Fluorescent probe

Abstract

The present invention belongs to the technical field of biomedical functional dyes and probes, and relates to a fluorescent probe, specifically a cyclodextrin-based near-infrared fluorescent probe. The near-infrared fluorescent probe is a conjugate in which a fluorescent molecule is encapsulated by cyclodextrin. In the near-infrared fluorescent probe of the present invention, the cyclodextrin is embedded onto a carbon chain of the fluorescent molecule, which improves the water solubility, histocompatibility and safety, enhances the fluorescence lifetime and photostability, and achieves rapid delivery and response in ureters, lymphatic vessels and lymph nodes in vivo. The near-infrared fluorescent probe can be used for NIR-I and NIR-II imaging. In addition, the near-infrared fluorescent probe can be labeled with various tumor-targeting molecules to form targeted near-infrared fluorescent probes, thereby achieving precise targeted fluorescence imaging in both NIR-I and NIR-II windows for different types of tumors such as genitourinary tumors, head and neck cancer, breast cancer and cervical cancer, and metastatic lymph nodes. The near-infrared fluorescent probe can be used for the navigation of fluorescence imaging during clinical surgeries.

Description

A fluorescent probe Technical Field

[0001] This invention belongs to the field of biomedical functional dyes and probes, and particularly relates to a fluorescent probe. Background Technology

[0002] Near-infrared (NIR) fluorescent dyes / probes have made significant progress in fluorescence-guided surgery (FGS), particularly in improving visualization of tumor tissue during surgery, which helps achieve precise resection. For example, NIR fluorescent dyes such as Indocyanine Green (ICG) can be used to more accurately identify tumor boundaries during surgery, thereby reducing the risk of residual tumor tissue. However, currently FDA-approved NIR fluorescent dyes / probes for clinical use, such as ICG and Cytalux, still have shortcomings in tumor targeting and spatial resolution, which limits their widespread clinical application.

[0003] Although ICG has been widely used in recent years in various fields such as blood perfusion assessment, anatomical visualization, tumor localization, and lymph node localization / imaging, it still has some significant limitations. These include signal interference caused by leakage from target tissue, inability to link with target molecules for tumor-targeted imaging, and inability to image the ureteral system. Furthermore, because the emission wavelength of ICG is located in the near-infrared region I (~814 nm), its imaging resolution, contrast, and depth are limited by scattering.

[0004] In recent years, although Cytalux fluorescent targeting agents have been introduced, their targeting is poor, with low fluorescence signal between tumors and normal tissues (T / NT ratio of only 3-4 times), resulting in unclear tumor boundaries and hindering tumor resection during surgery. Furthermore, Cytalux is only indicated for ovarian cancer, limiting its widespread clinical application. Existing fluorescent conjugates, such as ICG, fluorescein sodium (FLS), and 5-aminolevulinic acid (5-ALA), lack specific targeting properties, making it impossible to achieve highly personalized treatment plans, especially for precise localization and treatment of specific tumor types. Summary of the Invention

[0005] To address the shortcomings of the existing technology, this invention provides a near-infrared fluorescent probe based on cyclodextrin, its preparation method, and its application. The specific technical solution is as follows:

[0006] The first objective of this invention is to provide a near-infrared fluorescent probe (NIR Dye-CD complex) based on cyclodextrin, wherein the near-infrared fluorescent probe is a conjugate in which fluorescent molecules are coated with cyclodextrin; wherein the fluorescent molecules are cyanine dyes or electron donor-electron acceptor-electron donor dyes.

[0007] Furthermore, the cyanine dyes include, but are not limited to, Cyanine7, Cyanine7.5, Cyanine9, Cyanine9.5, Cyanine11, Cyanine11.5, Cyanine13, Cyanine13.5, Cyanine 15, Cyanine15.5, Flab 7, FD-1080, IR-1048, IR-26, IR-1061, and IR-1051.

[0008] Furthermore, the electron donor-electron acceptor-electron donor dyes include, but are not limited to, CH1055, Fluorene-based DAD dyes, IR-FGP, IR-FEP, IR-FEPC, IR-FTAP, CH4T, IR-pFE, IRT, IR-BGP6, IR-BEMC6P, IR-1048-MZ, IR-FE, IR-FTP, IR-BBE, IR-BBEP, IR-FTX, IR-FTXP, IR-FX, IR-FXP, SXH1100, Q-FITBBTTFI, IR-FT, IR-FP8P, 2TT-oC6B, 2TT-oC26B, TT3-oCB, and HL3.

