following structure:

[0021]

[0022] The first targeting molecule, N3-PEG4-ALKADK, has the following structure:

[0023]

[0024] In one embodiment of the present invention, the catalyst is a mixture of CuSO4·5H2O, sodium L-ascorbate, and THPTA.

[0025] In one embodiment of the present invention, the molar ratio of CuSO4·5H2O, L-ascorbic acid sodium salt, and THPTA (tris(3-hydroxypropyltriazolylmethyl)amine) in the mixture is 1:2:1.

[0026] In one embodiment of the present invention, when the first targeting molecule is modified on a modifiable alkyne end of the fluorescent molecule, step two is as follows: first, the fluorescent molecule G0 and the catalyst are dissolved in a solvent to obtain a reaction system, and then the first targeting molecule N3-PEG4-ALKADK is added dropwise to the reaction system to carry out a condensation reaction to obtain a molecular probe.

[0027] When the first targeting molecule is modified on the two modifiable alkyne ends of the fluorescent molecule, step two is as follows: first, the first targeting molecule N3-PEG4-ALKADK and the catalyst are dissolved in a solvent to obtain a reaction system, and then the fluorescent molecule G0 is added dropwise to the reaction system to carry out a condensation reaction to obtain a molecular probe.

[0028] When the first and second target molecules are respectively modified on the two modifiable alkyne ends of the fluorescent molecule, step two is as follows: first, the fluorescent molecule G0 and the catalyst are dissolved in a solvent to obtain a reaction system, and then the first target molecule N3-PEG4-ALKADK is added dropwise to the reaction system to carry out a condensation reaction to obtain intermediate QS-1. Step three is as follows: intermediate QS-1, the second target molecule 2-azido-2-deoxy-D-glucose, the solvent and the catalyst are mixed and then carried out a condensation reaction to obtain a molecular probe.

[0029] In one embodiment of the present invention, when the first targeting molecule is modified on a modifiable alkyne end of the fluorescent molecule, in step two, the molar ratio of the fluorescent molecule G0 to the first targeting molecule N3-PEG4-ALKADK is 1:0.6.

[0030] When the first targeting molecule is modified on the two modifiable alkyne ends of the fluorescent molecule, in step two, the molar ratio of the fluorescent molecule G0 to the first targeting molecule N3-PEG4-ALKADK is 1:2.2.

[0031] When the first and second target molecules are respectively modified on the two modifiable alkyne ends of the fluorescent molecule, in step two, the molar ratio of the fluorescent molecule G0 to the first target molecule N3-PEG4-ALKADK is 1:0.6, and in step three, the molar ratio of the intermediate QS-1 to the second target molecule 2-azido-2-deoxy-D-glucose is 1:1.

[0032] In one embodiment of the present invention, the solvent is a mixture of an organic solvent and water.

[0033] In one embodiment of the present invention, the volume ratio of organic solvent to water in the mixed solution is 5:1.

[0034] In one embodiment of the present invention, the organic solvent is DMF (N,N-dimethylformamide) or DMSO (dimethyl sulfoxide).

[0035] In one embodiment of the present invention, the condensation reaction is carried out at a temperature of 45°C for a time of 1.5 hours.

[0036] The present invention also provides an NRP-1 targeting peptide, the amino acid sequence of which is shown in SEQ ID NO.1.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] In one embodiment of the present invention, the imaging drug is used for early diagnosis and efficacy evaluation of tumors.

[0041] 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.

[0042] 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.

[0043] In one embodiment of the present invention, the imaging drug is used for early diagnosis and efficacy evaluation of tumors.

[0044] 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.

[0045] The technical solution of this invention has the following advantages:

[0046] 1. This invention provides a molecular probe, which is composed of a fluorescent molecule G0 and a first targeting molecule N3-PEG4-ALKADK. The fluorescent molecule G0 is an anthocyanin dye containing two modifiable alkynyl ends. The first targeting molecule N3-PEG4-ALKADK is composed of the NRP-1 targeting peptide ALKADK (as shown in SEQ ID NO.1) and a PEG chain. Furthermore, the first targeting molecule N3-PEG4-ALKADK modifies one or both modifiable alkynyl ends of the fluorescent molecule G0. The molecular probe has the following advantages:

[0047] First, the lipid-water partition coefficients of fluorescent molecule G0, molecular probe QS-1, and molecular probe QS-2 are 1.893±0.229, -0.454±0.023, and -1.117±0.036, respectively, indicating that the introduction of the first targeting molecule N3-PEG4-ALKADK significantly increased the hydrophilicity of molecular probes QS-1 and QS-2. Furthermore, the absorption and fluorescence spectra show that the introduced hydrophilic first targeting molecule N3-PEG4-ALKADK not only improved the hydrophilicity of molecular probes QS-1 and QS-2, but also significantly improved their optical properties. Therefore, the molecular probes have the advantages of good hydrophilicity, low aggregation tendency, and good optical properties.

[0048] Second, neurocilia protein-1 (NRP-1) is highly expressed in various tumor tissues and immune cells. The molecular probes have shown targeting specificity for NRP-1-highly expressing tumor cells in both in vitro cell experiments and in vivo animal experiments. In particular, in in vivo animal experiments, the molecular probes have the advantage of rapid uptake and long-term retention at the tumor site in tumor-bearing mice. This indicates that the introduction of the first targeting molecule N3-PEG4-ALKADK, which targets NRP-1, has indeed significantly increased the targeting specificity of molecular probes QS-1 and QS-2 for NRP-1-highly expressing tumors. Therefore, the molecular probes have the advantage of strong targeting specificity for NRP-1-highly expressing tumors.

[0049] In summary, the molecular probes described above show great promise for the detection of NRP-1-highly expressed tumors (e.g., breast cancer, glioma, lung cancer, or pancreatic cancer) and for the visualization guidance of subsequent tumor resection.

[0050] 2. This invention provides a molecular probe, which is composed of a fluorescent molecule G0, a first targeting molecule N3-PEG4-ALKADK, and a second targeting molecule 2-azido-2-deoxy-D-glucose. The fluorescent molecule is an anthocyanin dye containing two modifiable alkynyl ends. The first targeting molecule N3-PEG4-ALKADK is composed of the NRP-1 targeting peptide ALKADK (as shown in SEQ ID NO. 1) and a PEG chain. The first targeting molecule N3-PEG4-ALKADK and the second targeting molecule 2-azido-2-deoxy-D-glucose are respectively modified on the two modifiable alkynyl ends of the fluorescent molecule G0. The molecular probe has the following advantages:

[0051] First, the absorption and fluorescence spectra show that the introduction of the first targeting molecule N3-PEG4-ALKADK and the second targeting molecule 2-azido-2-deoxy-D-glucose significantly increases the hydrophilicity of the molecular probe DT-G0. Furthermore, after the introduction of the first targeting molecule N3-PEG4-ALKADK and the second targeting molecule 2-azido-2-deoxy-D-glucose improves the hydrophilicity of the molecular probe DT-G0, the optical properties of the molecular probe DT-G0 are also improved. Therefore, the molecular probe has the advantages of good hydrophilicity, low aggregation tendency and good optical properties.

[0052] Second, neurocilia protein-1 (NRP-1) is highly expressed in various tumor tissues and immune cells. The molecular probe has shown targeting specificity for tumor cells with high NRP-1 expression in both in vitro cell experiments and in vivo animal experiments. In particular, in in vivo animal experiments, the molecular probe has the advantage of rapid uptake and long retention at the tumor site in tumor-bearing mice. This indicates that the introduction of the first targeting molecule N3-PEG4-ALKADK and the second targeting molecule 2-azido-2-deoxy-D-glucose, which target NRP-1, has indeed significantly increased the targeting specificity of the molecular probe DT-G0 for tumors with high NRP-1 expression. Therefore, the molecular probe has the advantage of strong targeting specificity for tumors with high NRP-1 expression.

