Targeting vegr-3 polypeptides, uses, tumor-targeting imaging probes, and methods of making

Abstract

The present application relates to the technical field of bioengineering, and particularly relates to a VEGFR-3 targeting polypeptide, application, a tumor targeting imaging probe and a preparation method. The targeting polypeptide is TMVP1446 / GS5, the amino acid sequence of the TMVP1446 / GS5 is shown as SEQ ID NO:1, and the tumor targeting imaging probe is TMVP1446 / GS5-Cy7. The targeting VEGFR-3 polypeptide has good targeting capability for primary tumors and metastatic lymph nodes; the tumor targeting imaging probe has excellent metabolic stability, can specifically target primary tumors in the body, has excellent targeting capability for lymph node metastasis, and is a good reagent for visualizing lymph node metastasis; the preparation method has the advantages of simple steps, mild conditions, easy operation, high efficiency and low cost.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to a VEGFR-3 targeted polypeptide, its application, a tumor-targeted imaging probe and a preparation method. Background Art

[0002] Most cancer patients die from tumor metastasis rather than the primary tumor itself. Tumor metastasis is a complex and multi-stage process involving many key steps: cancer cells migrate from the primary tumor, invade the blood or lymphatic system, extravasate to distant organs, and form metastatic lesions. Studies have shown that tumor cells can stimulate the formation of lymphatic vessels and promote tumor metastasis. Tumor-induced new lymphatic vessels can occur both within the tumor and in the surrounding area of ​​the tumor and are associated with tumor metastasis and poor patient prognosis. Therefore, early tracking of lymphangiogenesis is an ideal strategy for tumor diagnosis and prognosis assessment.

[0003] Vascular endothelial growth factor receptor 3 (VEGFR-3) is the primary receptor for the best-known lymphangiogenic growth factors, vascular endothelial growth factor C (VEGF-C) and VEGF-D, mediating lymphangiogenesis. During the embryonic stage, VEGFR-3 is highly expressed in vascular endothelial cells, but expression decreases significantly during lymphatic sprouting from existing embryonic blood vessels. However, VEGFR3 is highly expressed in developing lymphatic endothelial cells (LECs). In adult lymphatic vessels, VEGFR-3 expression is low or absent, but it is upregulated during lymphangiogenesis in pathological conditions such as wound healing, inflammation, and tumor metastasis. VEGFR-3 is a potential target protein for molecular imaging and therapy of tumors.

[0004] Compared to conventional imaging techniques, which primarily include ultrasound, computed tomography (CT), and magnetic resonance imaging (MRI), optical molecular imaging based on high-affinity binding offers higher spatial, temporal, and superior quantitative resolution. Therefore, optical molecular imaging offers solutions for early tumor detection and image-guided surgery.

[0005] Currently, TMVP1446 has been confirmed to be a peptide with good binding ability to VEGFR-3 protein and tumor targeting ability. The chemical structure of TMVP1446 is as follows:

[0006]

[0007] However, peptides are easily degraded by proteases, limiting the application of TMVP1446 as a target molecule for molecular imaging probes. While conventional chelation can be used to improve it, the resulting chelates can lead to decreased affinity and sensitivity, as well as complex preparation processes and high costs. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide a VEGFR-3 targeting polypeptide, its application, a tumor targeting imaging probe and a preparation method, so as to solve the problems of low affinity, low sensitivity, complex preparation process and high cost of the polypeptide in the prior art.

[0009] The technical solution of the present invention to solve the above technical problems is as follows:

[0010] The present invention provides a VEGFR-3 targeting polypeptide, wherein the targeting polypeptide is TMVP1446 / GS5, and the amino acid sequence of the TMVP1446 / GS5 is shown in SEQ ID NO: 1.

[0011] The present invention also provides a use of the above-mentioned VEGFR-3 targeting polypeptide, which can be used to prepare a targeted imaging preparation for primary tumors and / or tumor metastatic lymph nodes.

[0012] The present invention also provides a tumor-targeted imaging probe, which comprises a fluorescent dye molecule with near-infrared luminescence ability and the above-mentioned TMVP1446 / GS5.

[0013] Furthermore, the probe is TMVP1446 / GS5-Cy7, wherein the fluorescent dye molecule is Cy7, and Cy7 is connected to the TMVP1446 / GS5 via lysine.

[0014] The present invention also provides a use of the above-mentioned tumor-targeted imaging probe, which can be used for targeted determination of primary tumors and / or tumor metastatic lymph nodes.

[0015] The present invention also provides a method for preparing the tumor targeting imaging probe as described above, comprising first synthesizing the TMVP1446 / GS5, and then coupling the fluorescent dye molecule to the TMVP1446 / GS5.