[0009] Furthermore, the fluorescent molecule comprises the following structure:

[0010] Among them, R1, R2, R3, R4, R5, R6, R7, R8, R 26 R 27 R 28 R 29 Each group is independently selected from hydrogen, amino, hydroxy, dimethylamino, sulfonic acid, methoxy, methylthio, fluorine, chlorine, bromine, iodine, ester or carboxyl, and one or more of substituted or unsubstituted straight-chain or branched alkyl, triphenylamine, straight-chain or branched alkoxy, or substituted or unsubstituted aryl groups.

[0011] R9, R 10 R 11 R 12 Each group is independently selected from one or more of the following groups: substituted or unsubstituted straight-chain or branched alkyl, alkoxy, phenyl, benzyl, sulfonic acid, carboxyl, alkynyl, mercapto, and N-hydroxysuccinimide ester groups.

[0012] R 15 R 16 R 17 R 18 R 19 R 20 R 21 R 22 R23 R 24 R 25 Each of the following is independently selected from one or more of hydrogen, substituted or unsubstituted straight-chain or branched alkyl groups, fluorine, chlorine, bromine, and iodine;

[0013] R 13 R 14 Each group is independently selected from one or more of the following groups: amino, hydroxyl, dimethylamino, sulfonic acid, methoxy modified group, methylthio, fluorine, chlorine, bromine, iodine, ester, carboxyl, alkynyl, mercapto, and N-hydroxysuccinimide ester group.

[0014] L and L', as linking groups, are independently selected from one or more of the following: substituted or unsubstituted straight-chain or branched alkyl groups, straight-chain or branched alkoxy groups, phenyl groups, and benzyl groups.

[0015] X - As anionic groups, they are independently selected from chloride ions, bromide ions, iodide ions, fluoride ions, acetate ions, etc.

[0016] Furthermore, the cyclodextrin-based near-infrared fluorescent probe comprises the following structural formula:

[0017] in, It is a cyclodextrin.

[0018] Furthermore, the molecular weight of the cyclodextrin is between 500 Da and 5000 Da, such as 900 Da, 3000 Da, etc. Changing the molecular weight of the probe can alter its distribution and excretion pathway in vivo. Probes with a molecular weight less than 40 kDa are excreted by the kidneys through urine and can be used for ureteral imaging.

[0019] Furthermore, the cyclodextrin is selected from one or more of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, hydroxypropyl-α-cyclodextrin (HP-α-cyclodextrin), hydroxypropyl-β-cyclodextrin (HP-β-cyclodextrin), hydroxypropyl-γ-cyclodextrin (HP-γ-cyclodextrin), methyl-α-cyclodextrin (Mα-cyclodextrin), methyl-β-cyclodextrin (Mβ-cyclodextrin), and methyl-γ-cyclodextrin (Mγ-cyclodextrin).

[0020] A second objective of this invention is to provide a method for preparing the aforementioned cyclodextrin-based near-infrared fluorescent probe, comprising the following steps:

[0021] (1) Dissolve (1E,3E)-5-oxopram-1,3-diene-1-acetate, pentaenal diphenylamine hexafluorophosphate, heptaenal diphenylamine hexafluorophosphate or nonadienal diphenylamine hexafluorophosphate in solvent one to obtain solution one;

[0022] (2) Dissolve cyclodextrin, indolon halide and potassium acetate in the first solution, remove oxygen and heat and stir at 25℃-110℃ for 40-50 hours to obtain the reaction solution;

[0023] (3) The reaction solution was diluted with water, washed with dichloromethane, and the aqueous solution was purified by dialysis and column chromatography to obtain a near-infrared fluorescent probe based on cyclodextrin.

[0024] The solvent is water, or a mixture of water and a water-miscible organic solvent, wherein the organic solvent includes, but is not limited to, acetonitrile, N,N-dimethylformamide, N,N-dimethylacetamide, acetone, methanol, ethanol, isopropanol, DMSO, etc.