[0053] In summary, the molecular probes described above show great promise for the detection of NRP-1-highly expressed tumors (e.g., breast cancer, glioma, lung cancer, or pancreatic cancer) and for the visualization guidance of subsequent tumor resection. Attached Figure Description

[0054] Figure 1 Synthetic route of the first target molecule N3-PEG4-ALKADK.

[0055] Figure 2 Synthetic routes of molecular probes QS-1 and QS-2.

[0056] Figure 3 ESI-MS analysis of fluorescent molecule G0.

[0057] Figure 4 HPLC chromatogram of fluorescent molecule G0.

[0058] Figure 5 ESI-MS analysis of the first targeting molecule N3-PEG4-ALKADK.

[0059] Figure 6 HPLC analysis chromatogram of the first target molecule N3-PEG4-ALKADK.

[0060] Figure 7ESI-MS analysis of molecular probe QS-1.

[0061] Figure 8 HPLC analysis chromatogram of molecular probe QS-1.

[0062] Figure 9 : The proton NMR spectrum of molecular probe QS-1.

[0063] Figure 10 : Carbon NMR spectrum of molecular probe QS-1.

[0064] Figure 11 ESI-MS analysis of molecular probe QS-2.

[0065] Figure 12 HPLC analysis chromatogram of molecular probe QS-2.

[0066] Figure 13 : The proton NMR spectrum of molecular probe QS-2.

[0067] Figure 14 : Carbon NMR spectrum of molecular probe QS-2.

[0068] Figure 15 Absorption spectra, fluorescence spectra, NIR fluorescence images, and quantitative analysis results of average fluorescence intensity of fluorescent molecule G0, molecular probe QS-1, and molecular probe QS-2 (concentration 4 μM) in different solvents; where a is the absorption spectrum of the molecular probe using methanol as solvent, b is the absorption spectrum of the molecular probe using water as solvent, c is the fluorescence spectrum of the molecular probe using methanol as solvent, d is the fluorescence spectrum of the molecular probe using water as solvent, e is the NIR fluorescence image of the molecular probe using water as solvent, and f is the quantitative analysis result of average fluorescence intensity of the molecular probe using water as solvent.

[0069] Figure 16 The absorption spectra and absorbance curves of molecular probes QS-1 and QS-2 (concentrations of 0.5, 1, 2, 3, and 4 μM) in different solvents at different concentrations are shown. Among them, a is the absorption spectrum of molecular probe QS-1 using methanol as solvent, b is the absorption spectrum of molecular probe QS-1 using water as solvent, c is the absorption spectrum of molecular probe QS-2 using methanol as solvent, d is the absorption spectrum of molecular probe QS-2 using water as solvent, e is the fitted curve of molecular probe QS-1, and f is the fitted curve of molecular probe QS-2.

[0070] Figure 17NRP-1 expression in MDA-MB-231 and MDA-MB-468 cells (GAPDH as reference); where a represents the NRP-1 expression results obtained by Western blotting, and b represents the quantitative analysis of the NRP-1 expression results obtained by Western blotting.

[0071] Figure 18 The toxicity of different concentrations of molecular probes QS-1 and QS-2 (1.25, 2.5, 5, 10, and 20 μM, respectively) to MDA-MB-231 and MDA-MB-468 cells was analyzed. Among them, a represents the cell viability of MDA-MB-231 cells after incubation in the molecular probes for 48 h, and b represents the cell viability of MDA-MB-468 cells after incubation in the molecular probes for 48 h.

[0072] Figure 19 Fluorescence confocal images of molecular probes QS-1 and QS-2 (at a concentration of 2 μM) after incubation for 0.5 h in MDA-MB-231 and MDA-MB-468 cells; where Bright is the cell imaging image under bright field, DAPI is the cell nuclear imaging image, Probe is the molecular probe imaging image, and Merge is the common imaging image.

[0073] Figure 20 Flow cytometry analysis was performed on the cell uptake results of different concentrations of molecular probes QS-1 and QS-2 (0, 0.125, 0.25, 0.5, 1, and 2 μM, respectively) in MDA-MB-231 cells after incubation for 1 h.

[0074] Figure 21 The uptake results of molecular probes QS-1 and QS-2 in normal mice; where a is the NIR image of the molecular probe at different time points in normal mice, b is the quantitative result of the fluorescence intensity of the molecular probe in the kidney of normal mice at different time points, c is the hemolysis test result of QS-1, and d is the hemolysis test result of QS-2.

[0075] Figure 22 The uptake results of molecular probes QS-1 and QS-2 in MDA-MB-231 tumor-bearing mice are shown in Figure 1. Figure 2(a) shows NIR images of the molecular probes at different time points in MDA-MB-231 tumor-bearing mice; Figure 3(b) shows the quantitative fluorescence intensity of molecular probe QS-1 in the tumors and muscles of MDA-MB-231 tumor-bearing mice at different time points; Figure 4(c) shows the quantitative fluorescence intensity of molecular probe QS-2 in the tumors and muscles of MDA-MB-231 tumor-bearing mice at different time points; and Figure 5(d) shows the fluorescence intensity ratio (T / M) of the molecular probes in the tumors and muscles of MDA-MB-231 tumor-bearing mice at different time points.

[0076] Figure 23 Synthetic route of molecular probe DT-G0.

[0077] Figure 24 ESI-MS analysis of molecular probe DT-G0.

[0078] Figure 25 HPLC analysis chromatogram of molecular probe DT-G0.

[0079] Figure 26 Absorption spectra of molecular probe DT-G0 at different concentrations (0.5, 1, 2, 3, and 4 μM) in different solvents; where A represents the absorption spectra of molecular probe DT-G0 at different concentrations using methanol as solvent, and B represents the absorption spectra of molecular probe DT-G0 at different concentrations using water as solvent. The inset of Figures A and B is the fluorescence spectrum of 4 μM DT-G0. The horizontal axis of the inset is Wavelength (nm), and the vertical axis is Fluorescence Intensity 10. 3 au.

[0080] Figure 27 Fluorescence confocal images of molecular probe DT-G0 (concentration of 2 μM) and MDA-MB-231 and NCI-H1299 cells after co-incubation for 0.5 h; where Bright is the cell imaging image under bright field, DAPI is the cell nuclear imaging image, DT-G0 is the molecular probe imaging image, and Merge is the co-imaging image.

[0081] Figure 28 Cell uptake results after co-incubating different concentrations of molecular probe DT-G0 (0, 0.125, 0.25, 0.5, 1, 2 μM) with MDA-MB-231 and NCI-H1299 cells for 1 h; where the horizontal axis of the inlay is Concentration (μM) and the vertical axis is Relative Fluorescent Intensity.

[0082] Figure 29 NIR images of molecular probe DT-G0 at different time points in MDA-MB-231 tumor-bearing mice. Detailed Implementation

[0083] 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.

[0084] 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.

[0085] The 5-iodo-1-pentyne, 2,3,3-trimethylindoline, and various amino acids used in the following examples were purchased from Anaiji Chemical; the azide-tetraethylene glycol-carboxyl group was purchased from Jiangsu Aikon Company.

