VEGFR2 (vascular endothelial growth factor receptor 2) targeting polypeptide, fluorescent probe and application

Abstract

The invention provides a polypeptide targeting VEGFR2, a fluorescent probe and application. The amino acid sequence of the polypeptide targeting VEGFR2 is as shown in SEQ ID No: 1, or the polypeptide targeting VEGFR2 is a derivative polypeptide obtained by modifying the amino acid sequence as shown in SEQ ID No: 1. The polypeptide targeting VEGFR2 is coupled with ICG-NHS to obtain the fluorescent probe ICG-VTP, and the fluorescent probe is stable in structure and long in plasma half-life period, can specifically recognize and combine a VEGFR2 receptor, can be effectively enriched at a tumor part, is suitable for accurate fluorescence imaging of tumors, especially ovarian cancer, is beneficial to accurate recognition of a cancer tissue boundary in an ovarian cancer resection operation, and has a good application prospect. And tumors, especially ovarian cancer, can be diagnosed.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and in particular to a polypeptide targeting VEGFR2, a fluorescent probe and application. BACKGROUND

[0002] Ovarian cancer (OC) is one of the most common malignant tumors in the female reproductive system, and it faces many challenges in early diagnosis, including lack of effective screening methods, atypical clinical symptoms, etc. In addition, during the clinical operation, it is still difficult to accurately define the tumor boundary, which directly affects the formulation of intraoperative decision-making and the accuracy of pathological sampling. In view of the close relationship between the overexpression of specific receptors in the tumor occurrence process and disease progression, it is of great clinical significance and application value to develop a new type of molecular probe that can be used for real-time navigation and precise treatment of ovarian cancer during operation.

[0003] Vascular endothelial growth factor receptor 2 (VEGFR2) is a core molecule that regulates tumor angiogenesis. In solid tumors, VEGFR2 significantly enhances endothelial cell proliferation, migration and vascular permeability by mediating the VEGF signaling pathway, supporting tumor growth and metastasis. In the occurrence and development of ovarian cancer, VEGFR2 promotes tumor growth, invasion and metastasis by mediating tumor angiogenesis and lymphangiogenesis. VEGFR2 is highly expressed on the surface of endothelial cells in many tumors, making it an ideal molecular target.

[0004] In the field of tumor targeted diagnosis and treatment, imaging probes or anti-tumor drugs constructed using ligands such as antibodies, affibodies, and polypeptides that can specifically recognize tumor-associated antigens can significantly improve the detection sensitivity of tumor tissue. In the development of targeting molecules, antibodies have long been dominant, but their large molecular weight, high immunogenicity, and high preparation cost have limited their promotion in some clinical application scenarios. In contrast, polypeptides not only have high affinity and high specificity, but also have low immunogenicity, simple molecular structure, small size, and rapid clearance in the body. In addition, polypeptides have better flexibility in chemical modification and coupling, which is suitable for modification strategies that antibodies cannot achieve. Various polypeptides have been confirmed to be high-specificity targeting ligands for recognizing and binding tumor-related biomarkers, and have shown important value in the design of targeted probes and drugs. Therefore, it is of great significance to develop a polypeptide with high specificity for VEGFR2 receptors.

[0005] Near-infrared two region (NIR-II) fluorescent dye indocyanine green (ICG) exhibits significant advantages of strong tissue penetration and low tissue autofluorescence background in in vivo imaging. In addition, the dye has excellent biocompatibility and low toxicity characteristics. The emission spectrum of ICG can be extended to the near-infrared two region, which helps to further improve the quality of in vivo imaging. However, ICG itself does not have tumor targeting and cannot specifically recognize cancer cells. Due to the characteristics of its molecular structure being easy to modify, it can provide a structural basis for constructing targeted fluorescent probes, thereby having potential application prospects in precise tumor imaging. SUMMARY

[0006] The purpose of the present application is to provide a polypeptide targeting VEGFR2, a fluorescent probe and a preparation method and application thereof. The fluorescent probe is prepared by coupling a polypeptide specifically targeting VEGFR2 receptor with near-infrared fluorescent dye indocyanine green (ICG). The probe can specifically recognize tumor cells and lesions with high expression of VEGFR2 under in vivo and in vitro conditions, and is suitable for precise fluorescent imaging and surgical navigation of tumors.