[0016] Further, the following steps are included:

[0017] S1, synthesis of TMVP1446 / GS5 peptide containing protective groups;

[0018] S2. removing the protecting groups of the TMVP1446 / GS5 polypeptide to obtain the TMVP1446 / GS5;

[0019] S3, performing a first purification of the TMVP1446 / GS5;

[0020] S4. Mixing and reacting the TMVP1446 / GS5, dimethylformamide solution, fluorescent dye molecular coupling agent and diisopropylethylamine, and purifying the reaction product for a second time to obtain the probe.

[0021] Furthermore, in the TMVP1446 / GS5 polypeptide containing a protecting group, the amino acid sequence of the polypeptide is shown in SEQ ID NO: 1. In the SEQ ID NO: 1, the side chain protecting group of lysine is tert-butyloxycarbonyl, the side chain protecting group of arginine is 2,2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl, the side chain protecting groups of threonine and serine are tert-butyl, and the side chain protecting group of cysteine ​​is trityl.

[0022] Furthermore, in step S2, the TMVP1446 / GS5 polypeptide is mixed with trifluoroacetic acid, water, triisopropylsilane, and 3-mercaptopropionic acid to obtain a precipitate, and the precipitate is separated, washed, and dried to obtain the TMVP1446 / GS5.

[0023] Furthermore, in step S4, the fluorescent dye molecular coupling agent is Cy7 monofunctional N-hydroxysuccinimide ester.

[0024] The beneficial effects of the present invention are:

[0025] (1) The VEGFR-3 targeting polypeptide of the present invention has high affinity for binding to VEGFR-3 and can specifically bind to VEGFR-3, thereby having good targeting ability to primary tumors and metastatic lymph nodes;

[0026] (2) The targeted VEGFR-3 polypeptide of the present invention can be used to prepare a targeted imaging preparation for primary tumors and / or tumor metastatic lymph nodes;

[0027] (3) The tumor-targeting imaging probe of the present invention has excellent metabolic stability, can specifically target primary tumors in vivo, and also has excellent targeting ability for lymph node metastasis, making it a good reagent for visualizing lymph node metastasis;

[0028] (4) The tumor-targeted imaging probe of the present invention can be used to target and measure primary tumors and / or tumor-metastatic lymph nodes, thereby achieving visualization of primary tumor and / or tumor-metastatic lymph node testing with good accuracy, specificity, sensitivity, and safety;

[0029] (5) The method for preparing the tumor targeting imaging probe of the present invention has the advantages of simple steps, mild conditions, easy operation, high efficiency and low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1This is the first HPLC result diagram of the VEGFR-3 targeting polypeptide of the present invention, Figure 1 A in the figure is the HPLC chromatogram of TMVP1446 / GS5-Cy7. Figure 1 Figure B is the LC-MS chromatogram of TMVP1446 / GS5-Cy7. Figure 1 Figure C in the figure is the LC-MS mass spectrum of TMVP1446 / GS5-Cy7. Figure 1 D in the figure is the LC-MS mass spectrum of TMVP1446 / GS5-5-TAMRA. Figure 1 Figure E in the figure is the absorption spectrum of TMVP1446 / GS5 5-TAMRA, TMVP1446 5-TAMRA, and free 5-TAMRA. Figure 1 Figure F is the absorption spectra of TMVP1446 / GS5-Cy7, TMVP1446-Cy7 dye, and free Cy7 dye;

[0031] Figure 2 This is the second HPLC result diagram of Example 1 for the VEGFR-3 targeting polypeptide of the present invention; Figure 2 A in the middle is the HPLC chromatogram of TMVP1446 / GS5-5-TAMRA; Figure 2 Middle B is the LC-MS chromatogram of TMVP1446 / GS5-5-TAMRA; Figure 2 Middle C is the HPLC chromatogram of TMVP1446-5-TAMRA; Figure 2 D in the figure is the LC-MS chromatogram of TMVP1446-5-TAMRA; Figure 2 Figure E is the LC-MS mass spectrum of TMVP1446-5-TAMRA; Figure 2 Figure F is the HPLC chromatogram of TMVP1446-Cy7; Figure 2 G in the figure is the LC-MS chromatogram of TMVP1446-Cy7. Figure 2 H in the middle is the LC-MS mass spectrum of TMVP1446-Cy7;

[0032] Figure 3 This is a graph showing the in vitro specific binding ability test of the VEGFR-3 targeting polypeptide of the present invention in Example 2; Figure 3 Figure A is the SPR result of TMVP1446 and TMVP1446 / GS5 peptides and human VEGFR-3 protein. Figure 3 Middle B is a comparison of the cytotoxicity of TMVP1446 / GS5 at gradient concentrations of 0, 10, 20, 40, and 80 μM in HaCaT, 4T1, HeLa S3 CON, and HeLa S3 OE cells; Figure 3Middle C shows the expression of VEGFR-3 in 4T1, HeLa S3 CON and HeLa S3 OE cells. Figure 3 Middle D shows representative fluorescence images of free 5-TAMRA, TMVP1446-5-TAMRA, and TMVP1446 / GS5-5-TAMRA in HeLa S3 CON and HeLa S3 OE cells;