[0086] The absorption spectra of the molecular probes involved in the following examples were obtained using a UV-Vis spectrometer (Lambda 25; PerkinElmer), the emission spectra were obtained using steady-state and transient fluorescence spectrometers (QM / TM; PTI), and the spectra of the compounds were obtained using high-performance liquid chromatography (HPLC). The HPLC system consisted of an HPLC pump (Waters 1525; USA), a dual-wavelength absorption UV / Vis detector (Waters 2487; USA), and a C18 column (10 μm, 6 mm × 250 mm; elitehlc). The nuclear magnetic resonance spectra were recorded on a fully digital nuclear magnetic resonance spectrometer (AvanceⅢ 400 mHZ; Bruker). The mass-to-charge ratio of the compounds was verified using a quadrupole tandem mass spectrometer (SQ-detect2; Waters) and an electrospray ionization mass spectrometer (ESI-MS). NIR fluorescence images of normal mice and tumor-bearing mice were obtained using an IVIS fluorescence in vivo imaging system (CaliperLifeSciences; PerkinElmer).

[0087] Example 1: A molecular probe QS-1 targeting NRP-1

[0088] This embodiment provides a molecular probe QS-1 targeting NRP-1. The NRP-1 molecular probe QS-1 consists of a fluorescent molecule G0 and a first targeting molecule N3-PEG4-ALKADK. The fluorescent molecule G0 is an anthocyanin dye containing two modifiable alkynyl ends. The first targeting molecule N3-PEG4-ALKADK consists of the NRP-1 targeting peptide ALKADK (as shown in SEQ ID NO. 1) and a PEG chain. Furthermore, the first targeting molecule N3-PEG4-ALKADK modifies one of the modifiable alkynyl ends of the fluorescent molecule G0. The NRP-1 molecular probe QS-1 has the following structure:

[0089]

[0090] Example 2: A method for preparing the molecular probe QS-1 targeting NRP-1

[0091] This embodiment provides a method for preparing the molecular probe QS-1 targeting NRP-1 as described in Example 1 (synthetic route see...). Figures 1-2 The specific steps are as follows:

[0092] Step 1: 5-Iodo-1-pentyne (678 mg, 3.495 mmol, 1.5 eq) and 3,3-trimethylindoline (370 mg, 2.33 mmol, 1 eq) were dissolved in ultra-dry acetonitrile (10 mL) to obtain a solution. The solution was reacted at 86 °C for 24 h under nitrogen protection to obtain a reaction solution. After the reaction solution was cooled to room temperature (25 °C), the acetonitrile was first evaporated to dryness by vacuum distillation, then precipitated by diethyl ether, and finally recrystallized by acetone to obtain reaction product A (340 mg, yield 41%, white solid). Reaction product A (200 mg, 0.57 mmol, 1 eq) and 2-chloro-1-formyl 3-(hydroxymethylene)-1-cyclohexene (48.7 mg, 0.28 mmol, 0.5 eq) was dissolved in anhydrous acetonitrile (10 mL) to obtain a mixture. Acetic anhydride (78.5 μL) and sodium acetate (23.2 mg, 0.28 mmol, 0.5 eq) were added to the mixture, and the mixture was stirred at 85 °C and 150 rpm for 5 h to obtain reaction product B. Reaction product B was purified by column chromatography (mobile phase: dichloromethane solution containing 5 vt% methanol, vt% represents volume percentage) to obtain fluorescent molecule G0 (162 mg, yield 65%, dark green solid). The ESI-MS results of fluorescent molecule G0 are shown in [Figure number missing]. Figure 3 The HPLC analysis results are shown in the figure. Figure 4 C 40 H 44 ClN2 + ([M] + The calculated ESI-MS value was 588.2, and the measured value was 587.91. The purity of the product was greater than 98% according to HPLC analysis.

[0093] Step 2: Rinse the sand core funnel twice with dichloromethane, then dry it. Add 2-chlorotriphenylmethyl chloride resin (loading amount of 1.106 mmol / g, 361.66 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.

[0094] Step 3: Add amino acid Fmoc-Lys(Boc)-OH (0.5 mmol) to the sand core funnel obtained in Step 2, and dissolve it with 10 mL of DMF (N,N-dimethylformamide) to obtain a solution; add DIPEA (N,N-diisopropylethylamine) (175 μL, 1 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; 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, and shake for 10 h. After 2 min, filter under vacuum, repeating the operation once to remove unreacted amino acids; then wash twice with 10 mL DMF (HPLC type), shake for 2 min, and filter under vacuum; add 10 mL of DMF solution containing 20 vt% piperidine to the sintered glass funnel, shake for 10 min, and filter under vacuum, repeating the operation three times to remove the FMOC protecting group on the amino acid; then wash the filter cake five times with 10 mL DMF (HPLC type) to wash away excess piperidine; after washing, dry the solvent, take a sample for Kaiser test, the reagent color shows a deep purple, indicating that the FOMC group has been removed, exposing the amino group, which can be linked to the next amino acid;

[0095] Step 4: Based on Step 3, replace Fmoc-Lys(Boc)-OH (0.5mmol) sequentially with Fmoc-Asp(OtBu)-OH (0.5mmol), Fmoc-Ala-OH (0.5mmol), Fmoc-Lys(Boc)-OH (0.5mmol), Fmoc-Leu-OH (0.5mmol), and Fmoc-Ala-OH (0.5mmol);

[0096] Step 5: Add N3-PEG4-COOH (0.5 mmol) and HBTU (benzotriazole-tetramethylurea hexafluorophosphate) (1 mmol) to the sintered sand funnel obtained in Step 4, 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 a mixed solution of DMF / CH3OH / DIPEA (DMF / CH3OH / DIPEA = 7:2:1, v / v / v) to the sintered sand funnel to wash the filter cake, shake for 10 min, and filter by vacuum filtration. Repeat the operation once to remove unreacted N3-PEG4-COOH; add 10 mL of DMF / CH3OH / DIPEA to the sintered sand funnel. Wash twice with DMF (HPLC type), shake for 2 min, and filter. Add 10 mL of CH2Cl2 solution containing 1 vt% TFA (trifluoroacetic acid) to a sintered glass funnel to obtain a mixture. Shake the mixture at 25 °C for 10 min. After shaking, filter out the filtrate containing compound T (N3-PEG4-COOH). Repeat this operation until the 2-chlorotriphenylmethyl chloro resin turns wine red and does not fade. Remove the solvent from the collected filtrate using a rotary evaporator, precipitate with cold diethyl ether (4 °C), and transfer to a 5-cell precipitate. Centrifuge in 0 mL centrifuge tubes, remove supernatant by centrifugation; dry the precipitate to obtain compound T(N3-PEG4-COOH); dissolve compound T(N3-PEG4-COOH) in 2 mL of a mixed solution of DCM / TFA (DCM / TFA = 1:1, v / v), and stir at room temperature (25℃) and 150 rpm for 1 h to obtain the reaction product; remove the solvent from the reaction product under vacuum, and wash with cold (4℃) diethyl ether to obtain the first targeting molecule N3-PEG4-ALKADK (white solid). The ESI-MS results of the first targeting molecule N3-PEG4-ALKADK are shown below. Figure 5 The HPLC analysis results are shown in the figure. Figure 6 The first target molecule, N3-PEG4-ALKADK, can be used directly in subsequent reactions without further purification.

