Novel targeting VEGFR-3 polypeptide, application, tumor targeting imaging probe and preparation method

Abstract

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

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to novel VEGFR-3 targeting polypeptides, applications, tumor targeting imaging probes and preparation methods. 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 metastases. Previous 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 inside the tumor and in the area surrounding 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 main receptor for the best known lymphangiogenic growth factors, vascular endothelial growth factor C (VEGF-C) and VEGF-D, mediating lymphangiogenesis. During the embryonic period, VEGFR-3 is highly expressed in vascular endothelial cells, but the expression decreases significantly when lymphatic vessels sprout from existing embryonic blood vessels, while 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 in lymphangiogenesis under pathological conditions such as wound healing, inflammation, and tumor metastasis. VEGFR-3 is a potential target protein for tumor molecular imaging and treatment.

[0004] Compared with traditional imaging technologies, which mainly include ultrasound, computed tomography (CT) and magnetic resonance imaging (MRI), optical molecular imaging based on high-affinity binding has higher spatial, temporal and better quantitative resolution. Therefore, optical molecular imaging provides a solution for early detection of tumors 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, due to the action of proteases, peptides are easily degraded, which limits the application of TMVP1446 as a target molecule to construct molecular imaging probes. Although it can be improved through general chelation, the resulting chelator may lead to problems such as decreased affinity and reduced sensitivity. At the same time, there are also problems such as complex preparation process and high cost. Summary of the invention

[0008] The technical problem to be solved by the present invention is to provide a novel VEGFR-3 targeting polypeptide, application, tumor targeting imaging probe and preparation method, so as to solve the problems of low polypeptide affinity, low sensitivity, complex preparation process and high cost 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 novel 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 novel VEGFR-3 targeting polypeptide as described above, wherein the targeting polypeptide can be used to prepare a targeting imaging preparation for primary tumors and / or tumor metastatic lymph nodes.

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

[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 targeting imaging probe, wherein the tumor targeting imaging probe 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 with the TMVP1446 / GS5.

[0016] Further, the following steps are included:

[0017] S1, synthesizing TMVP1446 / GS5 peptide containing protecting groups;

[0018] S2, removing the protecting group of the TMVP1446 / GS5 polypeptide to obtain the TMVP1446 / GS5;

[0019] S3, purifying the TMVP1446 / GS5 for the first time;

[0020] S4, mixing and reacting the TMVP1446 / GS5, dimethylformamide solution, fluorescent dye molecular coupling agent and diisopropylethylamine, and purifying the product obtained by the reaction 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 as 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] Further, 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 novel 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 novel VEGFR-3 targeted polypeptide of the present invention can be used to prepare targeted imaging preparations for primary tumors and / or tumor metastatic lymph nodes;

[0027] (3) The tumor-targeted 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, and is 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, and realize 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 novel VEGFR-3 targeting polypeptide of the present invention, Figure 1 A in the figure is the HPLC chromatogram of TMVP1446 / GS5-Cy7. Figure 1 B is the LC-MS chromatogram of TMVP1446 / GS5-Cy7. Figure 1 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 is the absorption spectra of TMVP1446 / GS5 5-TAMRA, TMVP1446 5-TAMRA, and free 5-TAMRA. Figure 1 F in the middle 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 the novel VEGFR-3 targeting polypeptide of the present invention, Example 1; Figure 2 A in the figure is the HPLC chromatogram of TMVP1446 / GS5-5-TAMRA; Figure 2 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 E in the figure is the LC-MS mass spectrum of TMVP1446-5-TAMRA; Figure 2 F in the figure 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 figure 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 novel VEGFR-3 targeting polypeptide of the present invention in Example 2; Figure 3 Figure A shows the SPR results of TMVP1446 and TMVP1446 / GS5 peptides and human VEGFR-3 protein. Figure 3 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 3C shows the expression of VEGFR-3 in 4T1, HeLa S3 CON and HeLa S3 OE cells. Figure 3 D is a representative fluorescence image 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 biodistribution and metabolic stability test results of TMVP1446 / GS5-Cy7 in vivo in Example 3, a novel VEGFR-3 targeting polypeptide of the present invention; Figure 4 A in the middle is a representative near-infrared fluorescence image 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 average fluorescence intensity of the main organs in vitro at different time points after injection; Figure 4 D in the middle is the plasma concentration-time curve of normal Sprague-Dawley rats, including free Cy7, TMVP1446-Cy7 and TMVP1446 / GS5-Cy7;