[0033] Figure 4 This is a graph showing the in vivo biodistribution and metabolic stability test results of TMVP1446 / GS5-Cy7 in Example 3, a VEGFR-3 targeting polypeptide of the present invention; Figure 4 Center A shows representative near-infrared fluorescence images of BALB / c mice at different time points after intravenous injection of TMVP1446 / GS5-Cy7; Figure 4 Middle B is a representative near-infrared fluorescence image of major organs in vitro at different time points after injection; Figure 4 Middle C is the mean fluorescence intensity of major organs in vitro at different time points after injection; Figure 4 Figure D shows the plasma concentration-time curves of free Cy7, TMVP1446-Cy7, and TMVP1446 / GS5-Cy7 in normal Sprague-Dawley rats;

[0034] Figure 5 This is a graph showing the results of the targeting test of TMVP1446 / GS5 on primary tumors in mice in Example 4 for the VEGFR-3 targeting polypeptide of the present invention; Figure 5 Middle A is the in vivo imaging of subcutaneous tumor model mice after intravenous injection of 100 μl, 100 μM free Cy7, TMVP1446-Cy7 and TMVP1446 / GS5-Cy7. Figure 5 Middle B is a near-infrared fluorescence image of the tumor and major organs in vitro 6 hours later; Figure 5 Middle C is a comparison of the fluorescence intensity ratio of tumor and normal tissue. Figure 5 Middle D is a comparison of the mean fluorescence intensity of in vitro tumors and major organs after 6 hours; Figure 5 Middle E shows the changes in the mean fluorescence intensity of tumor tissue at different time points;

[0035] Figure 6 This is a graph showing the results of the in vivo lymph node metastasis targeting ability test of TMVP1446 / GS5 in Example 5 for the VEGFR-3 targeting polypeptide of the present invention; Figure 6 A in the middle is a schematic diagram of establishing a mouse popliteal lymph node metastasis model. Figure 6 Figure B is a schematic diagram of the experimental process for establishing a mouse popliteal lymph node metastasis model. Figure 6Figure 6C is a representative fluorescent image of the free Cy7, TMVP1446-Cy7 and TMVP1446 / GS5-Cy7 of the mouse tumor lymph node metastasis at 1, 2, 6, 12 and 24 hours after injection, Figure 6 Figure 6D is a representative near-infrared fluorescent image of the major organs (N-LN and T-LN) in vitro after 6 hours and 24 hours, Figure 6 Figure 6E is a graph showing the expression of VEGFR-3 in the normal lymph nodes (N-LN) and metastatic lymph nodes (T-LN) of the animal model of Figure A; Figure 6 Figure 6F is a comparison graph of the fluorescence intensity ratio of T-LN to N-LN;

[0036] Figure 7 Figure 6G is a representative HE staining image of each major organ in Example 6 of the VEGFR-3 targeting polypeptide of the present application,

[0037] Figure 8 Figure 6H is a comparison graph of the levels of each index of each major organ in Example 6 of the VEGFR-3 targeting polypeptide of the present application, Figure 8 Figure 6A is ALT, Figure 8 Figure 6B is AST, Figure 8 Figure 6C is BUN, Figure 8 Figure 6D is CREA. DETAILED DESCRIPTION

[0038] The principles and features of the present application are described below in conjunction with the accompanying drawings, which are only used to explain the present application and are not intended to limit the scope of the present application.

[0039] The VEGFR-3 targeting polypeptide of the present application is TMVP1446 / GS5, and the amino acid sequence of TMVP1446 / GS5 is shown in SEQ ID NO: 1.

[0040] The amino acid sequence of SEQ ID NO: 1 is Ac-CRRAibLGGGTTSR(Ahx)-NH2, and the chemical structure of TMVP1446 / GS5 is shown in formula (1):

[0041]

[0042] The TMVP1446 / GS5 of the present application, compared with the existing TMVP1446 amino acid sequence (CRRALGGGTTSR), the alanine (A) at the 4th position is replaced by the non-natural amino acid 2-aminoisobutyric acid (Aib), the arginine at the 12th amino acid is modified by 6-aminocaproi c acid (6-Aminocaproi c Ac id), and at the same time, the N-terminal of TMVP1446 / GS5 is acetylated and the C-terminal is amidated.

[0043] The TMVP1446 / GS5 of the present invention has high affinity for binding to VEGFR-3 and can specifically bind to VEGFR-3, thereby having good targeting ability to primary tumors and metastatic lymph nodes.

[0044] The targeted VEGFR-3 polypeptide of the present invention can be used to prepare a targeted imaging preparation for primary tumors and / or tumor metastatic lymph nodes.

