Near-infrared fluorescent probe as well as preparation method and application thereof

Abstract

The invention provides a near-infrared fluorescent probe, and relates to the technical field of biomedical functional dyes. According to the fluorescent probe, multi-arm polymers with different molecular weights are coupled with near-infrared or infrared fluorescent molecules, so that the fluorescent probe has better water solubility, higher histocompatibility and safety, longer fluorescence lifetime and higher light stability, and meanwhile, rapid transmission and response in ureters, lymphatic vessels and lymph nodes in vivo are realized; the method is used for imaging of NIR-I (850 nm to 1000 nm) and NIR-II (1000 nm to 1700 nm). The fluorescent probe is further marked with various tumor targeting molecules to obtain a targeted near-infrared fluorescent probe, so that accurate targeting fluorescence imaging of different types of tumors such as urogenital tumors, head and neck cancers, breast cancers, cervical cancers and ovarian cancers and metastatic lymph nodes in NIR-I and NIR-II windows can be realized; the method is applied to clinical intraoperative fluorescence imaging navigation.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical functional dyes and probes, and in particular relates to a near-infrared fluorescent dye / probe and a preparation method and application thereof. Background Art

[0002] The application of near-infrared (NIR) fluorescent dyes / probes in fluorescence-guided surgery (FGS) is rapidly developing, primarily to enhance visualization of tumor tissue during surgery for precise resection. The use of NIR fluorescent dyes, such as Indocyanine Green (ICG), can improve the identification of liver cancer tumor boundaries during surgery and reduce the likelihood of residual tumor tissue. Currently, the only FDA-approved NIR fluorescent dyes / probes for clinical use are ICG and Cytalux. However, these technologies still have numerous limitations, such as poor targeting of non-liver cancer tumors and insufficient spatial resolution, which limits their widespread clinical application.

[0003] In recent years, ICG has been widely used for blood perfusion assessment, anatomical visualization, tumor localization, and lymph node mapping / imaging. However, it exhibits significant functional limitations, such as: It easily leaks from target tissues, resulting in large-scale interference signals, which can affect clinical diagnosis and surgery; it cannot be modified and difficult to link with target molecules, which makes positioning and tracking difficult, making it impossible to achieve tumor-targeted imaging, identify tumor metastases, and target neural imaging; because it is excreted through the liver, it cannot image the ureteral system; its emission peak is in the near-infrared region (~814nm), and existing clinical medical imaging equipment can only operate in the near-infrared region of 800-900nm. Due to the strong scattering effect in this band, its imaging resolution, contrast, and depth are fundamentally limited, affecting the accuracy of tumor boundary detection and target observation.

[0004] These limitations reduce the potential application efficiency of NIR imaging in tumor-targeted imaging. In addition, although the Cytalux fluorescent targeting agent has been launched in recent years, the dye has poor targeting properties, and the fluorescence signal ratio between tumor and normal tissue is relatively low (T / NT), only 3-4 times, resulting in unclear tumor boundary distinction, which is not conducive to intraoperative tumor resection in clinical applications. The orphan drug indication of Cytalux is only ovarian cancer, which greatly limits its promotion and application in clinical practice. Moreover, the above fluorescent dyes all belong to the NIR-I imaging window (700nm-900nm), which determines the low tissue resolution, low tissue penetration and high background signal of this type of fluorescent dye. Existing fluorescent conjugates such as ICG, sodium fluorescein (FLS), and 5-aminolevulinic acid (5-ALA) lack specific targeting and cannot be linked to specific targeting molecules, resulting in the inability to accurately couple specific tumors or lesions, let alone target distant metastatic lesions or metastatic lymph nodes. This makes it impossible to achieve highly personalized diagnosis and treatment plans, especially in the precise positioning and treatment of specific types of tumors.

[0005] Dyes such as ICG have strong tissue interactions and slow transmission. When performing lymph node imaging, it is often necessary to wait 40-50 minutes after injection into the tumor. The surgeon may even complete the operation with the naked eye under white light, but the ICG has not yet been infused into the target lymph nodes. In clinical surgery where every second counts, this undoubtedly greatly increases the operation time and patient risk. In terms of ureteral imaging, due to the limitations of the excretion method of the above dyes, they need to be slowly excreted through the liver-intestine pathway rather than quickly excreted through the kidneys-ureters. They cannot be used for effective imaging of specific organs such as the ureters. This makes it difficult to effectively avoid accidental damage to the ureters in certain critical surgical procedures, such as abdominal and pelvic surgery.

[0006] Compared to the visible light region and NIR-I, the wavelength of NIR-II is further increased, significantly suppressing the scattering effect of biological tissue on photons, and the autofluorescence of biological tissue is almost negligible. Fluorescence imaging in this window has unprecedented tissue penetration depth (~3cm) and spatiotemporal resolution, which is more advantageous than NIR-I in clinical applications. Based on this, it is of great clinical significance to find an NIR-II fluorescent dye / probe that can achieve rapid response of the ureter, lymphatic vessels and lymph nodes in the body, as well as more accurate tumor localization and imaging. Summary of the Invention

[0007] The present invention addresses the challenges of existing technologies by providing a near-infrared fluorescent probe with excellent water solubility, high tissue compatibility and safety, and enhanced fluorescence lifetime and photostability. This probe enables high-resolution in vivo imaging of ureters, lymph nodes, and lymphatic vessels, as well as rapid imaging within minutes of injection. This probe can be further coupled with specific targeting molecules to produce a tumor-targeting NIR probe, achieving highly targeted and high-resolution imaging of tumor boundaries. This addresses the lack of specificity of existing NIR fluorescent dyes / probes and provides a powerful surgical navigation tool for precision medicine.

[0008] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0009] On the one hand, the present invention provides a near-infrared fluorescent probe (n-arm-PEG-NIR Dye complex), which is a conjugate of a NIR fluorescent molecule and a multi-arm polymer.

[0010] Furthermore, the multi-arm polymer is multi-arm polyethylene glycol, dextran or a dendrimer.

[0011] Furthermore, the NIR fluorescent molecules are cyanine dyes, electron donor-electron acceptor-electron donor (donor-acceptor-donor, DAD) dyes, boron-dipyrromethene (BODIPY) dyes, rhodamine dyes, metal-macrocycles complexes and gold cluster dyes.

[0012] Furthermore, the cyanine dyes include but are not limited to ICG-NHS, IRDye800CW, IR820-NHS, IR783-NHS, IR-12-NHS, CF790, CF800, CF820, CF850, CF850, CF870, ifluor 750, ifluor 790, ifluor 800, ifluor 810, ifluor 820, ifluor 840, ifluor 860, Zwitterioniccyanine dyes (ZW800, ZW800-1), Dylight 755, Dylight 800, Cy-7-NHS dyes, Alexa Fluor 750, Alexa Fluor 790, Alexa Fluor 800, NovaFluor Yellow 700, NovaFluor Yellow 730, NovaFluor Yellow 755, NovaFluor Red 75, Brilliant Ultra Violet 737, BrilliantUltra Violet 805, Brilliant Violet 711, Brilliant Violet 786, PE-Cyanine7, Qdot705Probe, Qdot 800Probe, Super Bright 780, Cy7, Cy7.5, Cy9 anthocyanin dyes, quantum dots and rare earth down-conversion nanoparticles are solubilized in aqueous phase by encapsulation or complexing with hydrophilic polyethylene glycol (PEG), carbohydrates, dextran, cyclodextrin or other molecules.