[0097] Step Six: Dissolve CuSO4·5H2O (7.88 mg, 0.032 mmol, 0.5 eq), L-ascorbic acid sodium salt (12.48 mg, 0.063 mmol, 1 eq), and THPTA ligand (13.69 mg, 0.032 mmol, 0.5 eq) in H2O (0.3 mL) to obtain a catalyst solution; add fluorescent molecule G0 (45 mg, 0.063 mmol, 1 eq) and DMF (1.5 mL) to the catalyst solution to obtain a reaction system; add compound N3-PEG4- ALKADK (34.67 mg, 0.038 mmol, 0.6 eq) was dissolved in a mixed solution (0.5 mL) of DMF and H2O (DMF:H2O = 5:1, v / v) to obtain a solution. The solution was added dropwise to the reaction system, and the mixture was stirred at 45 °C and 150 rpm for 1.5 h under nitrogen protection to obtain the reaction product. The reaction product was first purified by semi-preparative HPLC, and then dried using a vacuum freeze dryer to obtain the NRP-1-targeting molecular probe QS-1 (25 mg, yield 40.3%, dark green solid). The ESI-MS results of the NRP-1-targeting molecular probe QS-1 are shown below. Figure 7 The HPLC analysis results are shown in the figure. Figure 8 C 79 H 115 ClN 13 O 14 + ([M + The calculated ESI-MS value of the product was 1506.32, and the measured value was 1504.42. The purity of the product was greater than 98% according to HPLC analysis.

[0098] The nuclear magnetic resonance spectrum (NMR) and proton NMR spectrum of the molecular probe QS-1 targeting NRP-1 were tested (see [reference]). Figure 9 ) and carbon spectrum (see Figure 10 The data results are as follows:

[0099] Hydrogen spectrum analysis: 1HNMR (600 MHz, DMSO-d6) δ 8.27 (dd, J=17.4, 14.0 Hz, 2H), 8.16 (d, J=7.8 Hz, 1H), 8.10 (d, J=7.2 Hz, 1H), 7.99–7.93 (m, 2H), 7.93–7.86 (m, 3H), 7.81 (t, J=5.8 Hz, 6H), 7.64 (dd, J=14.2, 7.4 Hz, 2H), 7.49 (d, J=8.0 Hz, 1H), 7.45 (td, J=6.8, 6.0, 2.3 Hz, 3H), 7.35–7.26 (m, 2H), 6.37 (dd, J=24.8, 14.1 Hz, 2H), 4.56 (td, J=7.9, 5.3 Hz, 2H), 4.49 (t, J=5.3 Hz, 2H), 4.33 (t, J=7.6 Hz, 2H), 4.25 (dddd, J=19.1, 15.7, 7.8, 4.6 Hz, 6H), 4.16 (td, J=8.5, 4.9 Hz, 1H), 3.81 (t, J=5.3 Hz, 2H), 3.61–3.54 (m, 2H), 3.52 (dd, J=6.0, 3.4 Hz, 2H), 3.47 (dd, J=5.9, 3.5 Hz, 10H), 3.00 (t, J=2.6 Hz, 1H), 2.85–2.64 (m, 11H), 2.36 (ddt, J=15.0, 11.0, 7.9 Hz, 4H), 2.12 (p, J=7.4 Hz, 2H), 1.93 (p, J=7.1 Hz, 2H), 1.87 (p, J=6.3 Hz, 2H), 1.68 (s, 14H), 1.59 (dt, J=13.3, 6.8 Hz, 2H), 1.53 (h, J=5.0 Hz, 5H), 1.46 (t, J=7.0 Hz, 2H), 1.37–1.27 (m, 4H), 1.19 (dd, J=15.2, 7.0 Hz, 6H), 0.85 (dd, J=26.4, 6.5 Hz, 6H).

[0100] Carbon spectrum analysis: 13CNMR(151MHz,DMSO)δ173.68,173.24,172.86,172.55,172.51,172.42,172.05,171.54,170.93,170.51,148.61,146.00,144.03,143.21,142.5 5,142.44,141.69,141.44,129.13,129.07,126.96,126.70,125.87,125 .50,123.03,123.02,112.09,111.60,102.57,101.55,84.01,72.73,70. 20,70.18,70.12,70.07,70.01,69.94,69.21,67.17,52.59,52.11,51.60,49.79,49.75,49.62,49.39,48.68,48.53,43.93,43.17,40.94,36.46,36.26,31.56,30.91,27.98,27.92,27.05,26.98,26.85,26.42,26.35,26.31,24.61,23.47,22.59,22.46,22.04,20.85,18.58,18.46,15.77.

[0101] Example 3: A molecular probe QS-2 targeting NRP-1

[0102] This embodiment provides a molecular probe QS-2 targeting NRP-1. The NRP-1-targeting molecular probe QS-2 consists of a first targeting molecule N3-PEG4-ALKADK and a fluorescent molecule G0. The fluorescent molecule G0 is an anthocyanin dye containing two modifiable alkynyl ends. The first targeting molecule N3-PEG4-ALKADK consists of the NRP-1 targeting peptide ALKADK (as shown in SEQ ID NO. 1) and a PEG chain. Furthermore, the first targeting molecule N3-PEG4-ALKADK modifies the two modifiable alkynyl ends of the fluorescent molecule G0. The NRP-1-targeting molecular probe QS-1 has the following structure:

[0103]

[0104] Example 4: A method for preparing the molecular probe QS-2 targeting NRP-1

[0105] This embodiment provides a method for preparing the NRP-1-targeting molecular probe QS-2 described in Example 3 (synthetic route see...). Figures 1-2 The specific steps are as follows:

[0106] Based on Example 3, step six was replaced as follows: CuSO4·5H2O (2.79 mg, 0.011 mmol, 0.5 eq), L-ascorbic acid sodium salt (4.42 mg, 0.022 mmol, 1 eq), and THPTA ligand (4.85 mg, 0.011 mmol, 0.5 eq) were dissolved in H2O (0.3 mL) to obtain a catalyst solution; compound N3-PEG4-ALKADK (45 mg, 0.049 mmol, 2.2 eq) and DMF (1.5 mL) were added to the catalyst solution. The reaction system was obtained; G0 (15.92 mg, 0.022 mmol, 1 eq) was dissolved in a mixed solution (0.5 mL) of DMF and H2O (DMF:H2O = 5:1, v / v) to obtain a solution; the solution was added dropwise to the reaction system, and the mixture was stirred at 45 °C and 150 rpm for 1.5 h under nitrogen protection to obtain the reaction product; the reaction product was first separated and purified by semi-preparative HPLC, and then dried by vacuum freeze dryer to obtain the NRP-1 targeting molecular probe QS-2 (20 mg, yield 35%, dark green solid). The ESI-MS results of the NRP-1 targeting molecular probe QS-2 are shown in the figure. Figure 11 The HPLC analysis results are shown in the figure. Figure 12 C 118 H 186 ClN 24 O 28 + ([M+H)) 2+ The calculated ESI-MS value was 1212.18, and the measured value was 1212.82. The HPLC analysis showed that the product purity was greater than 98%.