[0045] The tumor targeting imaging probe of the present invention comprises a fluorescent dye molecule with near-infrared luminescence ability and TMVP1446 / GS5.

[0046] Preferably, the probe of the present invention is TMVP1446 / GS5-Cy7, wherein the fluorescent dye molecule is Cy7, and Cy7 is linked to the TMVP1446 / GS5 via lysine.

[0047] Specific experimental verification has shown that TMVP1446 / GS5-Cy7 has excellent metabolic stability and can specifically target primary tumors in the body. It also has excellent targeting ability for lymph node metastasis, making it an excellent reagent for visualizing lymph node metastasis. Furthermore, TMVP1446 / GS5-Cy7 has a good safety profile.

[0048] The probe of the present invention can be used for targeted determination of primary tumors and / or tumor metastatic lymph nodes, and realizes visualization of primary tumor and / or tumor metastatic lymph node testing, with good accuracy, specificity, sensitivity and safety.

[0049] The preparation method of the tumor targeting imaging probe of the present invention comprises the following steps: firstly synthesizing TMVP1446 / GS5, and then coupling the fluorescent dye molecule with TMVP1446 / GS5.

[0050] Specifically, the method of the present invention comprises the following steps:

[0051] S1. Using Rin Amide MBHA resin and Fmoc strategy, the TMVP1446 / GS5 peptide containing protective groups was synthesized.

[0052] Preferably, in the TMVP1446 / GS5 polypeptide containing a protecting group, the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 1. In SEQ ID NO: 1, the side chain protecting group of lysine is tert-butyloxycarbonyl, the side chain protecting group of arginine is 2,2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl, the side chain protecting groups of threonine and serine are tert-butyl, and the side chain protecting group of cysteine ​​is trityl.

[0053] S2. Remove the protecting groups of the TMVP1446 / GS5 polypeptide to obtain TMVP1446 / GS5.

[0054] Preferably, the TMVP1446 / GS5 polypeptide is mixed with trifluoroacetic acid, water, triisopropylsilane, and 3-mercaptopropionic acid to obtain a precipitate, and the precipitate is separated, washed, and dried to obtain TMVP1446 / GS5.

[0055] S3. Perform the first purification of TMVP1446 / GS5; the purification method may be reverse phase liquid chromatography.

[0056] S4. The purified TMVP1446 / GS5, dimethylformamide solution, fluorescent dye molecular coupling agent, and diisopropylethylamine are mixed and reacted, and the reaction product is purified a second time to obtain a probe. The second purification can also be performed using reversed-phase liquid chromatography.

[0057] Preferably, the fluorescent dye molecular coupling agent is Cy7 monofunctional N-hydroxysuccinimide ester.

[0058] The present invention is specifically described below by way of examples.

[0059] Example 1 Design, synthesis and characterization of molecular fluorescent probes

[0060] In this example, the peptide (TMVP1446 / GS5) was synthesized using the following steps:

[0061] (1) Using Rinke Amide MBHA resin and the Fmoc strategy, the partially protected peptide Ac-Cys(Trt)-Arg(Pbf)-Arg(Pbf)-Aib-Leu-Gly-GlyGly-Thr(tBu)-Thr(tBu)-Ser(tBu)-Arg(Pbf)-Ahx-Lys(Boc)-NH2 was synthesized.

[0062] The side chain protecting group of lysine is tert-butyloxycarbonyl (Boc), the side chain protecting group of arginine is 2,2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf), the side chain protecting group of threonine and serine is tert-butyl (tBu), and the side chain protecting group of cysteine ​​is trityl (Trt).

[0063] (2) Removal of Protecting Groups: The protected peptide was treated with a solution containing 90.0% trifluoroacetic acid (TFA), 2.5% water, 2.5% triisopropylsilane, and 5% 3-mercaptopropionic acid at room temperature for 2.0 hours. The mixture was precipitated, washed with cold tert-butyl methyl ether, and dried under vacuum to obtain the crude peptide TMVP1446 / GS5.

[0064] (3) The crude peptide TMVP1446 / GS5 was purified by reverse phase high performance liquid chromatography (RP-HPLC). The mobile phases of RP-HPLC were as follows: mobile phase A was a 0.075% TFA solution in water, and mobile phase B was acetonitrile.

[0065] (4) Cy7 Coupling and Purification: A 5 mL dimethylformamide (DMF) solution containing 0.033 mmol TMVP1446 / GS5 was added to Cy7 monofunctional N-hydroxysuccinimide ester (Cy7-NHS) and diisopropylethylamine (DIEA). After stirring at room temperature for 0.5 h under nitrogen, the pH was adjusted to 7 to obtain a reaction solution.

[0066] The reaction solution was purified by RP-HPLC, wherein the mobile phases were: mobile phase A was a 0.075% TFA solution in water, and mobile phase B was acetonitrile. During elution, the gradient of mobile phase B was 15-45% over 50 minutes.