[0013] The electron donor-electron acceptor-electron donor (donor-acceptor-donor, DAD) dyes include but are not limited to CH1055, Fluorene-based DAD dyes, IR-FGP, IR-FEP, IR-FEPC, IR-FTAP, CH4T, IR-pFE, Flav 7, FD-1080, IRT, IR-BGP6, IR-BEMC6P, IR-1048-MZ, etc.

[0014] The boron-dipyrromethene (BODIPY) dyes include but are not limited to TPA-BDP, Cz-BDP, TPACz-BDP, 3TPA-BDP, Protoporphyrin, Temoporfin, Verteporfin, Talaporfin, Photolon, Photofrin, and the like.

[0015] The rhodamine dyes include but are not limited to ECXa-j, CX-1-3, Rh824, Rh926, Rh1029, NIRII-RT3, NIRII-RT4, VIX-1-4, RhIndz, etc.

[0016] The metal-macrocycles complexes dyes include but are not limited to Pd-1, Pd-2, Pd-3, Pt-1, F-Yb and the like.

[0017] The gold cluster dye includes but is not limited to Au 25 , Au 28 , Au7Cd1, AuNCs, etc.

[0018] Furthermore, the multi-arm polyethylene glycol is selected from one or more of two-arm polyethylene glycol, four-arm polyethylene glycol, six-arm polyethylene glycol, eight-arm polyethylene glycol, and ten-arm polyethylene glycol;

[0019] Furthermore, the multi-arm polyethylene glycol is 8-arm-PEG-NH2, 8-arm-PEG-SH, 8-arm-PEG-alkyne, 8-arm-PEG-N3 or 8-arm-PEG-COOH.

[0020] Furthermore, the dextran is selected from one or more of dextran methoxypolyethylene glycol, dextran ester, diethylaminoethyl dextran, dextran polyethylene glycol, Dextran-NH2, Dextran-NHS, and DEAE-Dextran;

[0021] Furthermore, the dextran polyethylene glycol is Dextran-PEG-NH2, Dextran-PEG-COOH, Dextran-PEG-Alkyne, Dextran-PEG-N3 or Dextran-PEG-SH.

[0022] Furthermore, the dendrimer is selected from one or more of Dendrimer-NH2, polypropyleneimine dendrimer, polyamidoamine dendrimer, and polylysine dendrimer;

[0023] Furthermore, the molecular weight of the multi-arm polymer is 3kDa to 200kDa, such as 10kDa, 40kDa, 60kDa, 100kDa, etc. By changing the molecular weight of the probe, the distribution and excretion route of the probe in the body can be changed. Probes less than 30kDa are excreted by the kidneys through urine and can be used for ureteral imaging. Probes greater than 40kDa can effectively target and enrich into tumors due to their longer blood circulation time, and are used for efficient tumor imaging.

[0024] In one embodiment of the present invention, the structure of the near-infrared fluorescent probe is as shown in the attached Figure 1 As shown, it takes n-arm-PEG as the core, and n-arm-PEG is coupled with m NIR fluorescent molecules through its n arms, where n can be 2, 4, 6, 8, 10, and m is the number of NIR fluorescent molecules on each n-arm-PEG, m≤n.

[0025] The present invention also provides a method for preparing a near-infrared fluorescent probe, comprising the following steps:

[0026] (1) dissolving the NIR fluorescent molecule in a solvent to obtain a NIR fluorescent molecule solution;

[0027] (2) adding the NIR fluorescent molecule solution and the multi-arm polymer to a buffer solution, adding or not adding a catalyst or coupling agent, adjusting the pH value, and reacting for a certain time;

[0028] (3) The reactant is centrifuged and washed to obtain the near-infrared fluorescent probe.

[0029] Furthermore, the solvent in step (1) is selected from DMSO, pure water or buffer solution.

[0030] Furthermore, the pH value in step (2) is 5-9.

[0031] Furthermore, the reaction time of step (2) is 1-3 hours.

[0032] Furthermore, the catalyst or coupling agent in step (2) is EDC or copper ions.

[0033] Furthermore, the reaction in step (2) is carried out in the dark at room temperature.

[0034] Furthermore, in step (3), according to the molecular weight of the multi-arm polymer, an ultrafiltration centrifuge tube of corresponding molecular weight is used for centrifugation.

[0035] The present invention also provides the use of the near-infrared fluorescent probe in preparing a fluorescent imaging agent.

[0036] Furthermore, the fluorescent imaging agent is used for imaging the ureters, lymph nodes, lymphatic vessels, gastrointestinal tract, blood vessels, and nerves of animals and humans.

[0037] The method for using the near-infrared fluorescent probe of the present invention is to dissolve the near-infrared fluorescent probe in a buffer solution with a final concentration of 1 mg / ml for injection into the vein (IV), subcutaneous (SC), tumor (IT) or around the tumor, muscle (IM) and other tissues.

[0038] The present invention also provides a method for fluorescent imaging of animal or human blood vessels, comprising intravenously injecting a near-infrared fluorescent probe with a molecular weight of 30kDa-200kDa according to the present invention, and then imaging the blood vessels in vivo using a NIR-I or NIR-II fluorescence endoscope, or imaging the blood vessels on the entire body surface using an in vitro imaging device in the NIR-I or NIR-II region. This method is used to visualize blood vessels during surgery, thereby preventing damage to the blood vessels. Preferably, the multi-arm polyethylene glycol in the near-infrared fluorescent probe is 8-arm-PEG, and the molecular weight of the 8-arm-PEG is approximately 40KD; the NIR fluorescent molecule is one or more of IRDye800-NHS, ICG-NHS, IR-12-NHS, IR783-NHS, IR820-NHS, or IR840-NHS.

[0039] The present invention also provides a method for fluorescent imaging of the ureter in animals or humans. The method comprises injecting a near-infrared fluorescent probe of the present invention having a molecular weight of less than 30 kDa intravenously, intraperitoneally, or subcutaneously, followed by excretion into the ureter via the kidneys. The ureter is then imaged within 2 minutes to 5 hours using a fluorescence endoscope in the NIR-I or NIR-II range or a surgical robot fluorescence imaging system. For open surgery, the ureter can be exposed by opening the abdominal cavity and imaging can be performed using an in vitro NIR-I or NIR-II imaging device. This method is used to visualize the ureter during surgery, thereby preventing damage. Preferably, the multi-arm polyethylene glycol in the near-infrared fluorescent probe is 8-arm-PEG, having a molecular weight of approximately 10 kDa; and the NIR fluorescent molecule is one or more of IRDye 800-NHS, ICG-NHS, IR-12-NHS, IR783-NHS, IR820-NHS, or IR840-NHS.

[0040] The present invention also provides a fluorescence imaging method for lymphatic vessels and / or lymph nodes in animals or humans, the method comprising injecting the near-infrared fluorescent probe of the present invention into the lymph nodes through the lymphatic vessels after being injected into the tissue or tumor edge, and imaging the lymphatic vessels and / or lymph nodes near the tissue or tumor using a fluorescence endoscope in the NIR-I or NIR-II region or a surgical robot fluorescence imaging system within 2 minutes to 5 hours after the injection. For open surgery, an in vitro imaging device in the NIR-I or NIR-II region can be used to image the lymphatic vessels and / or lymph nodes near the tissue or tumor.