[0107] The nuclear magnetic resonance spectrum (NMR) and proton NMR spectrum of the molecular probe QS-2 targeting NRP-1 were tested (see [reference]). Figure 13 ) and carbon spectrum (see Figure 14 The data results are as follows:

[0108] Hydrogen spectrum analysis: 1HNMR (600 MHz, DMSO-d6) δ 8.26 (d, J=13.9 Hz, 2H), 8.15 (d, J=7.8 Hz, 2H), 8.08 (d, J=7.2 Hz, 2H), 7.94 (dd, J=7.6, 4.3 Hz, 4H), 7.91 (d, J=4.4 Hz, 4H), 7.88 (d, J=7.9 Hz, 2H), 7.77 (t, J=5.8 Hz, 11H), 7.64 (d, J=7.3 Hz, 2H), 7.49–7.41 (m, 4H), 7.30 (td, J=7.1, 1.4 Hz, 2H), 6.35 (d, J=14.1 Hz, 2H), 4.56 (td, J=7.9, 5.3 Hz, 4H), 4.49 (t, J=5.3 Hz, 4H), 4.31 (t, J=7.5 Hz, 4H), 4.24 (ddt, J=18.5, 8.4, 6.0 Hz, 9H), 4.15 (td, J=8.4, 5.0 Hz, 2H), 3.80 (t, J=5.2 Hz, 4H), 3.60–3.53 (m, 4H), 3.52 (dd, J=5.8, 3.3 Hz, 4H), 3.47 (dd, J=5.8, 3.5 Hz, 20H), 2.82–2.63 (m, 18H), 2.42–2.29 (m, 4H), 2.11 (p, J=7.4 Hz, 4H), 1.86 (p, J=6.2 Hz, 2H), 1.68 (s, 16H), 1.59 (dt, J=12.7, 6.3 Hz, 4H), 1.52 (td, J=9.4, 8.7, 4.3 Hz, 10H), 1.47–1.41 (m, 4H), 1.37–1.25 (m, 8H), 1.19 (dd, J=15.4, 7.0 Hz, 12H), 0.85 (dd, J=26.2, 6.5 Hz, 12H).

[0109] Carbon spectrum analysis: 13CNMR(151MHz,DMSO)δ173.69,172.84,172.77,172.54,172.48,172.04,171.51,170.93,170.48,148.57,146.02,14 3.60,142.51,141.57,129.10,126.79,125.67,123.02,117.86,115.91,111.91,102.10,70.20,70.18,70.12,70.0 7,70.01,69.94,69.21,67.18,52.56,52.11,51.57,49.78,49.74,49.49,48.64,48.50,43.83,40.98,40.52,39.17,39.08,31.58,30.92,27.95,27.05,26.98,26.82,26.33,24.61,23.48,22.59,22.49,22.05,20.92,18.61,18.47.

[0110] Experimental Example 1: Physicochemical Properties of Molecular Probes Targeting NRP-1

[0111] This experimental example provides the physicochemical properties of a molecular probe targeting NRP-1, and the specific procedure is as follows:

[0112] Experiment 1: Determination of Absorption and Fluorescence Spectra

[0113] Methanol was used as the solvent to prepare 1 mM stock solutions of fluorescent molecule G0, molecular probe QS-1, and molecular probe QS-2. The stock solutions were diluted with methanol and water to different concentrations (4 μM, 3 μM, 2 μM, 1 μM, 0.5 μM) to obtain diluted solutions of fluorescent molecule G0, molecular probe QS-1, and molecular probe QS-2 at different concentrations. The absorption spectra of the diluted solutions of fluorescent molecule G0, molecular probe QS-1, and molecular probe QS-2 at different concentrations were measured, and the fluorescence spectrum of the 4 μM diluted solution of fluorescent molecule G0, molecular probe QS-1, and molecular probe QS-2 was also measured (the detection results are shown in [link to results]). Figures 15-16 ).

[0114] Depend on Figures 15-16It can be seen that in methanol, molecular probes QS-1 and QS-2 have absorption peaks at 782 nm and 783 nm, respectively, while the absorption peak of the fluorescent molecule G0 is at 780 nm; both molecular probes QS-1 and QS-2 have fluorescence emission peaks at 807 nm, while the emission peak of the fluorescent molecule G0 is at 804 nm. These subtle wavelength changes indicate that the introduction of the first targeting molecule N3-PEG4-ALKADK does not change the basic fluorescence properties of the fluorescent molecule G0. In methanol, the maximum absorbances of 4 μM dilutes of fluorescent molecule G0, molecular probe QS-1, and molecular probe QS-2 were 1.01, 1.08, and 1.04, respectively, with maximum fluorescence intensities of 12036 a.u., 12545 a.u., and 12048 a.u., respectively. In water, however, the maximum absorbances of the same 4 μM dilutes were 0.53, 0.67, and 0.75, respectively, with maximum fluorescence intensities of 2815 a.u., 4313 a.u., and 5564 a.u., respectively. These changes in absorption and fluorescence spectra indicate that the introduction of the hydrophilic first targeting molecule N3-PEG4-ALKADK to improve the hydrophilicity of the molecular probes also improved their optical properties. IVIS spectral imaging also showed that molecular probes QS-1 and QS-2 exhibited stronger fluorescence intensity in water compared to fluorescent molecule G0. In water, the fluorescence intensities of molecular probes QS-1 and QS-2 were 1.64 times and 1.79 times that of fluorescent molecule G0, respectively.

[0115] Experiment 2: Determination of the lipid-water partition coefficient

[0116] Fluorescent molecule G0, molecular probe QS-1, and molecular probe QS-2 were diluted to a concentration of 100 μM with water to obtain diluted solutions of fluorescent molecule G0, molecular probe QS-1, and molecular probe QS-2, respectively. 20 μL of each diluted solution was placed in a 5 mL centrifuge tube, and 1 mL of n-octanol and 1 mL of water were added to obtain a mixture. The mixture was shaken at room temperature (25 °C) for 3 min, and then centrifuged at 4000 g for 3 min to break the emulsion and separate the two phases. After static separation, 150 μL of the n-octanol phase was taken to measure its absorbance. The lipid-water partition coefficients of fluorescent molecule G0, molecular probe QS-1, and molecular probe QS-2 were calculated using the formula: LogP = log(Co / CW), where Co represents the concentration of the probe in n-octanol, and Cw represents the concentration of the probe in water.

[0117] The lipid-water partition coefficients of fluorescent molecule G0, molecular probe QS-1, and molecular probe QS-2 were 1.893±0.229, -0.454±0.023, and -1.117±0.036, respectively, indicating that the introduction of the first targeting molecule N3-PEG4-ALKADK significantly increased the hydrophilicity of molecular probes QS-1 and QS-2.

[0118] Experiment Example 2: Biocompatibility Experiment of Molecular Probes Targeting NRP-1

[0119] The expression of NRP-1 protein in triple-negative breast cancer cells MDA-MB-231 and MDA-MB-468 was determined by Western blot. The results are shown in the figure below. Figure 17 .from Figure 17 As can be seen, MDA-MB-231 cells highly express NRP-1 and can be used as NRP-1 positive tumor cells; MDA-MB-468 cells lowly express NRP-1 and can be used as NRP-1 negative tumor cells. These two cell types can be used as negative and positive controls for subsequent cell and animal experiments.

[0120] This experiment used MDA-MB-231 and MDA-MB-468 cells to demonstrate the biocompatibility of the molecular probe targeting NRP-1. The specific procedure is as follows:

[0121] Experiment 1: Determination of Cytotoxicity

[0122] The cytotoxicity of molecular probes QS-1 and QS-2 on MDA-MB-231 and MDA-MB-468 cells was investigated using the MTT assay. Molecular probes QS-1 and QS-2 were diluted to different concentrations (1.25, 2.5, 5, 10, 20 μM) with complete culture medium (purchased from BI), resulting in different concentrations of QS-1 and QS-2 dilutions. MDA-MB-231 and MDA-MB-468 cells were then incubated with 1×10⁻⁶ cells / mL. 4The cells were seeded at a density of 100 μL of complete culture medium into 96-well cell culture plates and cultured at 37°C in a 5% (v / v) CO2 incubator for 24 h to allow cell adhesion. After cell adhesion, using no molecular probe as a control, 200 μL of different concentrations of molecular probe QS-1 and molecular probe QS-2 dilutions were added to 96-well cell culture plates and incubated at 37°C in a 5% (v / v) CO2 incubator. After incubation for 48 h, 20 μL of MTT (3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide) aqueous solution (5.0 mg / mL) was added to each well and incubated at 37°C in a 5% (v / v) CO2 incubator. Incubation; after 4 hours of incubation, remove the medium containing MTT, add DMSO (150 μL) to each well and shake for 15 minutes to dissolve formazan; measure the average absorbance of each well at 490 nm using a microplate reader, and calculate the cell viability of MDA-MB-231 and MDA-MB-468 cells after incubation with different concentrations of molecular probes QS-1 and QS-2 for different times according to the formula: Cell viability (%) = Average absorbance of experimental wells / Average absorbance of control wells × 100%. This allows for the evaluation of the toxicity of different concentrations of molecular probes QS-1 and QS-2 to MDA-MB-231 and MDA-MB-468 cells (see results below). Figure 18 ).