[0067] The purified product was confirmed by liquid chromatography-mass spectrometry (LC-MS) (m / z: (M+3H)3+=732.8; m / z (M+2H)2+=1098.8; calculated molecular weight: 2195.65 g / mol); the retention time (Rt) of RP-HPLC was 9.536 minutes (phase B gradient: 20-50-20 minutes).

[0068] The synthesis process of the TMVP1446 / GS5-Cy7 probe is shown in the following reaction formula:

[0069]

[0070] The excitation wavelength of Cy7 is typically between 745 and 780 nm, and the emission wavelength is between 767 and 820 nm. Due to the strong penetration of near-infrared light in biological tissues and low background fluorescence, Cy7 dye is particularly suitable for in vivo and deep tissue imaging. However, the excitation wavelength of Cy7 is not included in the commonly used wavelength range of laser confocal fluorescence microscopy. To observe peptide fluorescence under laser confocal fluorescence microscopy, 5-TAMRA is used instead of Cy7 in cell experiments. 5-TAMRA has an excitation wavelength of 544 nm and an emission wavelength of 572 nm.

[0071] The same method as above was used to synthesize TMVP1446-Cy7, TMVP1446-5-TAMRA and TMVP1446 / GS5-5-TAMRA, respectively, for subsequent comparative experiments.

[0072] The absorption spectrum of each fluorescent-labeled peptide prepared above was detected.

[0073] like Figure 1 and Figure 2 The liquid chromatography results showed that the retention times of TMVP1446 / GS5-Cy7 and TMVP1446 / GS5-5-TAMRA were 9.536 minutes and 10.947 minutes, respectively, and the purities were 98.34% and 95.71%, respectively. Figure 1 A and Figure 2 A).

[0074] Liquid chromatography-mass spectrometry (LC-MS) analysis results showed that the liquid chromatography peak appeared at 1.537 minutes with a mass-to-charge ratio (m / z) of 1098.8 ([M+2H]2+) and 732.8 ([M+3H]3+), which was consistent with the expected molecular weight (chemical formula: C 97 H 155 N 27 O 25 S3, exact mass: 2195.62) Figure 1 B and 1C).

[0075] The LC-MS results of TMVP1446 / GS5-5-TAMRA showed that the peak appeared at 1.536 minutes with m / z ratios of 648.7 ([M+3H]3+) and 486.8 ([M+4H]4+), which was consistent with the expected molecular weight (accurate mass: 1943.24) ( Figure 2 B and Figure 2 D).

[0076] TMVP1446-Cy7 and TMVP1446-5-TAMRA were characterized by HPLC and LC-MS, respectively. Figure 2 As shown in C-2H.

[0077] like Figure 1 As shown in Figures E and 1F, 5-TAMRA and Cy7 dyes linked to TMVP1446 or TMVP1446 / GS5 peptides have absorption spectra similar to those of free Cy7 dye, which are slightly red-shifted to 550 nm to 555 nm compared with the absorption peak of free 5-TAMRA, indicating that TMVP1446 and TMVP1446 / GS5 do not change the optical properties of 5-TAMRA and Cy7.

[0078] Example 2 In vitro targeting ability test of TMVP1446 / GS5 to VEGFR-3

[0079] In this example, the binding (Ka) and dissociation (Kd) constants of TMVP1446 / GS5 and TMVP1446 peptides to vascular endothelial growth factor receptor-3 (VEGFR-3) protein were compared and evaluated by surface plasmon resonance (SPR) technology, and the equilibrium dissociation constant (Kd / Ka, KD) was calculated. Figure 3 A is the SPR result of TMVP1446 and TMVP1446 / GS5 peptides and human VEGFR-3 protein. The KD of VEGFR-3 and TMVP1446 is 8.97×10 -7 .

[0080] Prior to the cell surface binding assay, the cytotoxicity of TMVP1446 / GS5 was assessed in vitro using a CCK8 assay. HaCaT cells and several tumor cell lines (4T1, VEGFR-3 overexpressing (OE) or control (CON) HeLa S3 cells) were incubated with a gradient of TMVP1446 / GS5 peptide for 24 hours at concentrations of 0, 10, 20, 40, and 80 μM.

[0081] The results of in vitro cytotoxicity assessment were as follows Figure 3 As shown in Figure 2B, it can be seen that even at the highest concentration (80 μM), the TMVP1446 / GS5 peptide did not show significant cytotoxicity against all cell lines, indicating that TMVP1446 / GS5 has excellent biocompatibility.

[0082] To evaluate the in vitro binding ability of TMVP1446 / GS5 to VEGFR-3, the expression levels of VEGFR-3 in 4T1, HeLa S3 CON, and HeLa S3 OE cells were detected by Western blotting, and the results were shown in Figure 2 . Figure 3 As shown in C. Figure 3 C As can be seen, VEGFR-3 is highly expressed in HeLa S3 OE cells but not in 4T1 and HeLa S3 cells.