[0041] The present invention also provides a kit for fluorescence imaging, which comprises the above-mentioned near-infrared fluorescent probe.

[0042] On the other hand, the present invention also provides a near-infrared fluorescent probe (n-Arm-PEG-NIR Dye / targeting ligand) for tumor-specific targeted imaging, wherein the fluorescent probe is a conjugate of a NIR fluorescent molecule, a multi-arm polymer, and a specific targeting molecule (targeting ligand).

[0043] Furthermore, the multi-arm polymer is multi-arm polyethylene glycol, dextran or a dendrimer.

[0044] Furthermore, the near-infrared fluorescent probe for tumor-specific targeted imaging is formed by coupling a near-infrared fluorescent probe with a specific targeting molecule.

[0045] Furthermore, the specific targeting molecule is selected from any one of peptide, aptamers, affibodies, and antibodies.

[0046] Furthermore, the specific targeting molecules are directed against PSMA, αvβ3integrin, α4β1integrin, α6β1integrin, HER1-4, SSTR1-5, GnRH-R, VIP, NTSR1, CCK2R, EphA2, CD133, TD05, TE02, AS1411, TLS11a, Sgc8, 41t, TE17, KDED 2a-3, KCHA10, Sgd5, TTA1, MUC-1, A32, S11e, S6, J3, F3B, A10, VEGF165, folate receptor FOLR1, folate receptor FOLR2, folate receptor FOLR3, folate receptor FOLR4, EGFR, HER2, HER3, PD-L1, PDGFRβ, VEGFR, CAIX, LMP, LMP1, PD-L1, PD-1, PSMA, TNFα, IL-1, IL-6, IL-12, IL-13, IL-17A, IL-23, MCP-1, IGF-1, IFN-γ , CSF1R, DLL4, TGF-β, TUFM, VEGF, EGFR, FGFR2, ICAM-1, ARTC2, CAIX, CapG, CD11b, CD20, CD33, CD38, CD44, CD45, CD47, CD52, CD7, CD8, CD105, CEA, c-Met, CTLA-4, CXCL11, CXCL12, CXCR2, CXCR4, CXCR7, Fibronectin, BCMA, HER2, HGF, Ly-6C / Ly-6G, MHC-II, MMR, Viral GPCR, US28, MMP2, MMP3, MMP7, MMP9, MMP12 target any one of peptides, nucleic acid aptamers, affibodies, and antibodies.

[0047] Furthermore, the specific targeting molecule is any one of peptides, nucleic acid aptamers, affinity bodies, and antibodies targeting PSMA, αvβ3integrin, HER2, EGFR, VEGFR1, VEGFR2, VEGFR3, TROP-2, Nectin-4, CD8, CD4, CD105, CD133, PD-L1, or PD-1 targets.

[0048] Furthermore, the specific targeting molecule is any one of the small molecule vitamin B12, vitamin H, thiamine, riboflavin, adenosine, N-acetylglucosamine, folic acid, HS-PEG-Folic acid (PEG molecular weight is 1-20 kDa), methotrexate, mannose, carbohydrates, hyaluronic acid, fructose, cRGD and Bevacizumab, Cetuximab, Atezolizumab, CD105 monoclonal antibody, Andecaliximab monoclonal antibody, and TRC105 antibody.

[0049] In one embodiment of the present invention, the structure of the near-infrared fluorescent probe for tumor-specific targeted imaging is as shown in the attached Figure 2 As shown, it takes n-arm-PEG as the core, and n-arm-PEG is coupled with m NIR fluorescent molecules and w specific targeting molecules through its n arms, wherein n can be 2, 4, 6, 8, 10, m is the number of NIR fluorescent molecules, m≤n, and w is the number of specific targeting molecules, w≤nm.

[0050] Preferably, the multi-arm polyethylene glycol is 8-arm-PEG, and the molecular weight of the 8-arm-PEG is about 10KD or about 40KD; the NIR fluorescent molecule is one or more of IRDye 800-NHS, ICG-NHS, IR-12-NHS, IR783-NHS, IR820-NHS or IR840-NHS; and the specific targeting molecule is a folic acid molecule, an RGD polypeptide or a PSMA polypeptide.

[0051] The present invention also provides a method for preparing a near-infrared fluorescent probe for tumor-specific targeted imaging, comprising the following steps:

[0052] (1) dissolving the NIR fluorescent molecule in a solvent to obtain a NIR fluorescent molecule solution;

[0053] (2) adding the NIR fluorescent molecule solution and the multi-arm polymer to a buffer solution, adding or not adding an appropriate amount of catalyst or coupling agent, adjusting the pH value, and reacting for a certain time;

[0054] (3) centrifuging and washing the reactants;

[0055] (4) Resuspending the washed reactants in a buffer solution, adding or not adding a linker molecule, and reacting for a certain period of time;

[0056] (5) centrifuging and washing the reactants;

[0057] (6) resuspending the washed reactants in a buffer solution, adjusting the pH value of the buffer, adding a catalyst, a coupling agent, and a specific targeting molecule, and reacting for a certain time;

[0058] (7) The reactants are centrifuged and washed to obtain the tumor-specific targeted imaging near-infrared fluorescent probe.

[0059] Furthermore, the solvent in step (1) is selected from DMSO, pure water or buffer solution.

[0060] Furthermore, the pH value in step (2) is 5-9.

[0061] Furthermore, the reaction time of step (2) is 1-3 hours.

[0062] Furthermore, the catalyst or coupling agent in step (2) is EDC or copper ions.

[0063] Furthermore, the reaction in step (2) is carried out in the dark at room temperature.

[0064] Furthermore, in step (3), according to the molecular weight of the multi-arm polymer, an ultrafiltration centrifuge tube of corresponding molecular weight is used for centrifugation.

[0065] Furthermore, the pH value in step (4) is 5-9.

[0066] Furthermore, the reaction in step (4) is carried out in the dark at room temperature, and the reaction time is 1-3 hours.

[0067] Furthermore, the linking molecule in step (4) is SMCC, SPDP or SPDB.

[0068] Furthermore, in step (5), according to the molecular weight of the multi-arm polymer, centrifugation is performed using an ultrafiltration centrifuge tube of corresponding molecular weight.

[0069] Furthermore, the pH value in step (6) is 5-9.

[0070] Furthermore, the reaction in step (6) is carried out in the dark at room temperature, and the reaction time is 1-3 hours.

[0071] Furthermore, the catalyst and coupling agent in step (6) are EDC or copper ions.

[0072] Furthermore, in step (7), according to the molecular weight of the multi-arm polymer, centrifugation is performed using an ultrafiltration centrifuge tube of corresponding molecular weight.

[0073] The present invention also provides the use of the tumor-specific targeted imaging near-infrared fluorescent probe in the preparation of tumor tracers, tumor boundary judgment imaging agents, tumor resection surgery navigation imaging agents or tumor metastasis lymph node imaging agents.

[0074] Furthermore, the tumors include genitourinary system tumors (kidney cancer, renal pelvis cancer, ureter cancer, adrenal cancer, bladder cancer, prostate cancer, penile cancer, testicular cancer), gastrointestinal system tumors, skin cancer, head and neck cancer, breast cancer, etc.