[0025] Furthermore, in step (3), the column chromatography includes, but is not limited to, normal phase column chromatography, reversed phase column chromatography, ion exchange chromatography, size exclusion chromatography, hydrophilic interaction chromatography, and preparative high performance liquid chromatography.

[0026] Further, the indolon halides include indolon iodides, indolon bromides, and indolon chlorides; the indolon iodides include, but are not limited to, 1,2,3,3-tetramethyl-3H-indolon iodide, 1,1,2,3-tetramethyl-1H-benzo[e]indolon iodide, and 1,1,2,3-tetramethyl-6,8-disulfonic acid-1H-benzo[e]indolon iodide; the indolon bromides include, but are not limited to, 1,2,3,3-tetramethyl-3H-indolon bromide, 1,1,2,3-tetramethyl-1H-benzo[e]indolon bromide, and 1,1,2,3-tetramethyl-6,8-disulfonic acid-1H-benzo[e]indolon bromide.

[0027] The third objective of this invention is to provide the application of the above-mentioned cyclodextrin-based near-infrared fluorescent probe in imaging of the ureter, lymph nodes, lymphatic vessels, gastrointestinal tract, blood vessels, and nerves in animals and humans.

[0028] The method of using the near-infrared fluorescent probe of the present invention is as follows: the near-infrared fluorescent probe is dissolved in a buffer solution to a final concentration of 1 mg / ml, and is used for intravenous (IV), subcutaneous (SC), intratumoral (IT) or peritumoral, intramuscular (IM) and other tissue injections.

[0029] This invention provides a fluorescence imaging method suitable for animal or human blood vessels. The method involves intravenously injecting a near-infrared fluorescent probe with a molecular weight of 500 Da to 5000 Da, as described in this invention, followed by imaging of the blood vessels in vivo using a NIR-I or NIR-II fluorescence endoscope, or imaging of the entire body's surface blood vessels using an in vitro imaging device in the NIR-I or NIR-II region. This method can visualize blood vessels during surgery, thereby reducing the risk of vascular damage.

[0030] This invention provides a fluorescence imaging method for the ureter in animals or humans. The method involves injecting a near-infrared fluorescent probe with a molecular weight of less than 40 kDa into the ureter via intravenous, tissue, or subcutaneous injection, followed by renal excretion into the ureter. Imaging of the ureter is then performed over 2 minutes to 5 hours using a NIR-I or NIR-II fluorescence endoscope or a surgical robot fluorescence imaging system. In open surgery, imaging can be performed using an external imaging device in the NIR-I or NIR-II region after exposing the ureter in the open abdominal cavity. This method facilitates visualization of the ureter during surgery, thereby avoiding damage.

[0031] This invention provides a fluorescence imaging method applicable to lymphatic vessels and / or lymph nodes in animals or humans. The method includes injecting the near-infrared fluorescent probe described herein through the edge of tissue or tumor, followed by the probe entering the lymph node via a lymphatic vessel; imaging the lymphatic vessels and / or lymph nodes near the tissue or tumor using a NIR-I or NIR-II fluorescence endoscope or surgical robot fluorescence imaging system within 2 minutes to 5 hours after injection; in open surgery, in vitro imaging devices in the NIR-I or NIR-II zones can be used to image the lymphatic vessels and / or lymph nodes near the tissue or tumor.

[0032] Furthermore, the cyclodextrin-based near-infrared fluorescent probe includes, but is not limited to, the following structures:

[0033] A fourth objective of this invention is to provide a near-infrared fluorescent probe (Cyclodextrin-NIR Dye / targeting ligand) for tumor-targeting imaging, which is a conjugate of the fluorescent molecule, cyclodextrin, and a specific targeting molecule (targeting ligand).

[0034] Furthermore, the near-infrared fluorescent probe for tumor-targeted imaging is a conjugate of the aforementioned cyclodextrin-based near-infrared fluorescent probe and a specific targeting molecule.

[0035] Groups used for coupling include, but are not limited to, carboxyl groups, alkynyl groups, and N-hydroxysuccinimide groups that are present on the fluorescent probe molecule or obtained by modifying cyclodextrin.

[0036] Furthermore, the cyclodextrin-based near-infrared fluorescent probe for coupling with a specific target molecule includes, but is not limited to, the following structures:

[0037] Furthermore, the specific target molecule is any one of peptide, aptamers, affibodies, or antibody.