[0083] Representative fluorescence images of free 5-TAMRA, TMVP1446-5-TAMRA, and TMVP1446 / GS5-5-TAMRA in HeLa S3 CON and HeLa S3 OE cells are shown in Figure 4. Figure 3 As shown in D. For TMVP1446 / GS5-5-TAMRA blocking agent, cells were treated with 20 μM TMVP1446 for 10 minutes before adding TMVP1446 / GS5-5-TAMRA. Figure 3 The scale bar in D is 20 μm, OE indicates VEGFR-3 overexpression, and CON indicates control.

[0084] according to Figure 3 D shows that in HeLa S3 OE cells, the fluorescence signal of TMVP1446 / GS5-5-TAMRA is slightly stronger than that of TMVP1446-5-TAMRA, while the fluorescence signal of free 5-TAMRA is almost undetectable.

[0085] Furthermore, in HeLa S3 OE cells, a 10-fold concentration of the free linear peptide TMVP1446 effectively blocked the binding of TMVP1446 / GS5-5-TAMRA to VEGFR-3. Meanwhile, no fluorescent signal was observed in HeLaS3 CON cells treated with TMVP1446 / GS5-5-TAMRA. These results indicate that TMVP1446 / GS5 can specifically bind to the VEGFR-3 protein.

[0086] Example 3: Biodistribution and metabolic stability test of TMVP1446 / GS5-Cy7 in vivo

[0087] The test results of this embodiment are as follows Figure 4 The specific test method is as follows.

[0088] (1) Evaluation of the biodistribution of TMVP1446 / GS5 in vivo: TMVP1446 / GS5-Cy7 was injected into the tail vein of BALB / c normal mice and monitored continuously for 7 days using an IVI S spectral imaging system. The specific monitoring time points were 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, and 7 days after injection. The monitoring results are shown in the figure below. Figure 4 As shown in A.

[0089] pass Figure 4 As can be seen from A, 30 minutes after the injection of TMVP1446 / GS5-Cy7, the fluorescent signal rapidly distributed throughout the body, mainly accumulated in the head, liver and bladder, and gradually weakened over time.

[0090] The fluorescence signals in various organs were monitored and quantified for 7 consecutive days. The specific organs were heart, liver, spleen, lung and kidney. The monitoring images were as follows: Figure 4 B shows the quantitative results. Figure 4 C. The results showed that the fluorescence signal was strongest in the kidney, followed by the liver and lung, while the fluorescence signals in the heart, spleen, and intestine were negligible.

[0091] Notably, the fluorescence signal in the kidney gradually weakened after 2 hours, then increased again and reached a peak at 6 hours, indicating that TMVP1446 / GS5-Cy7 was mainly eliminated from the animals through the kidneys.

[0092] (2) Evaluation of the in vivo stability of TMVP1446 / GS5-Cy7: The stability of free Cy7, TMVP1446-Cy7, and TMVP1446 / GS5-Cy7 in normal Sprague-Dawley rats was investigated. The plasma concentration-time curves were shown in Figure 2. Figure 4 The pharmacokinetic parameters are shown in Table 1.

[0093] according to Figure 4 As can be seen in Figure 4, the peak plasma concentrations (Cmax) of free Cy7 and TMVP1446-Cy7 were 183.99±68.81nM and 190.97±76.02nM, respectively, and declined rapidly within 1 hour after injection. In contrast, the Cmax of TMVP1446 / GS5-Cy7 was 252.21±63.66nM and declined relatively slowly. The plasma half-life (t1 / 2) of TMVP1446 / GS5-Cy7 was 1.70±0.36 hours, significantly longer than that of free Cy7 (0.52±0.16 hours) and TMVP1446-Cy7 (1.00±0.55 hours).

[0094] Table 1 Pharmacokinetic parameters of free Cy7, TMVP1446-Cy7, and TMVP1446 / GS5-Cy7 probes

[0095]

[0096] *The data in Table 1 are expressed as mean ± SD, n = 3 per group

[0097] In addition, as shown in Table 1, the average area under the curve (AUC0-t) of TMVP1446 / GS5-Cy7 (364.95±31.98nmo l / L*h) was also greater than that of free Cy7 (108.81±29.65nmo l / L*h) and TMVP1446-Cy7 (173.69±75.55nmo l / L*h).

[0098] These results indicate that TMVP1446 / GS5-Cy7 has excellent metabolic stability compared with free Cy7 and TMVP1446-Cy7.

[0099] Example 4 Targeting of TMVP1446 / GS5 to Primary Tumors in Mice

[0100] In this example, the 4T1-LUC subcutaneous tumor model was used to evaluate the in vivo tumor targeting ability of TMVP1446 / GS5. The evaluation results are shown in Figure 2. Figure 5 shown.