[0075] The method for using the near-infrared fluorescent probe for tumor-specific targeted imaging of the present invention is to resuspend the near-infrared fluorescent probe for tumor-specific targeted imaging in a buffer solution to a final concentration of 1 mg / ml for injection into the vein (IV), subcutaneously (SC), inside or around the tumor (IT), muscle (IM) and other tissues.

[0076] The present invention also provides a method for targeted near-infrared fluorescence imaging of tumors in animals or humans. A 40kDa to 200kDa tumor-specific near-infrared fluorescent probe for targeted imaging is injected intravenously into the bloodstream, where it specifically enters the tumor and binds to cancer cells. Within 24-72 hours, the tumor is imaged using a fluorescence endoscope or surgical robot in the NIR-I or NIR-II range. For open surgery, in vitro fluorescence imaging equipment in the NIR-I or NIR-II range can be used. This imaging can be used to trace the boundary between tumor and normal tissue, enabling precise tumor resection and providing surgical navigation.

[0077] The present invention also provides a near-infrared fluorescence imaging method for targeting metastatic cancer cells in animals or humans. A tumor-specific near-infrared fluorescent probe for targeted imaging is injected intravenously or at the tumor margin. The fluorescent probe enters the sentinel lymph node of the tumor through the lymphatic vessels. Within 1-48 hours, the lymph node is imaged using fluorescence in the NIR-I or NIR-II regions. Lymph nodes with a long-lasting fluorescence signal are those where cancer cells have metastasized. Lymph nodes with metastatic cancer cells bind to the specifically targeted near-infrared fluorescent probe, resulting in an enhanced and sustained fluorescence signal. This specific targeted imaging technology can provide real-time intraoperative navigation for tumor-metastatic lymph nodes.

[0078] The present invention also provides a near-infrared fluorescence imaging method for targeted nerves in animals or humans. A near-infrared fluorescent probe for tumor-specific targeted imaging is injected intravenously, enters the blood circulation and binds to nerves in the body. Within 1-48 hours, the nerves are imaged using fluorescence in the NIR-I or NIR-II region to provide surgical navigation to avoid nerve damage.

[0079] Compared with the prior art, the present invention has the following beneficial effects:

[0080] The present invention modifies near-infrared fluorescent molecules with multi-arm polymers to produce near-infrared fluorescent probes. These probes have improved water solubility, higher tissue compatibility, longer fluorescence lifetime, and greater photostability. This allows these probes to be rapidly excreted through the kidneys after injection. Furthermore, local or subcutaneous injection into tumors can achieve lymphatic, lymph node, and nerve responses within minutes, enabling high-resolution imaging of the ureters, lymph nodes, and lymphatic vessels in vivo. The design of these probes, combined with targeting ligands, not only enhances the targeting of the fluorescent probes but can also be used for fluorescence imaging of ureteral tumors, tumor sentinel lymph nodes, and neural tissue. These lymph nodes exhibit longer-lasting fluorescence than those without metastatic cancer cells. This innovation effectively overcomes the limitation of existing fluorescent dyes, which cannot be rapidly excreted for imaging, particularly reducing the risk of damage to vital organs during abdominal surgery. It also provides new possibilities for lymph node and nerve imaging, enabling more precise tumor localization and imaging, greatly enhancing the potential and practicality of this technology in clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 Schematic diagram of the structure of the near-infrared fluorescent probe of the present invention.

[0082] Figure 2 Schematic diagram of the structure of the near-infrared fluorescent probe for tumor-specific targeted imaging of the present invention.

[0083] Figure 3 A. Figure 3 B are the absorption spectrum and emission spectrum of the near-infrared fluorescent probe prepared in Example 1 of the present invention.

[0084] Figure 4 A. Figure 4 B respectively represent the absorption spectrum and emission spectrum of the near-infrared fluorescent probe prepared in Example 2 of the present invention.

[0085] Figure 5 A. Figure 5 B respectively represent the absorption spectrum and emission spectrum of the near-infrared fluorescent probe for tumor-specific targeted imaging prepared in Example 3 of the present invention.

[0086] Figure 6 A. Figure 6 B respectively represent the absorption spectrum and emission spectrum of the near-infrared fluorescent probe for tumor-specific targeted imaging prepared in Example 4 of the present invention.

[0087] Figure 7 A. Figure 7B respectively represent the absorption spectrum and emission spectrum of the near-infrared fluorescent probe for tumor-specific targeted imaging prepared in Example 5 of the present invention.

[0088] Figure 8 This is fluorescence imaging of the ureter in mice using the near-infrared fluorescent probe prepared in Example 1 of the present invention.

[0089] Figure 9 This is fluorescence imaging of lymph nodes and lymphatic vessels in mice using the near-infrared fluorescent probe prepared in Example 1 of the present invention.

[0090] Figure 10 This is fluorescence imaging of the ureter in mice using the near-infrared fluorescent probe prepared in Example 2 of the present invention.

[0091] Figure 11 This is fluorescence imaging of lymph nodes and lymphatic vessels in mice using the near-infrared fluorescent probe prepared in Example 2 of the present invention.

[0092] Figure 12 The near-infrared fluorescent probe for tumor-specific targeted imaging prepared in Example 3-4 of the present invention was used for tumor-targeted imaging in mice.

[0093] Figure 13 The near-infrared fluorescent probe for tumor-specific targeted imaging prepared in Example 5 of the present invention was used for tumor-targeted imaging in mice. DETAILED DESCRIPTION

[0094] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of embodiments of the present invention, rather than all embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work premise belong to the scope of protection of the present invention. In addition, it is worth noting that the raw materials involved in the present invention are all common commercially available products unless otherwise specified.

[0095] Definition of terms

[0096] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the procedures in chemistry, molecular biology, biochemistry, cell biology, and the like used herein are conventional procedures widely used in the relevant fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0097] The term "near infrared" as used herein refers to infrared light (NIR), which is an electromagnetic wave between visible light (VIS) and mid-infrared light (MIR). The NIR region is conventionally divided into two regions: NIR 1 (750-900 nm) and NIR 2 (1000-1700 nm).

[0098] In the present invention, "conjugate" refers to a new compound formed by covalently linking (coupling) two or more compound molecules through a bivalent or multivalent compound molecule with a linking function. A conjugate can also be formed by direct coupling or condensation of two molecules.

[0099] The "fluorescent probe" in the present invention means a moiety that can be detected by colorimetry or fluorescence measurement.

[0100] Example 1

[0101] A method for preparing a near-infrared fluorescent probe comprises the following steps:

[0102] (1) Dissolve 20 mg of the NIR fluorescent molecule IRDye800CW in DMSO at a concentration of 5 mM;

[0103] (2) Add 100 μl of the above-mentioned NIR fluorescent molecule solution and 2.5 mg of 8-Arm-PEG (10 kD molecular weight) to 0.5 ml of PBS solution, adjust the reaction system to a pH of 9, and shake the reaction at room temperature in the dark for 2 h.

[0104] (3) Based on the molecular weight of 8-Arm-PEG, an ultrafiltration centrifuge tube with a molecular weight cutoff of 3 kDa was used for centrifugation at 14,000 rpm / min for 5 minutes. After adding PBS, the mixture was repeatedly washed 4 times to remove excess NIR fluorescent molecules and DMSO, thereby obtaining the near-infrared fluorescent probe.