[0007] The technical solutions of the present application are as follows.

[0008] In a first aspect, the present application provides a polypeptide targeting VEGFR2, wherein the amino acid sequence of the polypeptide targeting VEGFR2 is shown in SEQ ID No: 1, or the polypeptide targeting VEGFR2 is a derivative polypeptide obtained by modifying the amino acid sequence shown in SEQ ID No: 1. The modifier of the modification treatment is selected from polyethylene glycol and a protecting group. The protecting group is selected from at least one of Boc, Fmoc, Adpoc, Iboc, Poc, Z (OMe), Tmz, Cbz and Ddz.

[0009] Preferably, the amino acid sequence of the polypeptide targeting VEGFR2 is HTMYYHHYQHHLSSSDICLPRWGCLWED, as shown in SEQ ID No: 1, and the structural formula is as shown in Figure 1 .

[0010] In a second aspect, the present application provides a fluorescent probe targeting VEGFR2, wherein the preparation raw material of the fluorescent probe targeting VEGFR2 comprises the above-mentioned polypeptide targeting VEGFR2.

[0011] Preferably, the structural formula of the fluorescent probe targeting VEGFR2 is . Wherein, R1 is the above-mentioned polypeptide targeting VEGFR2.

[0012] The structure of the fluorescent probe targeting VEGFR2 (fluorescent probe ICG-VTP) is shown in the following formula: Figure 4

[0013] In a third aspect, the present application provides a preparation method of the polypeptide targeting VEGFR2, comprising: connecting an initial amino acid protected by Fmoc to a solid phase carrier, and removing the Fmoc protection; according to the amino acid sequence of the polypeptide targeting VEGFR2, repeating the following steps: coupling an amino acid protected by Fmoc, removing the Fmoc protection group, obtaining the polypeptide targeting VEGFR2 connected to the solid phase carrier, cleaving, and purifying to obtain the polypeptide targeting VEGFR2.

[0014] In a fourth aspect, the present application provides a preparation method of the fluorescent probe targeting VEGFR2, comprising: dissolving ICG-NHS in an organic solvent, adding the polypeptide targeting VEGFR2, N,N-diisopropyl ethylamine, and performing a light-avoiding reaction, and purifying to obtain the fluorescent probe targeting VEGFR2.

[0015] Preferably, the organic solvent is DMSO, and the time for the light-avoiding reaction is controlled to be 10-14 h.

[0016] In a fifth aspect, the present application provides an application of the polypeptide targeting VEGFR2, the fluorescent probe targeting VEGFR2, or the fluorescent probe targeting VEGFR2 prepared by the preparation method in the preparation of a biological imaging reagent.

[0017] Preferably, the biological imaging reagent is a biological imaging reagent for tumor diagnosis.

[0018] Preferably, the biological imaging reagent for tumor diagnosis is a biological imaging reagent for diagnosis of a tumor with high expression of VEGFR2.

[0019] Preferably, the biological imaging reagent for tumor diagnosis is a biological imaging reagent for diagnosis of ovarian cancer.

[0020] In a sixth aspect, the present application provides an application of the polypeptide targeting VEGFR2, the fluorescent probe targeting VEGFR2, or the fluorescent probe targeting VEGFR2 prepared by the preparation method in the preparation of a resection contour indicating reagent for tumor resection surgery.

[0021] Preferably, the resection contour indicating reagent for tumor resection surgery is a resection contour indicating reagent for ovarian cancer resection surgery.