[0101] After intravenous injection of free Cy7, TMVP1446-Cy7, or TMVP1446 / GS5-Cy7, the fluorescence signals of tumor-bearing mice were detected at designated time points using the IVIS imaging system. The images obtained are shown in Figure 2. Figure 5 As shown in A. Before the injection of TMVP1446 / GS5-Cy7, 10 times the amount of TMVP1446 peptide was injected intravenously as a competitive blocker. Figure 5The white circle in A indicates the location of the tumor. Figure 5 E is a quantitative comparison of the mean fluorescence intensity of in vivo tumors at different time points.

[0102] pass Figure 5 As shown in Figures A and 5E, TMVP1446 / GS5-Cy7 exhibits a bright fluorescence signal at the tumor site within the first 2 hours after injection, which then gradually diminishes. It then maintains a stable high level between 2 and 6 hours before gradually diminishing again, indicating that the drug is retained in the tumor. Notably, this bright fluorescence signal is eliminated by excess linear peptide TMVP1446. Furthermore, TMVP1446 / GS5-Cy7 exhibits a stronger fluorescence signal at the tumor site than both TMVP1446-Cy7 and free Cy7.

[0103] To further illustrate the distribution of free Cy7, TMVP1446-Cy7, and TMVP1446 / GS5-Cy7, tumors and major organs were collected for near-infrared imaging 6 h after injection, as shown in Figure 2 . Figure 5 As shown in B, the fluorescence intensity quantification comparison Figure 5 D. T is tumor, H is heart, Li is liver, S is spleen, Lu is lung, Ki is kidney, In is intestine, the fluorescence intensity ratio of tumor to normal tissue is expressed as T / N ratio, * indicates P < 0.05.

[0104] according to Figure 5 As can be seen in Figure 2, strong fluorescence signals were observed in the kidney for all probes, moderate fluorescence signals were observed in tumor tissue, liver, and lung, and the weakest fluorescence signals were observed in the heart, spleen, and intestine. Furthermore, TMVP1446 / GS5-Cy7 exhibited the strongest fluorescence signals not only in organs but also in tumors, a signal that could be partially offset by an excess of TMVP1446 peptide. Figure 5 These observations were further confirmed by quantification of the fluorescence intensity of D.

[0105] To further evaluate the targeting ability, this example measured and calculated the fluorescence intensity ratio of tumor to normal tissue (T / N). The results are shown in Figure 2. Figure 5 As shown in C. Figure 5As can be seen in Figure 3, the maximum T / N ratios of TMVP1446 / GS5-Cy7 and TMVP1446-Cy7 at 6 hours were 6.96 and 6.74, respectively. Although the T / N ratios of TMVP1446 / GS5-Cy7 and TMVP1446-Cy7 in vivo were similar, the fluorescence signal of TMVP1446 / GS5-Cy7 was significantly higher than that of TMVP1446-Cy7. More importantly, the fluorescence intensity and T / N ratio of TMVP1446-Cy7 and TMVP1446 / GS5-Cy7 were significantly higher than those of free Cy7.

[0106] The results of this example indicate that, similar to TMVP1446-Cy7, TMVP1446 / GS5-Cy7 can also specifically target tumors in vivo.

[0107] Example 5 Testing of the targeting ability of TMVP1446 / GS5 on lymph node metastasis in vivo

[0108] In this example, the popliteal lymph node (LN) metastasis model was used to evaluate the in vivo targeting ability of TMVP1446 / GS5 to tumor metastatic lymph nodes (T-LN).

[0109] like Figure 6 As shown in the schematic diagram of Figure A, this example specifically implemented mouse model construction, Cy7-peptide probe administration, near-infrared imaging, tumor metastasis lymph node resection, and pathological examination. Twenty-one days after 4T1 cell implantation into the hind footpad of mice, the popliteal lymph nodes on the treated side were significantly enlarged compared to the contralateral normal popliteal lymph nodes (N-LN), and H&E staining confirmed tumor metastasis.

[0110] Figure 6 E is the image of VEGFR-3 expression in N-LN and T-LN detected by immunohistochemistry (IHC), the scale bar is 20 μm, T-LN is the tumor metastasis lymph node, N-LN is the normal lymph node, ns indicates P>0.05, * indicates P<0.05. Figure 6 E As can be seen, tumor-metastatic lymph nodes highly express VEGFR-3, while normal lymph nodes do not express VEGFR-3. Mice with unilateral tumor-metastatic lymph nodes were injected with free Cy7, TMVP1446-Cy7, or TMVP1446 / GS5-Cy7 and then monitored for 24 hours.