[0038] Furthermore, the specific targeting molecules are those targeting 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, and Folate. Receptor (folate receptor), EGFR, HER2, HER3, Trop-2, Nectin-4, PD-L1, PDGFRβ, VEGFR, CAIX, LMP, LMP1, PD-L1, PD-1, PS MA, 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, Any one of the following: 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, HGF, Ly-6C / Ly-6G, MHC-II, MMR, Viral GPCR, peptides, nucleic acid aptamers, affinity molecules, or antibodies targeting US28.

[0039] Furthermore, the specific targeting molecule is any one of the following: peptide, nucleic acid aptamer, affinity, or antibody targeting PSMA, αvβ3integrin, HER2, EGFR, VEGF, Trop-2, Nectin-4, CD8, CD4, CD133, CD105, PD-L1, or PD-1.

[0040] Furthermore, the specific target molecules are vitamin B12, vitamin H, thiamine, riboflavin, adenosine, N-acetylglucosamine, folic acid, methotrexate, mannose, carbohydrates, and hyaluronic acid. Hyaluronic acid, fructose, cRGD (arginine aspartate peptide), cRGD, Bevacizumab, or Avastin, Cetuximab, Erbitux, Herceptin, trastuzumab, Sacituzumab, Ramucirumab, Enfortumab, Datopotamab, Atezolizumab, CD105 monoclonal antibody, Amivantamab-VMJM, or Rybrevant, Ivonescimab, or TRC105 antibody.

[0041] Furthermore, the preparation method of the tumor-targeting imaging near-infrared fluorescent probe includes the following steps:

[0042] (1) Dissolve fluorescent molecules with carboxyl or alkyne groups in a solvent to obtain a fluorescent molecule solution;

[0043] (2) Add the fluorescent molecule solution and cyclodextrin to the buffer solution, adjust the pH value, and react at room temperature in the dark for 2-4 hours;

[0044] (3) Centrifuge and wash the reactants;

[0045] (4) Resuspend the washed reactants in a buffer solution, add EDC or Sulfo-NHS or EDC and Sulfo-NHS together and specific targeting molecules with amino or azide, and react for 1-3 hours in the dark and at room temperature.

[0046] (5) Centrifuge and wash the reactants to obtain the tumor-targeting imaging near-infrared fluorescent probe.

[0047] Furthermore, in step (1), the solvent is selected from DMSO, pure water, or a buffer solution.

[0048] Furthermore, in step (2), the pH value is 5-9.

[0049] Furthermore, in step (3), a catalyst or coupling agent, such as EDC or Sulfo-NHS, or EDC and Sulfo-NHS used together, is added.

[0050] Furthermore, in step (3), centrifugation is performed using an ultrafiltration centrifuge tube with a corresponding molecular weight, based on the molecular weight of the cyclodextrin.

[0051] Furthermore, the near-infrared fluorescent probe for tumor-specific targeted imaging is used in the preparation of tumor tracers, tumor boundary determination imaging agents, tumor resection surgical navigation imaging agents, or tumor metastatic lymph node imaging agents.

[0052] Furthermore, the tumors include tumors of the genitourinary system (kidney cancer, renal pelvis cancer, ureter cancer, adrenal cancer, bladder cancer, prostate cancer, penile cancer, testicular cancer), tumors of the gastrointestinal system, skin cancer, head and neck cancer, breast cancer, etc.

[0053] The method of using the near-infrared fluorescent probe for tumor-specific targeted imaging described in this invention is as follows: the near-infrared fluorescent probe for tumor-specific targeted imaging is resuspended in a buffer solution, with a final concentration of 1 mg / ml, for intravenous (IV), subcutaneous (SC), intratumoral (IT) or peritumoral, intramuscular (IM) and other tissue injection.

[0054] Furthermore, this invention provides a near-infrared fluorescence imaging method suitable for targeting metastatic cancer cells in animals or humans. A tumor-specific near-infrared fluorescent probe for imaging is injected intravenously or into the tumor margin, allowing the probe to enter the sentinel lymph node via lymphatic vessels. Within 1-48 hours, the lymph node is imaged using fluorescence in the NIR-I or NIR-II regions. Lymph nodes with prolonged fluorescence signals are those with metastatic cancer cells. The combination of the specifically targeted near-infrared fluorescent probe with the metastatic lymph node results in enhanced and sustained fluorescence signals. This specific targeting imaging technique can provide real-time intraoperative navigation for metastatic lymph nodes.