[0022] The present application has the following beneficial effects: ​1. The present application provides a kind of polypeptide targeted to VEGFR2, the present application with polypeptide K237 targeted to VEGFR2 as targeting part, and it is combined with albumin binding peptide (Albumin Binding Peptide, ABP) to construct new polypeptide targeted to VEGFR2, and the polypeptide targeted to VEGFR2 obtained is coupled with ICG-NHS to construct new type of targeted fluorescent probe ICG-VTP, the fluorescent probe structure is stable, and plasma half-life is long, can specifically recognize and combine VEGFR2 receptor, realizes the targeted positioning of ovarian cancer tissue to the present application.The present application also provides the synthesis method of the above-mentioned polypeptide targeted to VEGFR2, and its synthesis cost is low.

[0023] 2. The present application provides a kind of fluorescent probe targeted to VEGFR2, the fluorescent probe includes the above-mentioned polypeptide targeted to VEGFR2, which can be effectively enriched in tumor site and realize longer retention, suitable for the precise fluorescence imaging of tumor, especially ovarian cancer, with higher signal-to-background ratio, help accurate identification of cancer tissue boundary in ovarian cancer resection surgery, so as to improve the accuracy of tumor resection, and maximum normal tissue is retained.

[0024] 3. The present application also provides the application of the above-mentioned polypeptide targeted to VEGFR2 and fluorescent probe in the preparation of biological imaging reagent. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 It is the structural formula of polypeptide VTP; Figure 2 It is the mass spectrum analysis result of polypeptide VTP; Figure 3 It is the SPR detection result of polypeptide VTP; Figure 4 It is the structural formula of fluorescent probe ICG-VTP; Figure 5 It is the mass spectrum analysis diagram of fluorescent probe ICG-VTP; Figure 6 It is the spectral characterization of fluorescent probe ICG-VTP;Wherein, (A) is the absorption spectrum of fluorescent probe ICG-VTP;(B) is the emission spectrum of fluorescent probe ICG-VTP; Figure 7 It is the structural formula and mass spectrum analysis of polypeptide K237; Figure 8 It is the fluorescent probe and mass spectrum analysis of fluorescent probe ICG-K237; Figure 9It is an imaging effect comparison chart of fluorescent probes ICG-VTP and ICG-K237; wherein, (A) is a fluorescence imaging chart of fluorescent probes ICG-VTP and ICG-K237 in femoral vein of Balb / c mice at different time nodes (5 min, 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 90 min) after injection; (B) is a blood clearance rate comparison chart of fluorescent probes ICG-VTP and ICG-K237; Figure 10 It is a NIR-II fluorescence imaging result of tumor-bearing mice after tail vein injection of fluorescent probe ICG-VTP; wherein, (A) is a NIR-II fluorescence image of experimental group (injection of fluorescent probe ICG-VTP) and blocking group (injection of fluorescent probe ICG-VTP and polypeptide VTP) at different time nodes; (B) is a result chart of tumor / background ratio of experimental group (injection of fluorescent probe ICG-VTP) and blocking group (injection of fluorescent probe ICG-VTP and polypeptide VTP) with time; Figure 11 It is a fluorescent probe ICG-VTP imaging mediated tumor resection surgery; Figure 12 It is a distribution of fluorescent probe ICG-VTP in vivo. DETAILED DESCRIPTION

[0026] The present application will be further described in conjunction with specific examples, but the following examples are only used to illustrate the present application, and should not be regarded as limiting the scope of the present application. In the following examples, the specific conditions are not specified, and the conventional conditions or the conditions recommended by the manufacturer are used, and the methods used are the conventional methods known in the art, and the consumables and reagents used are commercially available unless otherwise specified. Unless otherwise specified, the professional and scientific terms used in this text have the same meaning as those familiar to those skilled in the art. In addition, any method or material similar or equivalent to the described content can also be applied in the present application.