[0105] like Figure 3 Figure A shows the absorption spectra of IRDye800CW and IRDye800CW-8-Arm-PEG (10kDa) collected after filtration using an ultrafiltration tube with a molecular weight cutoff of 3kDa. The absorption peak is located at 775nm. Since the molecular weight of IRDye800CW is only 0.96kDa, the dye that is not coupled to 8-Arm-PEG will be filtered out, while the dye collected in the ultrafiltration tube is the successfully coupled IRDye800CW-8-Arm-PEG. The number of NIR fluorescent molecules on each 8-arm-PEG is 3. Its emission spectrum under 808nm laser excitation conditions is shown in Figure 4. Figure 3 B shows 890nm.

[0106] Example 2

[0107] A method for preparing a near-infrared fluorescent probe:

[0108] Referring to the preparation method of Example 1, the NIR fluorescent molecule IRDye800CW was replaced by ICG-NHS, and the remaining steps were the same.

[0109] like Figure 4 Figure A shows the absorption spectra of ICG-NHS and ICG-NHS-8-Arm-PEG (10kDa) collected after filtration using an ultrafiltration tube with a molecular weight cutoff of 3kDa. The absorption peak is located at 789nm. Since the molecular weight of ICG-NHS is only 0.82kDa, the dye that is not coupled to 8-Arm-PEG will be filtered out, while the dye collected in the ultrafiltration tube is the successfully coupled ICG-NHS-8-Arm-PEG. The number of NIR fluorescent molecules on each 8-arm-PEG is 3. The emission spectrum of ICG-NHS-8-Arm-PEG under 808nm laser excitation is shown in Figure 4. Figure 4 As shown in B, its emission peak is at 890nm.

[0110] Example 3

[0111] A method for preparing a near-infrared fluorescent probe for tumor-specific targeted imaging comprises the following steps:

[0112] (1) Dissolve 20 mg of the NIR fluorescent molecule IRDye800CW in DMSO at a concentration of 5 mM;

[0113] (2) Add 100 μl of the above-mentioned NIR fluorescent molecule solution and 10 mg of 8-Arm-PEG (40 kDa) to 0.5 ml of PBS solution, adjust the reaction system to a pH of 9, and shake the reaction at room temperature in the dark for 2 h.

[0114] (3) Based on the molecular weight of 8-Arm-PEG, use an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa and centrifuge at 14,000 rpm / min for 5 minutes. After adding PBS, wash repeatedly for 4 times to remove excess NIR fluorescent molecules and DMSO;

[0115] (4) The washed reactants were resuspended in 0.5 ml of PBS solution (pH 5), 2 mg of N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride was added, and after mixing, the specific targeting molecule Folic acid (FA) was added. The reaction was shaken in the dark at room temperature for 2 hours.

[0116] (5) According to the molecular weight of the specific targeting molecule, an ultrafiltration centrifuge tube with a molecular weight smaller than the molecular weight is used, centrifuged at 14,000 rpm / min for 5 minutes, and then washed repeatedly 4 times after adding PBS to obtain the near-infrared fluorescent probe for tumor-specific targeted imaging.

[0117] like Figure 5 A shows the absorption spectra of IRDye800CW and IRDye800CW-8-Arm-PEG (40kDa) and IRDye800CW-8-Arm-PEG-FA collected after filtration using an ultrafiltration tube with a molecular weight cutoff of 10kDa. The absorption peak is located at 775nm. Since the molecular weights of IRDye800CW and FA are only 0.96kDa and 0.44kDa, the dye that is not coupled to 8-Arm-PEG will be filtered out. What is collected in the ultrafiltration tube after two filtrations is the successfully coupled RDye800CW-8-Arm-PEG and IRDye800CW-8-Arm-PEG-FA. The number of NIR fluorescent molecules and targeting molecules on each 8-arm-PEG is 3 and 3. Its emission spectrum under 808nm laser excitation conditions is shown as follows: Figure 5 B shows 895nm.

[0118] Example 4

[0119] A method for preparing a near-infrared fluorescent probe for tumor-specific targeted imaging is described, referring to the preparation method of Example 3, except that the NIR fluorescent molecule IRDye800CW is replaced with 820NHS, and the remaining steps are the same.

[0120] like Figure 6 As shown in A, the absorption spectra of 820NHS and 820NHS-8-Arm-PEG (40kDa) and 820NHS-8-Arm-PEG-FA collected after filtration using an ultrafiltration tube with a molecular weight cutoff of 10kDa are shown. The absorption peak is located at 820nm. Since the molecular weights of 820NHS and FA are only 1.05kDa and 0.44kDa, the dye that is not coupled to 8-Arm-PEG will be filtered out. The dye collected in the ultrafiltration tube after two filtrations is the successfully coupled 820NHS-8-Arm-PEG and 820NHS-8-Arm-PEG-FA. The number of NIR fluorescent molecules and targeting molecules on each 8-arm-PEG is 3 and 3. Its emission spectrum under 808nm laser excitation conditions is shown in Figure 1. Figure 6 B shows 955nm.

[0121] Example 5

[0122] A method for preparing a near-infrared fluorescent probe for tumor-specific targeted imaging comprises the following steps:

[0123] (1) Dissolve 20 mg of the NIR fluorescent molecule IR-12-NHS in DMSO at a concentration of 2.5 mM; also dissolve the folic acid molecule with thiol modification in DMSO at a concentration of 2.64 mg / ml.

[0124] (2) Add 40 μl of the above-mentioned NIR fluorescent molecule and 0.5 mg of 8-Arm-PEG (10 kDa) to 0.8 ml of PBS solution, adjust the reaction system to a pH of 7.4, and shake the reaction at room temperature in the dark for 3 hours;

[0125] (3) Based on the molecular weight of 8-Arm-PEG, use an ultrafiltration centrifuge tube with a molecular weight cutoff of 3 kDa to centrifuge at 14,000 rpm / min for 10 minutes, add PBS and wash repeatedly 4 times to remove excess NIR fluorescent molecules and DMSO;

[0126] (4) The washed reaction product was resuspended in 0.8 ml of PBS solution (pH 6.5), 2 mg of succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate was added, mixed, and then 200 μl of the folic acid (FA) solution prepared in step (1) was added. The reaction was shaken in the dark at room temperature for 15 hours.

[0127] (5) According to the molecular weight of the specific targeting molecule, an ultrafiltration centrifuge tube with a smaller molecular weight is used, centrifuged at 14,000 rpm / min for 5 minutes, and then washed repeatedly 8 times after adding PBS. Finally, the mixture is resuspended in 2 ml of PBS solution with a pH value of 7.4 to obtain the near-infrared fluorescent probe for tumor-specific targeted imaging.

[0128] like Figure 7 A. Figure 7 B are the absorption and emission spectra of IR-12-NHS-8-Arm-PEG-FA, respectively. Figure 7 A is the absorption spectrum of IR-12-NHS-8-Arm-PEG and IR-12-NHS-8-Arm-PEG-FA collected after filtration using an ultrafiltration tube with a molecular weight cutoff of 10kDa. The absorption peak is located at 790nm. Since the molecular weight of IR-12-NHS is only 1.7kDa, the dye that is not coupled to 8-Arm-PEG will be filtered out. After two filtrations, the dye collected in the ultrafiltration tube is the successfully coupled IR-12-NHS-8-Arm-PEG and IR-12-NHS-8-Arm-PEG-FA. The average number of NIR fluorescent molecules and targeting molecules on each 8-arm-PEG is 1.8 and 3. Figure 7B is the emission spectrum of the IR-12-NHS-8-Arm-PEG-FA probe under 808 nm laser excitation, and the probe has obvious fluorescence signals.