[0055] The beneficial effects of this invention are as follows:

[0056] This invention modifies near-infrared fluorescent dyes with cyclodextrin and synthesizes them in an aqueous supramolecular system. The hydrophilic macrocyclic cyclodextrin is nested into the carbon chain of the fluorescent molecule in a host-guest molecule manner, and Fischer indole is attached to both ends of the carbon chain. Due to steric hindrance, the cyclodextrin is nested on the carbon chain to form a stable structure and will not leave, forming a single stable water-soluble near-infrared fluorescent probe that can be used for NIR-I (850-1000nm) and NIR-II (1000-1700nm) imaging.

[0057] The near-infrared fluorescent probe of this invention possesses excellent water solubility, high tissue compatibility and safety, as well as longer fluorescence lifetime and photostability. The behavior and photophysical properties of the fluorescent molecules in the near-infrared fluorescent probe are relatively less affected by the in vivo environment, facilitating the prediction of molecule metabolic behavior in vivo and precise quantification. Cyclodextrin is nested on the molecule, and due to the restriction of carbon chain molecular vibration by cyclodextrin, the possibility of excited-state molecules returning to the ground state through vibration is reduced, allowing more excited-state molecules to return to the ground state by emitting fluorescence, thereby improving the quantum yield and brightness of the molecule. Cyclodextrin coating significantly improves the photophysical stability of anthocyanin molecules, enabling use under longer and higher intensity excitation conditions, thus broadening its applicability and exhibiting higher redox stability.

[0058] The near-infrared fluorescent probe of this invention can achieve high-resolution imaging of the ureter, lymph nodes, and lymphatic vessels in vivo, and obtain imaging results rapidly within a short time after injection, thereby reducing operation time and patient risk, and greatly improving the potential and practicality of this technology in clinical applications; this type of near-infrared fluorescent probe can be rapidly excreted through the kidneys after injection, effectively overcoming the limitation of existing fluorescent dyes that cannot be rapidly excreted for imaging, especially reducing the risk of damage to vital organs in abdominal surgery.

[0059] Furthermore, the near-infrared fluorescent probes of this invention can also be labeled with various tumor-targeting molecules to form targeted near-infrared fluorescent probes, thereby achieving precise targeted fluorescence imaging of different types of tumors such as urogenital tumors, head and neck cancer, breast cancer, and cervical cancer, as well as metastatic lymph nodes in the NIR-I and NIR-II windows. These near-infrared fluorescent probes not only enhance the targeting of fluorescent probes, but can also be applied to fluorescence imaging of ureteral tumors, tumor sentinel lymph nodes, and nerve tissues. These lymph nodes will show more persistent fluorescence than lymph nodes without metastatic cancer cells. This achieves highly targeted and high-resolution imaging of tumor boundaries, which will overcome the shortcomings of existing NIR fluorescent dyes / probes in terms of specificity and provide an effective surgical navigation tool for precision medicine. Attached Figure Description

[0060] Figure 1 is a mass spectrum of the near-infrared fluorescent probe prepared in Example 1 of the present invention;

[0061] Figure 2 shows the absorption spectrum of the near-infrared fluorescent probe prepared in Example 1 of the present invention;

[0062] Figure 3 shows the emission spectrum of the near-infrared fluorescent probe prepared in Example 1 of the present invention;

[0063] Figure 4 shows the fluorescence images of the near-infrared fluorescent probe prepared in Example 1 of the present invention under different wavelength filters;

[0064] Figure 5 shows the absorption spectrum of the near-infrared fluorescent probe prepared in Example 2 of the present invention;

[0065] Figure 6 shows the emission spectrum of the near-infrared fluorescent probe prepared in Example 2 of the present invention;

[0066] Figure 7 shows the fluorescence images of the near-infrared fluorescent probe prepared in Example 2 of the present invention under different wavelength filters;

[0067] Figure 8 shows the fluorescence imaging of the near-infrared fluorescent probe prepared in Example 1 of the present invention under different wavelength filters in the ureter of a mouse.