[0027] Balb / c nude mice and Balb / c mice (female, 6-8 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.; human ovarian cancer SKOV3 cells were from the Chinese Academy of Sciences; acetonitrile, dimethyl sulfoxide (DMSO), N,N-diisopropyl ethylamine (DIEA), trifluoroacetic acid (TFA), dichloromethane (DCM), N,N-dimethylformamide (DMF) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; ICG-NHS was purchased from Xi'an Rixi Biological Technology Co., Ltd.; O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU), triisopropylsilane (TIS), piperidine (PIP) were purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd.; resins and amino acids were purchased from Shanghai Jier Biochemical Technology Co., Ltd.; DMEM medium was purchased from Thermo Fisher Scientific (China) Co., Ltd.

[0028] Example 1: Preparation of fluorescent probe ICG-VTP The amino acid sequence of the polypeptide VTP is HTMYYHHYQHHLSSSDICLPRWGCLWED, as shown in SEQ ID No: 1.

[0029] Preparation of polypeptide VTP (polypeptide targeting VEGFR2) by solid phase synthesis (1) On the polypeptide synthesizer, set the corresponding program according to the target polypeptide sequence. Weigh 1.26 g of MBHA resin into the reaction bottle, swell in dichloromethane (DCM) for 30 min, then remove the DCM and wash the resin with N,N-dimethylformamide (DMF) three times, and fully dry after each washing. Then add C-terminal first fluorenylmethoxycarbonyl (Fmoc) protected amino acid Fmoc-Asp(OtBu)-OH (3 mmol), activated reagent O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU) (4.5 mmol) in DMF solution, base catalyst N,N-diisopropyl ethylamine (DIEA) (9 mmol) in DMF solution, react for 45 min, and then wash with DMF, DCM and DMF to obtain Fmoc-Asp(OtBu)-resin.

[0030] (2) Remove Fmoc: Add a DMF solution containing 20% (v / v) piperidine to the reaction bottle to remove the Fmoc protecting group at the amino terminus of the amino acid. After the reaction is complete, wash with DMF, DCM and DMF in turn to obtain Asp(OtBu)-resin.

[0031] (3) Coupling: the second amino acid at the C-terminus of the target sequence is fixed on the Asp(OtBu)-resin obtained in step (2), and the Asp(OtBu)-resin obtained in step (2), Fmoc-protected amino acid Fmoc-Glu(OtBu)-OH (3 mmol), a solution of activating reagent O-benzotriazol-tetramethyluronium hexafluorophosphate (HBTU) (4.5 mmol) in DMF, a solution of base catalyst N,N-diisopropyl ethylamine (DIEA) (4.5 mmol) in DMF are added to the reaction bottle, and the reaction is carried out for 45 min. The product is washed with DMF, DCM and DMF to obtain Fmoc-Glu(OtBu)-Asp(OtBu)-resin. Then, Fmoc is removed according to the method for removing Fmoc in step (2) to obtain Glu(OtBu)-Asp(OtBu)-resin. The process of coupling amino acids and removing Fmoc is repeated according to the above method, and Glu(OtBu)-Asp(OtBu)-resin is sequentially coupled with Fmoc-Trp(Boc)-OH, Fmoc-Leu-OH, Fmoc-L-Cys(Trt)-OH, Fmoc-Gly-OH, Fmoc-Trp(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-L-Cys(Trt)-OH, Fmoc-Ile-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-His(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-His(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Met-OH, Fmoc-Thr(tBu)-OH, Fmoc-His(Trt)-OH, and Fmoc is removed. The product is washed and vacuum dried to obtain a polypeptide VTP containing resin.