[0123] Depend on Figure 18 The results showed that even after incubation for 48 hours at high concentrations of molecular probes QS-1 and QS-2 (20 μM), the survival rate of MDA-MB-231 and MDA-MB-468 cells remained above 80%. Compared with molecular probe QS-1, at the same concentration, molecular probe QS-2 exhibited lower toxicity to MDA-MB-231 and MDA-MB-468 cells. These results demonstrate that the biocompatibility of molecular probes QS-1 and QS-2 can be improved by introducing hydrophilic groups, thus validating the feasibility of using molecular probes QS-1 and QS-2 as biological probes for subsequent biological research.

[0124] Experiment 2: Determination of Hemolysis Rate

[0125] Molecular probes QS-1 and QS-2 were diluted to different concentrations (0.25, 0.5, 1, 2, and 4 μM) with physiological saline to obtain different concentrations of QS-1 and QS-2 dilutions. 0.5 mL of blood from healthy mice (Balb-nu strain, 8–9 weeks old, purchased from Changzhou Cavens Company) was added to heparin-coated tubes. Physiological saline was added to the tubes, and red blood cells were collected by centrifugation. The red blood cells were washed three times with physiological saline and resuspended to a volume percentage concentration of 2% to obtain a red blood cell suspension. 200 μL of the red blood cell suspension was then divided... Do not mix with 800 μL of water (positive control), physiological saline (negative control), or different concentrations of molecular probe QS-1 and molecular probe QS-2 dilutions to obtain a suspension; incubate the suspension at 37℃ for 4 h and then centrifuge; take 200 μL of the supernatant and measure the absorbance at 545 nm using an ELISA reader, and calculate the hemolysis rate of red blood cells according to the formula: hemolysis rate = ([experimental group - negative control] / [positive control - negative control]) × 100%, and then evaluate the hemolytic effect of different concentrations of molecular probe QS-1 and molecular probe QS-2 on red blood cells (the test results are shown in 21).

[0126] Depend on Figure 21 It can be seen that the hemolysis rates of molecular probes QS-1 and QS-2 are generally low, both below 1%, indicating that the hemolytic effect of molecular probes QS-1 and QS-2 on erythrocytes is not significant. The results show that molecular probes QS-1 and QS-2 have good blood compatibility and can be used for intravenous injection.

[0127] Experiment Example 3: Cell Imaging Experiment with Molecular Probes Targeting NRP-1

[0128] This experiment used MDA-MB-231 and MDA-MB-468 cells to provide cell imaging experiments using molecular probes targeting NRP-1. The specific procedure is as follows:

[0129] Experiment 1: Measurement of Fluorescence Images

[0130] MDA-MB-231 and MDA-MB-468 cells were cultured at a density of 2 × 10⁶ cells per well. 5Cells were seeded at a density of 1 mL in NEST laser confocal microscopy glass-bottomed culture dishes containing 1 mL of complete culture medium and cultured at 37°C in a 5% (v / v) CO2 cell culture incubator. After 16 h of culture, 1 mL of 4% (m / v, g / 100 mL) paraformaldehyde fixative containing 0.1% (m / v, g / 100 mL) Triton X-100 was added to the dish, and the cells were fixed at 37°C for 0.5 h. After fixation, the cells were washed three times with PBS buffer. After washing, 1 mL of fresh complete culture medium containing 2 μM molecular probe QS-1 or molecular probe QS-2 was added to the dish, and the cells were incubated at 37°C in the dark for 30 min. Then, the medium was replaced with an equal volume of DAPI aqueous solution (DAPI:water = 1:1000) for staining. After staining for 5 min, the cells were washed three times with PBS buffer to remove free dye, and then fluorescence images of the cells were recorded using a fluorescence confocal scanning microscope (see results below). Figure 19 ).

[0131] Depend on Figure 19 It was observed that both molecular probes QS-1 and QS-2 were extensively internalized into NRP-1-positive MDA-MB-231 cells, exhibiting strong fluorescence. However, only a small amount of fluorescence was observed in NRP-1-negative MDA-MB-468 cells. This demonstrates that both molecular probes QS-1 and QS-2 have a strong targeting effect on tumor cells highly expressing NRP-1, and their targeting is consistent with the expression level of NRP-1. Furthermore, the fluorescence intensity of molecular probe QS-2 was weaker than that of molecular probe QS-1 in MDA-MB-231 cells. This may be because the cell membrane is composed of phospholipids, which have lipophilic properties; the increased hydrophilicity of the probes, in turn, makes it more difficult for them to enter the cells.

[0132] Experiment 2: Measurement of Cellular Uptake

[0133] Molecular probes QS-1 and QS-2 were diluted to different concentrations (0.125, 0.25, 0.5, 1, 2 μM) using DMEM medium (purchased from BI), resulting in different concentrations of QS-1 and QS-2 dilutions. MDA-MB-231 cells in logarithmic growth phase were then divided into two groups of 2 × 10⁶ cells per tube. 5 The dose of each cell was suspended in sterile tubes with 1 mL of different concentrations of molecular probe QS-1 and molecular probe QS-2 dilutions, and then incubated in a 37°C, 5% (v / v) CO2 cell culture incubator. After 1 h of incubation, the cell fluorescence intensity was detected by flow cytometry. 20,000 target cells were taken for each sample (results are shown in [link to results]). Figure 20 ).

[0134] like Figure 20As shown, the fluorescence intensity increases with increasing concentrations of molecular probes QS-1 and QS-2, exhibiting a significant linear relationship (R0). 2 QS-1 =0.996, R 2 QS-2 =0.986), verifying that molecular probes QS-1 and QS-2 specifically bind to tumor cells with high NRP-1 expression in a concentration-dependent manner. Furthermore, the uptake of molecular probe QS-1 in MDA-MB-231 cells was higher than that of molecular probe QS-2, consistent with confocal imaging results.

[0135] Experiment Example 4: Mouse Imaging Experiment with Molecular Probes Targeting NRP-1

[0136] This experimental example provides a mouse fluorescence imaging experiment using a molecular probe targeting NRP-1. The specific procedure is as follows:

[0137] Experiment 1: Imaging Experiment of Normal Mice

[0138] Molecular probes QS-1 and QS-2 were diluted to a concentration of 25 μM using physiological saline to obtain diluents of QS-1 and QS-2, respectively. Normal mice (Balb-nu strain, 8–9 weeks old, purchased from Changzhou Cavens Biotechnology Co., Ltd.) were anesthetized with oxygen containing 2 wt% isoflurane at a flow rate of 2 L / min. After fixing the limbs and tails of the normal mice, 200 μL of the diluents of QS-1 and QS-2 were injected into the tail veins of the normal mice, respectively. After the probe injection, scans were performed at different time points using an IVIS small animal in vivo imaging system (scanning results are shown in [link to IVIS small animal in vivo imaging system]). Figure 21 ).