[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, a novel VEGFR-3 targeting polypeptide of the present invention; Figure 5 A in the middle 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 in vitro tumor and major organs after 6 hours; Figure 5 C in the middle is a comparison of the fluorescence intensity ratio between tumor and normal tissue. Figure 5 D in the middle 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 of mean fluorescence intensity of tumor tissue at different time points;

[0035] Figure 6 This is a graph showing the test results of the targeting ability of TMVP1446 / GS5 on lymph node metastasis in vivo in Example 5, which is a novel 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 6C shows representative fluorescence images of free Cy7, TMVP1446-Cy7, and TMVP1446 / GS5-Cy7 in tumor lymph node metastasis of mice at 1, 2, 6, 12, and 24 hours after injection. Figure 6 D is a representative near-infrared fluorescence image of the main organs (N-LN and T-LN) in vitro after 6 and 24 hours. Figure 6 Figure E is the expression of VEGFR-3 in the normal lymph nodes (N-LN) and metastatic lymph nodes (T-LN) of the animal model in Figure A; Figure 6 F in the middle is a comparison diagram of the fluorescence intensity ratio of T-LN and N-LN;

[0036] Figure 7 The novel VEGFR-3 targeting polypeptide of the present invention is a representative HE staining image of each major organ in Example 6;

[0037] Figure 8 This is a comparison chart of the levels of various indicators of various major organs in Example 6 of the novel VEGFR-3 targeting polypeptide of the present invention. Figure 8 A in the middle is ALT, Figure 8 B stands for AST. Figure 8 In the middle, C stands for BUN. Figure 8 D in it stands for CREA. DETAILED DESCRIPTION

[0038] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0039] The novel VEGFR-3 targeting polypeptide of the present invention 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] Compared with the existing TMVP1446 amino acid sequence (CRRALGGGTTSR), the alanine (A) at the 4th position of the TMVP1446 / GS5 of the present invention is replaced by the non-natural amino acid 2-aminoisobutyric acid (Aib), and the arginine at the 12th amino acid is modified by 6-aminohexanoic acid (6-Aminocaproic Acid). At the same time, the terminal of NTMVP1446 / GS5 is acetylated and the C-terminus 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 novel 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 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 connected to the TMVP1446 / GS5 via lysine.

[0047] After specific experimental verification, TMVP1446 / GS5-Cy7 has excellent metabolic stability, can specifically target primary tumors in the body, and has excellent targeting ability for lymph node metastasis, making it a good reagent for visualizing lymph node metastasis. At the same time, TMVP1446 / GS5-Cy7 also has good safety.

[0048] The above-mentioned probe of the present invention can be used for targeted determination of primary tumors and / or tumor metastatic lymph nodes, realizes visualization of primary tumor and / or tumor metastatic lymph node testing, and has 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. Use Rink Amide MBHA resin and Fmoc strategy to synthesize TMVP1446 / GS5 peptide containing protective groups.

[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 on 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 product obtained by the reaction is purified for the second time to obtain a probe. The second purification can also use reverse 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 phase of RP-HPLC was specifically that mobile phase A was a 0.075% TFA aqueous solution and mobile phase B was acetonitrile.

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

[0066] The reaction solution was purified by RP-HPLC, wherein the mobile phase of RP-HPLC was as follows: mobile phase A was a 0.075% TFA aqueous solution, and mobile phase B was acetonitrile. During elution, the gradient of mobile phase B was 15-45% in 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 (B phase gradient: 20-50-20 minutes).

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

[0069]

[0070] The excitation wavelength of Cy7 is usually between 745 and 780nm, and the emission wavelength is between 767 and 820nm. Since near-infrared light has strong penetration in biological tissues and low background fluorescence, Cy7 dye is particularly suitable for in vivo imaging and deep tissue imaging. However, the common wavelength of laser confocal fluorescence microscopy does not include the excitation wavelength of Cy7. In order to observe the fluorescence of peptides under laser confocal microscope, 5-TAMRA is used to replace Cy7 for cell experiments. The excitation wavelength of 5-TAMRA is 544nm, and the emission wavelength is 572nm.