[0032] (4) Cleavage: according to the amount of the polypeptide VTP containing resin, a corresponding volume of cleavage solution was prepared at a ratio of 10:1 (v / w) of the cleavage solution to the polypeptide VTP containing resin, and the cleavage solution was composed of 94% trifluoroacetic acid, 2% triisopropylsilane, 2% ethanedithiol and 2% water. The cleavage solution was added to the polypeptide VTP containing resin obtained in step (3); stirring was performed at room temperature for 3-4 hours; then, solid-liquid separation was performed, and the obtained filtrate was collected; pre-cooled anhydrous ether was added to the filtrate, and centrifugal separation was performed to obtain a solid product; the obtained solid was washed with pre-cooled anhydrous ether for three times, the precipitate was collected and vacuum dried to obtain a crude polypeptide VTP product. (5) Purification: the crude polypeptide VTP product obtained in step (4) was purified by high performance liquid chromatography (HPLC), and a reversed-phase C18 column (XBridge BEH C18 0BD Prep Column) was used for the HPLC, gradient elution was performed with acetonitrile and water as the mobile phase, the mobile phase A was acetonitrile (containing 0.1% TFA), the mobile phase B was water (containing 0.1% TFA), the gradient elution time was 15 min, the flow rate was 15 mL / min, the gradient elution conditions were as follows: 33% A + 67% B at 2 min; 80% A + 20% B at 12.5 min; 20% A + 80% B at 15 min, the detection wavelength was 250 nm, the mass spectrometry detection was used to determine that the molecular weight of the target component was 3514.93, and the corresponding eluate was collected. Then, acetonitrile was removed by concentration, and freeze-drying was performed to obtain a white powder target polypeptide, which was polypeptide VTP. The structural formula of the polypeptide VTP is shown in Figure 1 , the mass spectrometry analysis result of the polypeptide VTP is shown in Figure 2 , and the surface plasmon resonance (SPR) detection result of the polypeptide VTP is shown in Figure 3 .

[0033] Preparation of the fluorescent probe ICG-VTP (a fluorescent probe targeting VEGFR2) (1) 1 mg of ICG-NHS was dissolved in 600 μL of DMSO, 15.3 mg of polypeptide VTP and 3.3 μL of N, N-diisopropylethylamine (DIEA) were added, and reaction was performed at 37°C in the dark for 12 h to obtain a reaction solution containing the fluorescent probe ICG-VTP. (2) Purification of the reaction solution containing the fluorescent probe ICG-VTP: The reaction solution containing the fluorescent probe ICG-VTP was diluted by adding methanol, and was separated and purified by high performance liquid chromatography (HPLC) under the following conditions: the chromatographic column was a reversed-phase C18 column (XBridge BEH C18 0BD Prep Column), gradient elution, the mobile phase A was acetonitrile (containing 0.1% TFA), the mobile phase B was water (containing 0.1% TFA), the gradient elution time was 24.5 min, the flow rate was 8 mL / min, the gradient elution condition was: 10% A + 90% B at 2 min, 50% A + 50% B at 16 min, 95% A + 5% B at 18 min, 10% A + 90% B at 24.5 min, and the detection wavelength was 250 nm. The green product finally obtained was confirmed as the expected product ICG-VTP by HPLC and mass spectrometry analysis.

[0034] The structural formula of ICG-VTP is shown in Figure 4 The mass spectrometry analysis result of ICG-VTP is shown in Figure 5 The absorption and emission spectra are shown in Figure 6

[0035] Example 2: Preparation of the fluorescent probe ICG-K237 The amino acid sequence of the polypeptide K237 is HTMYYHHYQHHL, as shown in SEQ ID No: 2.

[0036] The polypeptide K237 was prepared by solid phase synthesis, including the following steps: (1) On the polypeptide synthesizer, the corresponding program was set according to the target polypeptide sequence. 1.26 g of MBHA resin was weighed into a reaction bottle, swelled in dichloromethane (DCM) for 30 min, then the DCM was removed, and the resin was washed with N,N-dimethylformamide (DMF) three times, and each time after washing, it was fully sucked dry. Fmoc-protected amino acid Fmoc-Leu-OH (3 mmol), activated reagent O-benzotriazol-tetramethyluronium hexafluorophosphate (HBTU) (4.5 mmol) in DMF solution, and base catalyst N,N-diisopropylethylamine (DIEA) (9 mmol) in DMF solution were added, reacted for 45 min, and then washed with DMF, DCM and DMF to obtain Fmoc-Leu-resin.