[0068] Figure 9 shows the signal homogenization diagram of the near-infrared fluorescent probe prepared in Example 1 of the present invention at various wavelengths in the ureter of a mouse.

[0069] Figure 10 shows the fluorescence imaging of the near-infrared fluorescent probe prepared in Example 1 of the present invention at different time points in mice during ureteral excretion.

[0070] Figure 11 shows the photostability test results of the near-infrared fluorescent probe prepared in Example 1 of the present invention;

[0071] Figure 12 shows fluorescence imaging of the near-infrared fluorescent probe prepared in Example 1 of the present invention at different time points after injection in the lymph nodes, lymphatic vessels, kidneys, and bladder of mice.

[0072] Figure 13 shows the fluorescence imaging of the near-infrared fluorescent probe prepared in Example 2 of the present invention under different wavelength filters in mouse lymph nodes, lymphatic vessels, and kidneys.

[0073] Figure 14 shows fluorescence imaging of the near-infrared fluorescent probe prepared in Example 2 of the present invention at different time points after injection in mouse lymph nodes, lymphatic vessels and kidneys.

[0074] Figure 15 is a fluorescence imaging image of the near-infrared fluorescent probe prepared in Example 2 of the present invention excreting through the ureter in mice.

[0075] Figure 16 shows the signal homogenization of the near-infrared fluorescent probe prepared in Example 2 of the present invention at various wavelengths in the ureter of a mouse. Detailed Implementation

[0076] The principles and features of the present invention are described below with reference to embodiments. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Unless otherwise specified, all raw materials involved in the invention are commercially available products.

[0077] In this invention, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0078] In this invention, "near-infrared" refers to near-infrared light (NIR), which is an electromagnetic wave between visible light (VIS) and mid-infrared light (MIR). Conventionally, the near-infrared region is further divided into two areas: near-infrared region I (750-900nm) and near-infrared region II (1000-1700nm).

[0079] In this invention, "coupling" refers to a new compound formed by the covalent connection (coupling) of two or more compound molecules through bivalent or multivalent compound molecules with connecting functions. Couplings can also be formed directly from two molecules through coupling or condensation; "fluorescent probe" refers to a module that can be detected by colorimetry or fluorescence assay.

[0080] Example 1

[0081] A method for preparing a near-infrared fluorescent probe includes the following steps:

[0082] (1) Dissolve 1.22g heptadenal diphenylamine hexafluorophosphate, 14.0g α-cyclodextrin, 1.50g 1,2,3,3-tetramethyl-3H-indolium iodide and 4.10g sodium acetate in 50ml of water. After deoxygenation, heat the solution to 50℃ and stir for two days to obtain the reaction solution.

[0083] (2) After the reaction solution cools to room temperature, dilute it with 100 ml of water and wash the aqueous solution thoroughly with dichloromethane.

[0084] (3) After purification by column chromatography, the near-infrared fluorescent probe Cy9-αCD was obtained. The structural formula is shown below and the mass spectrum is shown in Figure 1.

[0085] As shown in Figure 2, the absorption peak of Cy9-αCD is located at 860 nm; as shown in Figure 3, its emission spectrum peak under 860 nm laser excitation is 890 nm; as shown in Figure 4, after being excited by 860 nm laser, Cy9-αCD is imaged under different long-pass filters (900 nm, 1000 nm, 1200 nm, 1300 nm and 1500 nm), and the probe has obvious fluorescence signals in all of them.

[0086] Example 2

[0087] A method for preparing a near-infrared fluorescent probe includes the following steps:

[0088] (1) Dissolve 1.22 g of pentadienal diphenylamine hydrochloride hexafluorophosphate, 14.0 g of α-cyclodextrin, 1.50 g of 1,1,2,3-tetramethyl-1H-benzo[e]indole iodide and 4.10 g of sodium acetate in 50 mL of water. After deoxygenation, heat the solution to 50 °C and stir for two days to obtain the reaction solution.

[0089] (2) After the reaction solution cools to room temperature, dilute it with 100 ml of water and wash the aqueous solution thoroughly with dichloromethane.

[0090] (3) After purification by column chromatography, the near-infrared fluorescent probe Cy7.5-αCD was obtained from the aqueous solution, and its structural formula is shown below.