[0139] like Figure 21 As shown, significant fluorescent signals were observed in the kidneys of normal mice injected with both molecular probes QS-1 and QS-2, indicating that the kidney is one of the main metabolic organs for both molecular probes QS-1 and QS-2 in normal mice. 24 hours after injection, molecular probe QS-2 was almost completely cleared from normal mice. 36 hours after injection, significant fluorescent signals were still observed in molecular probe QS-1, and 48 hours after injection, molecular probe QS-1 was also almost completely cleared from normal mice. The higher clearance rate of molecular probe QS-2 is mainly due to its high hydrophilicity. These results indicate that molecular probes QS-1 and QS-2 have relatively rapid clearance rates.

[0140] Experiment 2: Imaging Experiment of Tumor-Bearing Mice

[0141] Molecular probes QS-1 and QS-2 were diluted to a concentration of 25 μM using physiological saline to obtain dilutions of molecular probes QS-1 and QS-2, respectively. MDA-MB-231 cells (NRP-1 positive cells, purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences) were then cultured at a concentration of 1 × 10⁻⁶ cells / cell. 7 A dose (suspended in 100 μL PBS buffer) was subcutaneously injected into the right forelimb axilla of normal mice (Balb-nu strain, 6–7 weeks old, purchased from Changzhou Cavens Biotechnology Co., Ltd.); the tumor diameter was monitored every other day, and when the tumor volume reached 200.0 ± 25.0 mm... 3 (Formula for calculating tumor volume: 1 / 2 × major diameter × minor diameter) 2 During the procedure, tumor-bearing 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 tumor-bearing mice, 200 μL of diluted molecular probes QS-1 and QS-2 were injected into the tail vein of the tumor-bearing mice, respectively. After the probe injection, scans were performed at different time points using an IVIS small animal in vivo imaging system (scanning results are shown in [link to IVIS small animal in vivo imaging system]). Figure 22 ).

[0142] like Figure 22As shown, 1 hour after injection, both molecular probes QS-1 and QS-2 exhibited fluorescence signals at the tumor site, demonstrating their rapid tumor-targeting ability. However, QS-2 was almost completely metabolized and cleared from non-target organs 24 hours after injection, while QS-1 remained cleared from non-target organs for up to 60 hours. This is consistent with findings in normal mice, attributed to differences in their hydrophilicity; the improved hydrophilicity of QS-2 accelerated its clearance in the animal body. Furthermore, both QS-1 and QS-2 demonstrated long-term tumor retention. QS-1 still showed fluorescence at the tumor site 132 hours after injection, while QS-2 maintained its fluorescence signal for up to 72 hours even with increased clearance rates. Such extended tumor imaging is beneficial for effective disease detection. Over time, the uptake trends of QS-1 and QS-2 in tumors and muscle were roughly the same. Furthermore, both molecular probes QS-1 and QS-2 reached their maximum tumor uptake around 24 hours after injection. However, QS-1 showed a more gradual accumulation and clearance at the tumor site, leading to a gradual increase in its tumor-to-muscle ratio (T / M) in the latter half of the timeframe. In contrast, QS-2 exhibited strong uptake at the tumor site within a very short time and was rapidly cleared after reaching its peak. Even so, its T / M remained above 3 throughout the subsequent timeframe. In conclusion, both probes demonstrated rapid tumor-specific targeting and long-term retention in tumor-bearing mice, fully demonstrating their application potential in tumor detection and the feasibility of further extended research.

[0143] Example 5: A molecular probe DT-G0 targeting NRP-1

[0144] This embodiment provides a molecular probe DT-G0 targeting NRP-1. DT-G0 consists of a fluorescent molecule G0, a first targeting molecule N3-PEG4-ALKADK, and a second targeting molecule 2-azido-2-deoxy-D-glucose. The fluorescent molecule G0 is an anthocyanin dye containing two modifiable alkynyl ends. The first targeting molecule N3-PEG4-ALKADK consists of the NRP-1 targeting peptide ALKADK (as shown in SEQ ID NO. 1) and a PEG chain. The first targeting molecule N3-PEG4-ALKADK and the second targeting molecule 2-azido-2-deoxy-D-glucose are respectively modified at the two modifiable alkynyl ends of the fluorescent molecule G0. The molecular probe DT-G0 targeting NRP-1 has the following structure:

[0145]

[0146] Example 6: A method for preparing DT-G0, a molecular probe targeting NRP-1.

[0147] This embodiment provides a method for preparing the NRP-1-targeting molecular probe DT-G0 described in Example 5 (synthetic route see...). Figure 23 The specific steps are as follows:

[0148] Using the NRP-1-targeting molecular probe QS-1 prepared in Example 2 as intermediate QS-1; CuSO4·5H2O (1.53 mg, 0.006 mmol, 0.5 eq), L-ascorbic acid sodium salt (2.43 mg, 0.012 mmol, 1 eq) and THPTA ligand (2.66 mg, 0.006 mmol, 0.5 eq) were dissolved in H2O (0.3 mL) to obtain a catalyst solution; intermediate QS-1 (20 mL) was added to the catalyst solution. After adding g (0.012 mmol, 1 eq), 2-azido-2-deoxy-D-glucose (2.51 mg, 0.012 mmol, 1 eq) and DMF (1.5 mL), the mixture was stirred at 45 °C and 150 rpm for 1.5 h under nitrogen protection to obtain the reaction product. The reaction product was first separated and purified by semi-preparative HPLC, and then dried by vacuum freeze dryer to obtain the NRP-1 targeting molecular probe DT-G0 (10 mg, yield 47.6%, dark green solid). The ESI-MS results of the NRP-1 targeting molecular probe DT-G0 are shown in the figure. Figure 24 The HPLC analysis results are shown in the figure. Figure 25 C 85 H 126 ClN 16 O 19 + ([M] + The calculated ESI-MS value was 1711.49, and the measured value was 1711.36. The HPLC analysis showed that the product purity was greater than 98%.

[0149] Experimental Example 5: Physicochemical Properties of DT-G0, a Molecular Probe Targeting NRP-1

[0150] This experimental example provides the physicochemical properties of the NRP-1-targeting molecular probe DT-G0, and the specific procedure is as follows:

[0151] Methanol was used as the solvent to prepare 25 mM stock solutions of fluorescent molecule G0 and molecular probe DT-G0. The stock solutions were then diluted with methanol and water to different concentrations (4 μM, 3 μM, 2 μM, 1 μM, 0.5 μM) to obtain diluted solutions of fluorescent molecule G0 and molecular probe DT-G0 at different concentrations. The absorption spectra of the diluted solutions of fluorescent molecule G0 and molecular probe DT-G0 at different concentrations were measured (see [Results]). Figure 26 The fluorescence spectra of the fluorescent molecule G0 dilution and the molecular probe DT-G0 dilution at a concentration of 4 μM were measured (see the detection results). Figure 26 Embedded image).

[0152] Depend on Figure 26 It can be seen that in methanol, the molecular probe DT-G0 has an absorption peak at 783 nm, while the fluorescent molecule G0 has an absorption peak at 780 nm; the molecular probe DT-G0 has a fluorescence emission peak at 807 nm, while the fluorescent molecule G0 has an emission peak at 804 nm. These small wavelength changes indicate that the introduction of the first targeting molecule N3-PEG4-ALKADK and the second targeting molecule 2-azido-2-deoxy-D-glucose does not change the basic fluorescence properties of the fluorescent molecule G0. In methanol, the maximum absorbance of the 4 μM molecular probe DT-G0 dilution is 1.06, and the maximum fluorescence intensity is 12365 a.u., which is not significantly different from that of the fluorescent molecule G0 dilution. However, in water, the maximum absorbance of the 4 μM molecular probe DT-G0 dilution is 0.72, and the maximum fluorescence intensity is 5374 a.u. (G0≈3000 a.u.), showing a significant enhancement in both maximum absorbance and fluorescence intensity. These changes in the absorption and fluorescence spectra show that the optical properties of the molecular probe DT-G0 are improved by introducing a hydrophilic targeting molecule to enhance its hydrophilicity. These experimental results demonstrate that the molecular probe DT-G0 is a cyanin probe with strong hydrophilicity and superior optical properties.