[0037] (2) Removal of Fmoc: A DMF solution containing 20% (v / v) piperidine was added to the reaction bottle to remove the Fmoc protecting group at the amino terminus of the amino acid. After the reaction was completed, DMF, DCM and DMF were used for washing in turn to obtain Leu-resin.

[0038] ​(3) According to the method in step (1) and (2), the process of coupling amino acid and removing Fmoc protecting group is repeated, and Leu-resin is coupled with Fmoc-His(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-His(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Met-OH, Fmoc-Thr(tBu)-OH, Fmoc-His(Trt)-OH, respectively, and Fmoc is removed, washed, and vacuum dried to obtain resin-containing polypeptide K237.

[0039] (4) Cleavage: according to the amount of resin-containing polypeptide K237, a cleavage solution is prepared in a volume ratio of 10:1 (v / w) of the cleavage solution to the crude polypeptide K237, and the cleavage solution is composed of 94% trifluoroacetic acid, 2% triisopropylsilane, 2% ethanedithiol, and 2% water. The cleavage solution is added to the resin-containing polypeptide K237 obtained in step (3); stirring is performed at room temperature for 3-4 hours; then, solid-liquid separation is performed, and the obtained filtrate is collected; pre-cooled anhydrous ether is added to the filtrate, and the solid product is obtained by centrifugal separation; the obtained solid is washed three times with pre-cooled anhydrous ether, the precipitate is collected, and vacuum drying treatment is performed to obtain the crude polypeptide K237.

[0040] (5) Purification: the crude polypeptide K237 is purified, and the purification method of the crude polypeptide K237 is the same as the purification method of the crude polypeptide VTP in Example 1, and the white powdery target polypeptide, i.e., polypeptide K237, is obtained.

[0041] The structural formula and mass spectrum result of the polypeptide K237 are shown in Figure 7

[0042] The preparation method of the fluorescent probe ICG-K237 comprises the following steps: (1) 1 mg of ICG-NHS is dissolved in 600 μL of DMSO, 6.03 mg of polypeptide K237 and 3.3 μL of DIEPA are added, and reaction is performed at 37°C in the dark for 12 hours to obtain a reaction solution containing the fluorescent probe ICG-K237.

[0043] (2) The purification method of the reaction solution containing the fluorescent probe ICG-K237 is the same as the purification method of the reaction solution containing the fluorescent probe ICG-VTP in Example 1. The finally prepared green product is confirmed as the expected product, the fluorescent probe ICG-K237, by HPLC and mass spectrum analysis.

[0044] ​The structural formula and mass spectrometry results of the fluorescent probe ICG-K237 are as follows: Figure 8 As shown.

[0045] Example 3: Testing of fluorescent probes ICG-VTP and ICG-K237 1) Blood half-life assay of fluorescent probes ICG-VTP and ICG-K237 in Balb / c mice To determine the plasma half-life of two fluorescent probes (ICG-VTP and ICG-K237), Balb / c mice were injected with equal doses (20 nmol) of each probe via the tail vein. Fluorescence imaging was performed on the femoral vein region of the mice's abdomen, and the clearance rate of the probes in vivo was calculated based on changes in fluorescence signal. Results are as follows: Figure 9 As shown, Figure 9 (A) shows the fluorescence imaging of the femoral vein of Balb / c mice at different time points (5 min, 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, and 90 min) after injection in different experimental groups (fluorescent probe ICG-VTP and ICG-K237 groups). Figure 9 (B) in the figure is a comparison of the blood clearance rates of fluorescent probes ICG-VTP and ICG-K237. Figure 9 The normalized fluorescence intensity in (B) was calculated as 100% of the vascular fluorescence intensity at 5 min. Figure 9 As shown in Figure (A), both fluorescent probes could clearly image the femoral vein at 5 minutes post-injection; however, the femoral vein signal of fluorescent probe ICG-K237 became blurred at 40 minutes and almost undetectable at 60 minutes, indicating a faster plasma clearance rate. In contrast, fluorescent probe ICG-VTP could still clearly display the femoral vein structure at 60 minutes and maintained good visibility until signal blurring occurred at 90 minutes. Figure 9 The quantitative analysis results in (B) further confirmed that the blood half-life of the fluorescent probe ICG-VTP was significantly longer than that of the fluorescent probe ICG-K237. The specific plasma half-lives of the fluorescent probes ICG-VTP and ICG-K237 were calculated using the following formulas: the plasma half-life of the fluorescent probe ICG-K237 was 10.84 min; the plasma half-life of the fluorescent probe ICG-VTP was 30.24 min.