[0111] Representative fluorescence images of free Cy7, TMVP1446-Cy7, and TMVP1446 / GS5-Cy7 in tumor lymph node metastases of mice at 1, 2, 6, 12, and 24 hours after injection are shown. Figure 6As shown in FIG. C, also, before intravenous injection of TMVP1446 peptide, a ten-fold amount of TMVP1446 peptide was injected through tail vein as competitive blocker, white arrow indicates tumor. According to Figure 6 As can be seen, 12 hours after injection of TMVP1446 / GS5-Cy7, a more bright fluorescence was observed from the popliteal tumor metastatic lymph node in normal tissue, and lasted for 12 hours.

[0112] In addition, after injection of TMVP1446 / GS5-Cy7, the fluorescence signal of tumor metastatic lymph node was significantly stronger than that of normal lymph node. In contrast, in the free Cy7 group, there was no significant difference in fluorescence intensity between tumor metastatic lymph node and normal lymph node, indicating that free Cy7 had no targeting ability for tumor metastatic lymph node.

[0113] Representative near-infrared fluorescence images and average fluorescence intensity of major organs (N-LN and T-LN) in vitro after 6 hours and 24 hours are shown in FIG. D. Figure 6 As shown in FIG. D, compared with TMVP1446 / GS5-Cy7, TMVP1446-Cy7 showed a weaker fluorescence signal in tumor metastatic lymph node and was rapidly excreted in vivo. Notably, pre-injection of excess TMVP1446 can quench the bright fluorescence signal of TMVP1446 / GS5-Cy7 in tumor metastatic lymph node. Further in vitro imaging of organs 6 hours or 24 hours after injection proved that the accumulation of TMVP1446 / GS5-Cy7 in tumor metastatic lymph node was higher than that of the other three groups, including the TMVP1446 / GS5-Cy7 blocking group.

[0114] To further evaluate the feasibility of TMVP1446 / GS5-Cy7 as a fluorescent probe for tumor metastatic lymph node, the fluorescence intensity ratio of tumor metastatic lymph node to contralateral normal lymph node was calculated, and the results are shown in FIG. E. Figure 6 As shown in FIG. E, at 6 hours, the ratio of TMVP1446 / GS5-Cy7 was 7.53, and the ratio of TMVP1446-Cy7 was 6.61, and the ratio of TMVP1446 / GS5-Cy7 group was significantly higher than that of free Cy7 group or TMVP1446 / GS5-Cy7 blocking group. At 24 hours, the ratio of TMVP1446 / GS5-Cy7 was still higher than that of other groups, but the increase was not statistically significant.

[0115] The above test results of this embodiment show that TMVP1446 / GS5-Cy7 has excellent targeting ability for lymph node metastasis, and will be a good reagent for visualizing lymph node metastasis.

[0116] Example 6 In vivo safety evaluation of TMVP1446 / GS5-Cy7

[0117] To evaluate the safety of TMVP1446 / GS5-Cy7, an acute toxicity study was conducted in this example. At the end of two weeks, no mice had died. After sacrifice, the main organs were dissected for hematoxylin-eosin (H&E) staining, and blood was collected for liver and kidney function testing.

[0118] Figure 7 The HE staining images of various organs and tissues are shown in the figure. The scale bar is 100 μm. Figure 7 It can be seen that no toxic pathological changes were found in the heart, liver, lung, spleen and kidney of mice in the control group, free Cy7 group, TMVP1446 group and TMVP1446 / GS5 group.

[0119] Figure 8 The comparison chart of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN) and creatinine (CREA) levels in four groups of mice is shown in Figure 2. Figure 8 It can be seen that there is no significant difference among the indicators.

[0120] The above results of this example indicate that TMVP1446 / GS5-Cy7 is a safe visual fluorescent probe.

[0121] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A polypeptide targeting VEGFR-3, characterized in that: The VEGFR-3 targeting polypeptide is TMVP1446 / GS5, and the structural formula of TMVP1446 / GS5 is as follows: 。 2. A use of the VEGFR-3 targeting polypeptide according to claim 1, characterized in that: The targeted VEGFR-3 polypeptide is used to prepare a targeted imaging preparation for primary tumors and / or tumor metastatic lymph nodes with overexpression of VEGFR-3.

3. A tumor targeting imaging probe, characterized in that: The probe is TMVP1446 / GS5-Cy7, which is formed by the reaction of Cy7 monofunctional N-hydroxysuccinimide ester Cy7-NHS with TMVP1446 / GS5 in claim 1 through lysine; the carboxyl group of the lysine reacts with the amino group of 6-aminohexanoic acid in TMVP1446 / GS5, and the carbonyl group formed after the Cy7-NHS removes the NHS group is connected to the amino group at position 6 of lysine.

4. A use of the tumor targeting imaging probe according to claim 3, characterized in that: The tumor targeting imaging probe is used for preparing a targeting imaging preparation for primary tumors and / or tumor metastatic lymph nodes with overexpression of VEGFR-3.

Images