[0153] Experiment Example 6: Cell imaging experiment using the NRP-1-targeting molecular probe DT-G0

[0154] This experiment used MDA-MB-231 cells and NCI-H1299 cells to provide cell imaging experiments using the NRP-1-targeting molecular probe DT-G0. The specific procedure is as follows:

[0155] Experiment 1: Measurement of Fluorescence Images

[0156] The procedure for measuring the fluorescence image is the same as Experiment 1 in Example 3, and the detection results are shown in [see attached image]. Figure 27 .

[0157] Depend on Figure 27It can be seen that the molecular probe DT-G0 was internalized in large quantities into NRP-1 positive cells MDA-MB-231, and strong fluorescence was detected in the non-nuclear locations of the cells. In contrast, only a small amount of fluorescence was observed in NRP-1 negative cells NCI-H1299, and red fluorescence was observed in the nucleus. This proves that DT-G0 has a strong targeting effect on tumor cells that highly express NRP-1, and the targeting is consistent with the expression level of NRP-1.

[0158] Experiment 2: Measurement of Cellular Uptake

[0159] The procedure for measuring cellular uptake is the same as Experiment 2 in Example 3, and the detection results are shown below. Figure 28 .

[0160] like Figure 28 As shown, the fluorescence intensity increases with increasing concentration of the molecular probe DT-G0, exhibiting a significant linear relationship (R0). 2 =0.98), which verified that the molecular probe DT-G0 specifically binds to tumor cells with high NRP-1 expression and is concentration-dependent.

[0161] Experiment Example 7: Mouse Imaging Experiment of NRP-1 Targeting Molecular Probe DT-G0

[0162] This experimental example provides a mouse fluorescence imaging experiment using the NRP-1-targeting molecular probe DT-G0. The specific procedure is as follows:

[0163] The procedure for the mouse fluorescence imaging experiment is the same as Experiment 2 in Experiment 3. The scanning results are shown in […]. Figure 29 .

[0164] like Figure 29 As shown, DT-G0 exhibited a certain fluorescence signal at the tumor site 1 hour after injection, indicating its rapid tumor-targeting capability. In non-target organ sites, DT-G0 was completely cleared after 12 hours, while the fluorescence signal at the tumor site persisted until 72 hours. Therefore, after 12 hours, the fluorescence signal was primarily observed at the tumor site, fully demonstrating the excellent tumor-specific imaging capability of DT-G0 and its significant application potential in tumor diagnosis.

[0165] 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 consists of a first target molecule and a fluorescent molecule; the fluorescent molecule has two modifiable alkyne ends; the first target molecule is modified on one or both modifiable alkyne ends of the fluorescent molecule. Alternatively, the molecular probe is composed of a first targeting molecule, a second targeting molecule, and a fluorescent molecule; the fluorescent molecule has two modifiable alkyne ends; the first and second targeting molecules are respectively modified on the two modifiable alkyne ends of the fluorescent molecule; When the first targeting molecule is modified onto a modifiable alkyne terminus of the fluorescent molecule, the molecular probe has the following structure: ； When the first targeting molecule is modified at the two modifiable alkyne ends of the fluorescent molecule, the molecular probe has the following structure: ； When the first and second targeting molecules are respectively modified onto the two modifiable alkyne ends of the fluorescent molecule, the molecular probe has the following structure: 。 2. A method for preparing the molecular probe according to claim 1, characterized in that, When the first targeting molecule is modified on one or both modifiable alkyne ends of a fluorescent molecule, the method includes the following steps: Step 1: Synthesize the fluorescent molecule G0 and the first targeting molecule N3-PEG4-ALKADK; Step 2: The first targeting molecule N3-PEG4-ALKADK and the fluorescent molecule G0 are condensed in the presence of a catalyst to obtain a molecular probe; When the first and second targeting molecules are respectively modified onto the two modifiable alkyne ends of the fluorescent molecule, the method includes the following steps: Step 1: Synthesize the fluorescent molecule G0 and the first targeting molecule N3-PEG4-ALKADK; Step 2: The first targeting molecule N3-PEG4-ALKADK and the fluorescent molecule G0 are condensed under the action of a catalyst to obtain intermediate QS-1; Step 3: The intermediate QS-1 is condensed with the second target molecule 2-azido-2-deoxy-D-glucose under the action of a catalyst to obtain the molecular probe; The fluorescent molecule G0 has the following structure: ； The first targeting molecule, N3-PEG4-ALKADK, has the following structure: 。 3. The method as described in claim 2, characterized in that, The catalyst is a mixture of CuSO4·5H2O, sodium L-ascorbate, and THPTA; the molar ratio of CuSO4·5H2O, sodium L-ascorbate, and THPTA in the mixture is 1:2:

1.

4. The method as described in claim 2 or 3, characterized in that, When the first targeting molecule is modified on a modifiable alkyne end of the fluorescent molecule, step two is as follows: first, the fluorescent molecule G0 and the catalyst are dissolved in a solvent to obtain a reaction system, and then the first targeting molecule N3-PEG4-ALKADK is added dropwise to the reaction system to carry out a condensation reaction to obtain a molecular probe. When the first targeting molecule is modified on the two modifiable alkyne ends of the fluorescent molecule, step two is as follows: first, the first targeting molecule N3-PEG4-ALKADK and the catalyst are dissolved in a solvent to obtain a reaction system, and then the fluorescent molecule G0 is added dropwise to the reaction system to carry out a condensation reaction to obtain a molecular probe. When the first and second target molecules are respectively modified on the two modifiable alkyne ends of the fluorescent molecule, step two is as follows: first, the fluorescent molecule G0 and the catalyst are dissolved in a solvent to obtain a reaction system, and then the first target molecule N3-PEG4-ALKADK is added dropwise to the reaction system to carry out a condensation reaction to obtain intermediate QS-1. Step three is as follows: intermediate QS-1, the second target molecule 2-azido-2-deoxy-D-glucose, the solvent and the catalyst are mixed and then carried out a condensation reaction to obtain a molecular probe.

5. The method as described in claim 2 or 3, characterized in that, When the first targeting molecule is modified on a modifiable alkyne end of the fluorescent molecule, in step two, the molar ratio of the fluorescent molecule G0 to the first targeting molecule N3-PEG4-ALKADK is 1:0.

6. When the first targeting molecule is modified on the two modifiable alkyne ends of the fluorescent molecule, in step two, the molar ratio of the fluorescent molecule G0 to the first targeting molecule N3-PEG4-ALKADK is 1:2.

2. When the first and second target molecules are respectively modified on the two modifiable alkyne ends of the fluorescent molecule, in step two, the molar ratio of the fluorescent molecule G0 to the first target molecule N3-PEG4-ALKADK is 1:0.6, and in step three, the molar ratio of the intermediate QS-1 to the second target molecule 2-azido-2-deoxy-D-glucose is 1:

1.

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 claim 1 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 of claim 1.

9. The application of the molecular probe according to claim 1 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