[0046] The specific formula is as follows: T (1 / 2) = 0.693 / K (Formula 1) K=[ln(Int1)–ln(Int2)] / (t2-t1) (Formula 2) T(1 / 2) The plasma half-life is min; K = elimination rate constant; Int1 is the fluorescence intensity at t1; Int2 is the fluorescence intensity at t2; in this test, t1 is 5 min, and t2 is 60 min.

[0047] 2) Optical imaging of fluorescent probe ICG-VTP in SKOV3 tumor-bearing mice Establishment of SKOV3 tumor model: 5 x 10 6 SKOV3 cells were resuspended in 100 μL of DMEM basic medium, and 100 μL of the culture medium was injected subcutaneously into the right shoulder of each mouse. When the volume of the transplanted tumor grew to about 100-200 mm 3 The in vivo imaging experiment was performed on tumor-bearing mice.

[0048] Experimental group: The fluorescent probe ICG-VTP was prepared into a solution with a concentration of 125 μM using normal saline, and 200 μL of the fluorescent probe ICG-VTP solution was injected into the mice via the tail vein. Optical signal acquisition was performed at time points of 0.5, 1, 2, 4, 6, 8, 10, 12, 24, and 48 hours after injection to observe the distribution of the fluorescent probe ICG-VTP in the mice and its enrichment in the tumor area.

[0049] Blocking group: The fluorescent probe ICG-VTP was prepared into a solution with a concentration of 125 μM using normal saline, and polypeptide VTP was added to the solution so that the molar concentration of polypeptide VTP was 30 times that of the molar concentration of the fluorescent probe ICG-VTP, i.e., 3.75 mM, obtaining a mixed solution containing polypeptide VTP and fluorescent probe ICG-VTP. 200 μL of the mixed solution was injected into the mice via the tail vein. Optical signal acquisition was performed at time points of 0.5, 1, 2, 4, 6, 8, 10, 12, 24, and 48 hours after injection to observe the distribution of the fluorescent probe ICG-VTP in the mice and its enrichment in the tumor area.

[0050] The NIR-II fluorescence imaging results of the experimental group (injected with fluorescent probe ICG-VTP) and the blocking group (injected with fluorescent probe ICG-VTP and polypeptide VTP) at different time nodes are shown in Figures 2A-2D. Figure 10As shown in Figure (A), SKOV3 represents the experimental group, and Block represents the blocking group. The results show that in the experimental group, starting 4 hours after injection, the fluorescent probe ICG-VTP showed significant aggregation in the tumor area, and the tumor margin gradually became clearer; 48 hours after injection, the fluorescent probe ICG-VTP remained in the tumor area. Optical imaging results at various time points clearly show that after blocking with peptide VTP, no obvious fluorescent signal was observed at the tumor site, indicating that peptide VTP successfully competed for the binding site. The ability of the tumor site to take up the probe is due to the specific binding of the probe to VEGFR2. The changes in the tumor / background ratio (signal-to-background ratio) over time for the experimental group (injected with fluorescent probe ICG-VTP) and the blocking group (injected with both fluorescent probe ICG-VTP and peptide VTP) are shown in Figure (A). Figure 10 As shown in (B), SKOV3 represents the experimental group, and Block represents the blocking group. The experimental results show that the tumor / background signal ratio (signal-to-background ratio) reached its highest level in the experimental group 8 hours after injection. This demonstrates that the fluorescent probe ICG-VTP, used as a contrast agent, exhibits high-quality imaging, particularly in significantly improving the tumor-to-background signal ratio.