[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, TMVP1446 / GS5 peptide 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 evaluated in vitro using the 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 peptides at concentrations of 0, 10, 20, 40, and 80 μM for 24 h.

[0081] The results of in vitro cytotoxicity assessment were Figure 3 As shown in B, it can be seen that even at the highest concentration (80 μM), the TMVP1446 / GS5 peptide did not show significant cytotoxicity to 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 are shown in Figure 3 C. According to 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 3 D. For TMVP1446 / GS5-5-TAMRA blocking, cells were treated with 20 μM TMVP1446 for 10 min 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 It can be seen 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] In addition, in HeLa S3 OE cells, 10-fold concentration of free linear peptide TMVP1446 effectively blocked the binding of TMVP1446 / GS5-5-TAMRA to VEGFR-3. At the same time, no fluorescence signal was observed in HeLaS3 CON cells treated with TMVP1446 / GS5-5-TAMRA. These results indicate that TMVP1446 / GS5 can specifically bind to 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 As shown, 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 the 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. 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 h, then increased again and reached a peak at 6 h, 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 D It can be seen that the peak concentrations (Cmax) of free Cy7 and TMVP1446-Cy7 in plasma are 183.99±68.81nM and 190.97±76.02nM, respectively, and they drop rapidly within 1 hour after injection. In contrast, the Cmax of TMVP1446 / GS5-Cy7 is 252.21±63.66nM, and the rate of decline is relatively slow. The plasma half-life (t1 / 2) of TMVP1446 / GS5-Cy7 is 1.70±0.36 hours, which is significantly longer than 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 / Lh) was also greater than that of free Cy7 (108.81±29.65nmo l / Lh) 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 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 5 As shown in A. Before the injection of TMVP1446 / GS5-Cy7, 10 times the amount of TMVP1446 peptide was intravenously injected 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 can be seen in A and 5E, within the first 2 hours after injection, TMVP1446 / GS5-Cy7 showed a bright fluorescence signal at the tumor site and gradually weakened. Then, it remained at a stable high level between 2 and 6 hours and then gradually weakened again, indicating that a large amount of drug was retained at the tumor site. It is worth noting that its bright fluorescence signal was eliminated by excess linear peptide TMVP1446. In addition, compared with TMVP1446-Cy7 and free Cy7, TMVP1446 / GS5-Cy7 had a stronger fluorescence signal at the tumor site.

[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. Wherein, 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 B shows that for all probes, strong fluorescence signals were observed in the kidney, moderate fluorescence signals were observed in tumor tissues, liver and lungs, and the weakest fluorescence signals were observed in the heart, spleen and intestine. In addition, TMVP1446 / GS5-Cy7 not only has the strongest fluorescence signal in organs, but also has the strongest fluorescence signal in tumor sites, and its signal can be partially offset by excess 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), and the results are as follows: Figure 5 C. According to Figure 5C It can be seen that the maximum T / N 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 show 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 A, this embodiment specifically implements mouse model construction, Cy7-peptide probe administration, near-infrared imaging, tumor metastasis lymph node resection and pathological detection. Compared with the contralateral popliteal normal lymph nodes (N-LN), 21 days after the implantation of 4T1 cells in the hind foot pad of mice, the popliteal lymph nodes on the treated side were significantly enlarged, and tumor metastasis was confirmed by H&E staining.

[0110] Figure 6 E is the expression of VEGFR-3 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 means P>0.05, and * means 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. Figure 6As shown in C, similarly, before intravenous injection of TMVP1446 peptide, ten times the amount of TMVP1446 peptide was injected through the tail vein as a competitive blocker, and the white arrow indicates the tumor. Figure 6 As can be seen in C, 12 hours after injection of TMVP1446 / GS5-Cy7, brighter fluorescence was observed in the popliteal tumor metastatic lymph nodes compared with normal tissues and lasted until 12 hours.