[0129] Application Example 1

[0130] Application of the near-infrared fluorescent probe prepared in Example 1 in fluorescence imaging of the ureter in mice.

[0131] Take 100 μL of the IRDye800CW-8-Arm-PEG fluorescent probe prepared in Example 1. After completing anesthesia and disinfection, inject the probe into BABL / c mice via tail vein injection, intratumoral injection, or subcutaneous injection into the mouse tail. Under anesthesia, dissect the mouse skin, peritoneum, and intestines in sequence to expose the kidneys and ureters. Observe the excretion of the probe through the ureter by imaging under 808 nm laser irradiation and 900 nm, 1000 nm, 1100 nm, and 1200 nm wavelength filters.

[0132] like Figure 8 As shown, the IRDye800CW-8-Arm-PEG probe was injected into BABL / c mice via the tail vein, intratumoral, or subcutaneous injection. Within 2 minutes, the mice were dissected under anesthesia to expose the kidneys, ureters, and bladders. An 808 nm laser was used to excite the IRDye800CW-8-Arm-PEG fluorescent probe. Images of the IRDye800CW-8-Arm-PEG probe injected intravenously (A), intratumorally (B), and subcutaneously (C) are shown using different wavelength filters. The probe is rapidly excreted by the kidneys, with ureteral peristalsis expelling the probe from the kidneys into the bladder with urine. Within 2 minutes of injection, the kidneys, ureters, and bladder were clearly visualized, contrasting sharply with background tissue signals. Increasing the filter wavelength increased fluorescence imaging resolution, resulting in clearer images of the ureters and reduced tissue autofluorescence, highlighting the ureteral morphology.

[0133] Application of the near-infrared fluorescent probe prepared in Example 1 in fluorescence imaging of lymph nodes and lymphatic vessels in mice.

[0134] like Figure 9 Figures show fluorescence imaging of lymph nodes and lymphatic vessels in vivo at different time points using an 808nm laser excitation, following tumor injection (A) and subcutaneous injection (B) of the near-infrared fluorescent probe prepared in Example 1. (C) shows imaging of subcutaneous injection of the fluorescent probe using different wavelength filters. Increasing the filter wavelength increases the resolution of fluorescence imaging of lymph nodes and lymphatic vessels, resulting in clearer images and reduced tissue autofluorescence signals.

[0135] Application Example 2

[0136] Application of the near-infrared fluorescent probe prepared in Example 2 in fluorescence imaging of the ureter in mice.

[0137] Take 100 μL of the ICG-NHS-8-Arm-PEG fluorescent probe prepared in Example 2. After completing mouse anesthesia and disinfection, the probe was injected into BABL / c mice via tail vein injection, intratumoral injection, or subcutaneous injection into the mouse tail. Under anesthesia, the mouse skin, peritoneum, and intestine were dissected in sequence to expose the kidneys and ureters. Imaging was performed under 808 nm laser irradiation and 900 nm, 1000 nm, 1100 nm, and 1200 nm wavelength filters to observe the excretion of the probe through the ureter.

[0138] like Figure 10 As shown, the ICG-NHS-8-Arm-PEG probe was injected into BABL / c mice via the tail vein, intratumoral, or subcutaneous injection. Within 2 minutes of injection, the mice were dissected under anesthesia to expose the kidneys, ureters, and bladders. An 808 nm laser was used to excite the ICG-NHS-8-Arm-PEG fluorescent probe. Images of the ICG-NHS-8-Arm-PEG probe injected intravenously (A), intratumorally (B), and subcutaneously (C) are shown using different wavelength filters. The probe is rapidly excreted by the kidneys, with ureteral peristalsis expelling the probe from the kidneys into the bladder with urine. Within 2 minutes of injection, the kidneys, ureters, and bladder were clearly visualized, contrasting sharply with background tissue signals. Increasing the filter wavelength increased fluorescence imaging resolution, resulting in clearer ureteral imaging and reduced tissue autofluorescence, highlighting the ureteral morphology.

[0139] Application of the near-infrared fluorescent probe prepared in Example 2 in fluorescence imaging of lymph nodes and lymphatic vessels in mice.

[0140] like Figure 11 Figures show fluorescence imaging of lymph nodes and lymphatic vessels in vivo at different time points using an 808nm laser excitation, following tumor injection (A) and subcutaneous injection (B) of the near-infrared fluorescent probe prepared in Example 2. (C) shows imaging of subcutaneous injection of the fluorescent probe using different wavelength filters. Increasing the filter wavelength increases the resolution of fluorescence imaging of lymph nodes and lymphatic vessels, resulting in clearer images and reduced tissue autofluorescence signals.

[0141] Application Example 3

[0142] Application of the near-infrared fluorescent probe prepared in Example 3-4 in tumor-targeted imaging.

[0143] 100 μL of each of the IRDye800CW-8-Arm-PEG-FA and 820NHS-8-Arm-PEG-FA fluorescent probes prepared in Examples 3-4 was injected via the tail vein into mice bearing 4T1 tumors after anesthesia and disinfection. 48 hours later, imaging was performed using an 808 nm laser and filters at 1000 nm, 1100 nm, 1200 nm, and 1300 nm to observe probe targeting of the tumor.

[0144] like Figure 12 As shown in Figure A, after excitation by 808 nm laser, IRDye800CW-8-Arm-PEG-FA and 820NHS-8-Arm-PEG-FA were imaged under different long-pass filters (1000 nm, 1100 nm, 1200 nm and 1300 nm), and both probes had obvious fluorescence signals. Figure 12 As shown in Figure 2, 48 hours after tail vein injection, under the same conditions of 808 nm laser excitation, IRDye800CW-8-Arm-PEG-FA and 820NHS-8-Arm-PEG-FA were well targeted to the tumor when matched with different long-pass filters (1000 nm, 1100 nm, 1200 nm, and 1300 nm). As the filter wavelength was extended, the resolution of the tumor boundary increased, the imaging became clearer, and the tissue autofluorescence signal decreased.

[0145] Application Example 4

[0146] Application of the near-infrared fluorescent probe prepared in Example 5 in targeted imaging of breast cancer.

[0147] 100 μL of the IR-12-NHS-8-Arm-PEG-FA fluorescent probe prepared in Example 5 was injected into mice bearing 4T1 tumors via the tail vein after anesthesia and disinfection. Twenty-four hours later, imaging was performed using an 808 nm laser and filters at wavelengths of 850-1000 nm, 1000 nm, 1100 nm, 1200 nm, and 1300 nm to observe the probe targeting the tumor.

[0148] like Figure 13As shown in A, 24 hours after tail vein injection, under the same conditions of 808nm laser excitation, IR-12-NHS-8-Arm-PEG-FA was well targeted to the tumor with different long-pass filters (850nm-1000nm, 1000nm, 1100nm, 1200nm and 1300nm). As the filter wavelength was extended, the resolution of the tumor boundary increased, the imaging became clearer, and the tissue autofluorescence signal decreased. After removing the skin, as shown in Figure 13 As shown in Figure B, under the condition of 808nm laser excitation, by matching different long-pass filters (850nm-1000nm, 1000nm, 1100nm, 1200nm and 1300nm), it can be observed that the resolution of the tumor boundary is further increased and the tissue autofluorescence signal is further reduced.