[0051] 3) Tumor resection and in vivo distribution mediated by the fluorescent probe ICG-VTP like Figure 11 As shown, under the guidance of NIR-II fluorescence imaging using the fluorescent probe ICG-VTP, precise tumor identification and complete resection were achieved by differentiating the fluorescence signals of tumor tissue, surrounding skin, and muscle. While thoroughly removing the tumor tissue, the muscle tissue with weaker fluorescence signals was effectively preserved, thus achieving complete tumor removal while ensuring surgical safety. Mice were processed 48 hours after tumor resection imaging, and major organs and tissues were collected to evaluate the biodistribution of the fluorescent probe ICG-VTP in vivo. The results are as follows: Figure 12 As shown.

[0052] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the invention.

Claims

1. A polypeptide targeting VEGFR2, characterized in that, The amino acid sequence of the peptide targeting VEGFR2 is shown in SEQ ID No: 1, or the peptide targeting VEGFR2 is a derived peptide obtained by modifying the amino acid sequence shown in SEQ ID No:

1. The modifiers used in the modification treatment are selected from polyethylene glycol and protecting groups; The protecting group is selected from at least one of Boc, Fmoc, Adpoc, Iboc, Poc, Z(OMe), Tmz, Cbz and Ddz.

2. A fluorescent probe targeting VEGFR2, characterized in that, The raw materials for preparing the VEGFR2-targeting fluorescent probe include the VEGFR2-targeting peptide as described in claim 1.

3. The fluorescent probe targeting VEGFR2 according to claim 2, characterized in that, The structural formula of the fluorescent probe targeting VEGFR2 is as follows: ； Wherein, R1 is the peptide targeting VEGFR2 as described in claim 1.

4. A method for preparing the peptide targeting VEGFR2 as described in claim 1, characterized in that, include: Using Asp as the initial amino acid, the initial amino acid protected by Fmoc is linked to a solid support, and the Fmoc protection is removed. The following steps are repeated according to the amino acid sequence of the peptide targeting VEGFR2: coupling the Fmoc-protected amino acid, removing the Fmoc protecting group, to obtain the peptide targeting VEGFR2 linked to the solid support, cleaving, and purifying to obtain the peptide targeting VEGFR2.

5. A method for preparing a fluorescent probe targeting VEGFR2 as described in claim 2 or 3, characterized in that, include: ICG-NHS was dissolved in an organic solvent, and the peptide targeting VEGFR2 as described in claim 1 and N,N-diisopropylethylamine were added. The mixture was reacted in the dark and purified to obtain the fluorescent probe targeting VEGFR2.

6. The method for preparing a fluorescent probe targeting VEGFR2 according to claim 5, characterized in that, The organic solvent is DMSO, and the reaction time in the dark is controlled to be 10-14 hours.

7. The application of the peptide targeting VEGFR2 according to claim 1, the fluorescent probe targeting VEGFR2 according to claim 2 or 3, and the fluorescent probe targeting VEGFR2 obtained by the preparation method according to claim 5 or 6 in the preparation of bioimaging reagents.

8. An application as described in claim 7, characterized in that, The bioimaging reagent is a bioimaging reagent for tumor diagnosis.

9. The application according to claim 8, characterized in that, The bioimaging reagent for tumor diagnosis is a bioimaging reagent for ovarian cancer diagnosis.

10. The use of the VEGFR2-targeting polypeptide of claim 1, the VEGFR2-targeting fluorescent probe of claim 2 or 3, and the VEGFR2-targeting fluorescent probe obtained by the preparation method of claim 5 or 6 in the preparation of a resection contour indicator for tumor resection surgery.

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