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

[0113] Representative near-infrared fluorescence images and mean fluorescence intensity of major organs (N-LN and T-LN) in vitro after 6 and 24 h are shown in Figure 6 As shown in D, compared with TMVP1446 / GS5-Cy7, TMVP1446-Cy7 showed a weaker fluorescence signal in the tumor metastatic lymph nodes and was rapidly excreted from the body. Notably, pre-injection of an excess of TMVP1446 quenched the bright fluorescence signal of TMVP1446 / GS5-Cy7 in the tumor metastatic lymph nodes. Ex vivo imaging of the organs at 6 or 24 hours after injection further demonstrated that the accumulation of TMVP1446 / GS5-Cy7 in the tumor metastatic lymph nodes was higher than that in 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 nodes, the fluorescence intensity ratio of tumor metastatic lymph nodes to contralateral normal lymph nodes was calculated. Figure 6 As shown in F, it can be seen that at 6 hours, the ratio of TMVP1446 / GS5-Cy7 was 7.53, and the ratio of TMVP1446-Cy7 was 6.61. The ratio of the TMVP1446 / GS5-Cy7 group was significantly higher than that of the free Cy7 group or the TMVP1446 / GS5-Cy7 blocking group. At 24 hours, the ratio of TMVP1446 / GS5-Cy7 was still higher than that of the other groups, but the increase was not statistically significant.

[0115] The above test results of this example indicate 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 experiment was performed in this example. At the end of 2 weeks, all mice were alive, and after the mice were killed, the main organs were dissected for hematoxylin-eosin (H&E) staining, and blood was collected to detect liver and kidney function.

[0118] Figure 7 HE staining images of various organs and tissues, 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 the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN) and creatinine (CREA) in four groups of mice is shown in Figure 2. Figure 8 It can be seen that there are no significant differences in the indicators.

[0120] The above results of this example indicate that TMVP1446 / GS5-Cy7 is a safe visualized 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 principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A novel polypeptide targeting VEGFR-3, characterized in that: The targeting polypeptide is TMVP1446 / GS5, and the amino acid sequence of TMVP1446 / GS5 is shown in SEQ ID NO:

1.

2. A use of the novel VEGFR-3 targeting polypeptide as claimed in claim 1, characterized in that: The targeting polypeptide can be used to prepare a targeting imaging preparation for a primary tumor and / or tumor metastasis lymph node.

3. A tumor targeting imaging probe, characterized in that: The probe comprises a fluorescent dye molecule having near-infrared luminescence capability and the TMVP1446 / GS5 as claimed in claim 1.

4. A tumor targeting imaging probe according to claim 3, characterized in that: The probe is TMVP1446 / GS5-Cy7, wherein the fluorescent dye molecule is Cy7, and Cy7 is connected to the TMVP1446 / GS5 via lysine.

5. A use of the tumor targeting imaging probe as claimed in claim 3 or 4, characterized in that: The tumor-targeted imaging probe can be used for targeted determination of primary tumors and / or tumor metastatic lymph nodes.

6. A method for preparing a tumor targeting imaging probe as claimed in claim 3 or 4, characterized in that: The TMVP1446 / GS5 is first synthesized, and then the fluorescent dye molecule is coupled to the TMVP1446 / GS5.

7. The method for preparing a tumor targeting imaging probe according to claim 6, characterized in that: The following steps are involved: S1, synthesizing TMVP1446 / GS5 peptide containing protecting groups; S2, removing the protecting group of the TMVP1446 / GS5 polypeptide to obtain the TMVP1446 / GS5; S3, purifying the TMVP1446 / GS5 for the first time; S4, mixing and reacting the TMVP1446 / GS5, dimethylformamide solution, fluorescent dye molecular coupling agent and diisopropylethylamine, and purifying the product obtained by the reaction for a second time to obtain the probe.

8. The method for preparing a tumor targeting imaging probe according to claim 7, characterized in that: 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.

9. The method for preparing a tumor targeting imaging probe according to claim 7, characterized in that: 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.

10. The method for preparing a tumor targeting imaging probe according to claim 7, characterized in that: In step S4, the fluorescent dye molecular coupling agent is Cy7 monofunctional N-hydroxysuccinimide ester.

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