[0149] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A near-infrared fluorescent probe, which is a conjugate of a NIR fluorescent molecule and a multi-arm polymer, wherein the multi-arm polymer is multi-arm polyethylene glycol, dextran or a dendrimer.

2. The near-infrared fluorescent probe according to claim 1, characterized in that The NIR fluorescent molecules are selected from one or more of cyanine dyes, electron donor-electron acceptor-electron donor dyes, fluoroboron dipyrrole dyes, rhodamine dyes, metal macrocyclic complex dyes and gold cluster molecules.

3. The near-infrared fluorescent probe according to claim 1, characterized in that The multi-arm polyethylene glycol is selected from one or more of two-arm polyethylene glycol, four-arm polyethylene glycol, six-arm polyethylene glycol, eight-arm polyethylene glycol, and ten-arm polyethylene glycol; the dextran is selected from one or more of dextran methoxypolyethylene glycol, dextran ester, diethylaminoethyl dextran, dextran polyethylene glycol, Dextran-NH2, Dextran-NHS, and DEAE-Dextran; the dendrimer is one or more of Dendrimer-NH2, polypropyleneimine dendrimer, polyamidoamine dendrimer, and polylysine dendrimer; and the molecular weight of the multi-arm polymer is 3kDa to 200kDa.

4. The near-infrared fluorescent probe according to claim 3, characterized in that The multi-arm polyethylene glycol is 8-arm-PEG-NH2, 8-arm-PEG-SH, 8-arm-PEG-alkyne, 8-arm-PEG-N3 or 8-arm-PEG-COOH; the dextran polyethylene glycol is Dextran-PEG-NH2, Dextran-PEG-COOH, Dextran-PEG-Alkyne, Dextran-PEG-N3 or Dextran-PEG-SH; the molecular weight of the multi-arm polymer is 3kDa to 200kDa.

5. The method for preparing the near-infrared fluorescent probe according to any one of claims 1 to 4, comprising the following steps: (1) dissolving the NIR fluorescent molecule in a solvent to obtain a NIR fluorescent molecule solution; (2) adding the NIR fluorescent molecule solution and the multi-arm polymer to a buffer solution, adding or not adding a catalyst or coupling agent, adjusting the pH value, and reacting for a certain time; (3) The reactant is centrifuged and washed to obtain the near-infrared fluorescent probe.

6. The preparation method according to claim 5, characterized in that The solvent in step (1) is selected from DMSO, pure water or buffer solution, the pH value in step (2) is 5-9, the reaction time in step (2) is 1-3 hours, and the catalyst or coupling agent is EDC or copper ion.

7. Use of the near-infrared fluorescent probe according to any one of claims 1 to 4 in the preparation of a fluorescent imaging agent.

8. The use according to claim 7, wherein the fluorescent imaging agent is used for imaging ureters, lymph nodes, lymphatic vessels, gastrointestinal tract, blood vessels, and nerves in animals and humans.

9. A kit for fluorescence imaging, comprising the near-infrared fluorescent probe according to any one of claims 1 to 4.

10. A method for fluorescent imaging of animal or human blood vessels using the near-infrared fluorescent probe according to any one of claims 1 to 4, wherein the method is not intended for the diagnosis or treatment of a disease, the molecular weight of the near-infrared fluorescent probe being greater than 30 kDa, and the method comprising: The probe is injected intravenously into the blood vessels, and the blood vessels in the body are imaged using a NIR-I or NIR-II fluorescence endoscope, or the blood vessels on the surface of the body are imaged using an in vitro imaging device in the NIR-I or NIR-II area.

11. The fluorescence imaging method according to claim 10, wherein the multi-arm polyethylene glycol in the near-infrared fluorescent probe is 8-arm-PEG-NH2, and the molecular weight of the 8-arm-PEG-NH2 is about 40KD; and the NIR fluorescent molecule is one or more of IRDye 800-NHS, ICG-NHS, IR-12-NHS, IR783-NHS, IR820-NHS or IR840-NHS.

12. A method for fluorescent imaging of the ureter of an animal or human body using the near-infrared fluorescent probe according to any one of claims 1 to 4, wherein the method is not intended for the diagnosis or treatment of a disease, the molecular weight of the near-infrared fluorescent probe being less than 30 kDa, and the method comprising: The probe is injected intravenously or into the tissue and excreted through the kidneys into the ureter. The ureter is then imaged within 2 minutes to 5 hours using a fluorescence endoscope or surgical robot fluorescence imaging system in the NIR-I or NIR-II range, or using an in vitro imaging device in the NIR-I or NIR-II range.

13. The fluorescence imaging method according to claim 12, wherein the multi-arm polyethylene glycol in the near-infrared fluorescent probe is 8-arm-PEG-NH2, and the molecular weight of the 8-arm-PEG-NH2 is about 10KD; and the NIR fluorescent molecule is one or more of IRDye 800-NHS, ICG-NHS, IR-12-NHS, IR783-NHS, IR820-NHS or IR840-NHS.

14. A method for fluorescence imaging of lymphatic vessels and / or lymph nodes in animals or humans using the near-infrared fluorescent probe according to any one of claims 1 to 4, wherein the method is not intended for the diagnosis or treatment of a disease, and comprises: The probe is injected into the tissue or tumor edge and enters the lymph nodes through the lymphatic vessels. Within 2 minutes to 5 hours after injection, the lymphatic vessels and / or lymph nodes near the tissue or tumor are imaged using a fluorescence endoscope or surgical robot fluorescence imaging system in the NIR-I or NIR-II region, or the lymphatic vessels and / or lymph nodes near the tissue or tumor are imaged using an in vitro imaging device in the NIR-I or NIR-II region.

15. A near-infrared fluorescent probe for tumor-specific targeted imaging, wherein the fluorescent probe is a conjugate of a multi-arm polymer, a NIR fluorescent molecule and a specific targeting molecule, and the multi-arm polymer is multi-arm polyethylene glycol, dextran or a dendrimer.

16. The near-infrared fluorescent probe for tumor-specific targeted imaging according to claim 15, characterized in that: The specific targeting molecule is selected from any one of peptides, nucleic acid aptamers, affibodies, and antibodies.

17. The near-infrared fluorescent probe for tumor-specific targeted imaging according to claim 16, characterized in that: The specific targeting molecules are directed against PSMA, αvβ3integrin, α4β1integrin, α6β1integrin, HER1-4, SSTR1-5, GnRH-R, VIP, NTSR1, CCK2R, EphA2, CD133, TD05, TE02, AS1411, TLS11a, Sgc8, 41t, TE17, KDED2a-3, KCHA10, Sgd5, TTA1, MUC-1, A32, S11e, S6, J3, F3B, A10, VEGF165, VEGFR1, VEGFR2, VEGFR3, TROP-2, Nectin-4, folate receptor FOLR1, folate receptor FOLR2, folate receptor FOLR3, folate receptor FOLR4, EGFR, HER2, HER3, PD-L1, PDGFRβ, VEG FR, CAIX, LMP, LMP1, PD-L1, PD-1, PSMA, TNFα, IL-1, IL-6, IL-12, IL-13, IL-17A, IL-23, MCP-1 , IGF-1, IFN-γ, CSF1R, DLL4, TGF-β, TUFM, VEGF, EGFR, FGFR2, ICAM-1, ARTC2, CAIX, CapG, CD11b , CD20, CD33, CD38, CD4, CD44, CD45, CD47, CD52, CD7, CD8, CD105, CEA, c-Met, CTLA-4, CXCL11, CXCL12, CXCR2, CXCR4, CXCR7, Fibronectin, BCMA, HER2, HGF, Ly-6C / Ly-6G, MHC-II, MMR, Viral Any of peptides, aptamers, affibodies, and antibodies targeting GPCR, US28, MMP2, MMP3, MMP7, MMP9, and MMP12.

18. The near-infrared fluorescent probe for tumor-specific targeted imaging according to claim 17, characterized in that: The specific targeting molecule is any one of peptides, nucleic acid aptamers, affinity bodies, and antibodies targeting PSMA, αvβ3integrin, HER2, EGFR, VEGFR1, VEGFR2, VEGFR3, TROP-2, Nectin-4, CD8, CD4, CD105, CD133, PD-L1, or PD-1 targets.

19. The near-infrared fluorescent probe for tumor-specific targeted imaging according to claim 16, characterized in that: The specific targeting molecule is any one of vitamin B12, vitamin H, thiamine, riboflavin, adenosine, N-acetylglucosamine, folic acid, HS-PEG-Folic acid, methotrexate, mannose, carbohydrates, hyaluronic acid, fructose, cRGD, Cetuximab, Atezolizumab, CD105 monoclonal antibody, Andecaliximab monoclonal antibody, TRC105 antibody, and Bevacizumab.

20. The near-infrared fluorescent probe for tumor-specific targeted imaging according to claim 15, characterized in that: The NIR fluorescent molecules are selected from one or more of cyanine dyes, electron donor-electron acceptor-electron donor dyes, fluoroboron dipyrrole dyes, rhodamine dyes, metal macrocyclic complex dyes and gold cluster molecules.

21. The near-infrared fluorescent probe for tumor-specific targeted imaging according to claim 15, characterized in that: The multi-arm polyethylene glycol is selected from one or more of two-arm polyethylene glycol, four-arm polyethylene glycol, six-arm polyethylene glycol, eight-arm polyethylene glycol, and ten-arm polyethylene glycol; the dextran is selected from one or more of dextran methoxypolyethylene glycol, dextran ester, diethylaminoethyl dextran, dextran polyethylene glycol, Dextran-NH2, Dextran-NHS, and DEAE-Dextran; the dendrimer is one or more of Dendrimer-NH2, polypropyleneimine dendrimer, polyamidoamine dendrimer, and polylysine dendrimer; and the molecular weight of the multi-arm polymer is 3kDa to 200kDa.

22. The near-infrared fluorescent probe for tumor-specific targeted imaging according to claim 21, characterized in that: The multi-arm polyethylene glycol is 8-arm-PEG-NH2, 8-arm-PEG-SH, 8-arm-PEG-alkyne, 8-arm-PEG-N3 or 8-arm-PEG-COOH; the dextran is Dextran-PEG-NH2, Dextran-PEG-COOH, Dextran-PEG-Alkyne, Dextran-PEG-N3 or Dextran-PEG-SH; the molecular weight of the multi-arm polymer is 3kDa to 200kDa.

23. The near-infrared fluorescent probe for tumor-specific targeted imaging according to claim 22, characterized in that: The multi-arm polyethylene glycol is 8-arm-PEG-NH2, and the molecular weight of the 8-arm-PEG-NH2 is about 10KD or about 40KD; the NIR fluorescent molecule is one or more of IRDye 800-NHS, ICG-NHS, IR-12-NHS, IR783-NHS, IR820-NHS or IR840-NHS; the specific targeting molecule is a folic acid molecule, RGD polypeptide or PSMA polypeptide.

24. A method for preparing the near-infrared fluorescent probe for tumor-specific targeted imaging according to any one of claims 15 to 23, comprising the following steps: (1) dissolving the NIR fluorescent molecule in a solvent to obtain a NIR fluorescent molecule solution; (2) adding the NIR fluorescent molecule solution and the multi-arm polymer to a buffer solution, adding or not adding an appropriate amount of catalyst or coupling agent, adjusting the pH value, and reacting for a certain time; (3) centrifuging and washing the reactants; (4) Resuspending the washed reactants in a buffer solution, adding or not adding a linker molecule, and reacting for a certain period of time; (5) centrifuging and washing the reactants; (6) resuspending the washed reactants in a buffer solution, adjusting the pH value of the buffer, adding a catalyst or coupling agent and a specific targeting molecule, and reacting for a certain time; (7) The reactants are centrifuged and washed to obtain the tumor-specific targeted imaging near-infrared fluorescent probe.

25. The preparation method according to claim 24, characterized in that The solvent in step (1) is selected from DMSO, pure water or buffer solution; In step (2), the pH value is 5-9, the reaction time is 1-3 hours, and the catalyst or coupling agent is EDC or copper ion; The reaction time in step (4) is 1-3 hours, and the linker molecule is SMCC or SPDP or SPDB; The pH value in step (6) is 5-9, the reaction time is 1-3 hours, and the catalyst or coupling agent is EDC or copper ions.

26. Use of the near-infrared fluorescent probe for tumor-specific targeted imaging according to any one of claims 15 to 23 in the preparation of a tumor tracer, a tumor boundary determination imaging agent, a tumor resection surgery navigation imaging agent, or a tumor metastasis lymph node imaging agent.

27. The use according to claim 26, wherein the tumor comprises a urogenital system tumor, a gastrointestinal system tumor, skin cancer, head and neck cancer, cervical cancer, ovarian cancer or breast cancer.

28. A method for targeted near-infrared fluorescence imaging of tumors in animals or humans using the tumor-specific near-infrared fluorescent probe for targeted imaging according to any one of claims 15 to 23, wherein the method is not intended for diagnosis or treatment of a disease, and the molecular weight of the tumor-specific near-infrared fluorescent probe for targeted imaging is 40 kDa to 200 kDa, comprising: A near-infrared fluorescent probe for tumor-specific targeted imaging is injected into the bloodstream intravenously. The fluorescent probe enters the tumor in a targeted manner and binds to cancer cells. Within 24-72 hours, the tumor is imaged using an imaging system of a fluorescence endoscope or surgical robot in the NIR-I or NIR-II region, or using an in vitro fluorescence imaging device in the NIR-I or NIR-II region, to locate the tumor and determine its boundaries.

29. A method for near-infrared fluorescence imaging of metastatic cancer cells in animals or humans using the tumor-specific targeted imaging near-infrared fluorescent probe according to any one of claims 15 to 23, wherein the method is not intended for diagnosis or treatment of a disease, and comprises: The near-infrared fluorescent probe for tumor-specific targeted imaging is injected intravenously or at the edge of the tumor. The fluorescent probe enters the sentinel lymph node of the tumor through the lymphatic vessels, and the lymph node is imaged using fluorescence in the NIR-I or NIR-II region within 1-48 hours.

30. A method for near-infrared fluorescence imaging of targeted nerves in animals or humans using the near-infrared fluorescent probe for tumor-specific targeted imaging according to any one of claims 15 to 23, wherein the method is not intended for the diagnosis or treatment of a disease, and comprises: A near-infrared fluorescent probe for tumor-specific targeted imaging is injected intravenously. The fluorescent probe enters the blood circulation and binds to nerves in the body. The nerves are imaged using fluorescence in the NIR-I or NIR-II region within 1-48 hours.

Images