[0091] As shown in Figure 5, the absorption peak of Cy7.5-αCD is located at 808 nm; as shown in Figure 6, its emission peak under 808 nm laser excitation is 930 nm; as shown in Figure 7, after being excited by an 808 nm laser, Cy7.5-αCD is imaged under different long-pass filters (900 nm, 1000 nm, 1200 nm, 1300 nm and 1500 nm), and the probe has obvious fluorescence signals in all of them.

[0092] Example 3

[0093] The preparation of a near-infrared fluorescent probe includes the following steps:

[0094] (1) Dissolve 0.42 g of heptadenal diphenylamine hexafluorophosphate, 20.0 g of α-cyclodextrin, 0.48 g of 1,2,3,3-tetramethyl-3H-indolium iodide, 1.11 g of 1-(5-carboxypentyl)-2,3,3-trimethyl-3H-indolium-1-bromide and 4.90 g of potassium acetate in 50 mL of water. After deoxygenation, heat the solution to 50 °C and stir for two days to obtain the reaction solution.

[0095] (2) After the solution cools to room temperature, dilute it with 100 ml of water and wash the aqueous solution thoroughly with dichloromethane.

[0096] (3) The aqueous solution was purified by preparative high performance liquid chromatography to obtain near-infrared fluorescent probes Cy9-αCD-COOH-1, Cy9-αCD-COOH-2 and Cy9-αCD-2COOH.

[0097] Example 4

[0098] The preparation method of the near-infrared fluorescent probe Cy7-αCD-OMe is as follows:

[0099] (1) Mix 0.30 g of (1E,3E)-5-oxopentane-1,3-diene-1-acetate, 1.56 g of 5-methoxy-1,2,3,3-tetramethyl-3H-indolon iodide, 20.4 g of α-cyclodextrin, 4.91 g of anhydrous potassium acetate, 30 mL of water, and 3 mL of DMSO at room temperature. After deoxygenation, heat the solution to 80 °C and stir for 2 days.

[0100] (2) After the solution cools to room temperature, dilute it with water and wash the aqueous phase with dichloromethane.

[0101] (3) The aqueous solution was evaporated to dryness using a rotary evaporator, the crude product was dissolved in an appropriate amount of ethanol, and some inorganic salts were removed by filtration. The near-infrared fluorescent probe Cy7-αCD-OMe was obtained by column chromatography.

[0102] Example 5

[0103] The preparation method of the near-infrared fluorescent probe Cy7-αCD-NMe2 is as follows:

[0104] (1) Mix 0.30 g of (1E,3E)-5-oxopentane-1,3-diene-1-acetate, 1.62 g of 5-dimethylamino-1,2,3,3-tetramethyl-3H-indolium iodide, 20.4 g of α-cyclodextrin, 4.91 g of anhydrous potassium acetate, 30 mL of water, and 3 mL of DMSO at room temperature. After deoxygenation, heat the solution to 80 °C and stir for 2 days.

[0105] (2) After the solution cools to room temperature, dilute it with water and wash the aqueous phase with dichloromethane.

[0106] (3) The aqueous solution was evaporated to dryness using a rotary evaporator, the crude product was dissolved in an appropriate amount of ethanol, and some inorganic salts were removed by filtration. The near-infrared fluorescent probe Cy7-αCD-NMe2 was obtained by column chromatography.

[0107] Example 6

[0108] The preparation method of the near-infrared fluorescent probe Cy7.5-αCD is as follows:

[0109] (1) Mix 0.30 g of (1E,3E)-5-oxopentane-1,3-diene-1-acetate, 1.66 g of 1,1,2,3-tetramethyl-1H-benzo[e]indole-3-onium iodide, 20.4 g of α-cyclodextrin, 4.91 g of anhydrous potassium acetate, 30 mL of water, and 3 mL of DMSO at room temperature. After deoxygenation, heat the solution to 80 °C and stir for 2 days.

[0110] (2) After the solution cools to room temperature, dilute it with water and wash the aqueous phase with dichloromethane.

[0111] (3) The aqueous solution was evaporated to dryness using a rotary evaporator, the crude product was dissolved in an appropriate amount of ethanol, and some inorganic salts were removed by filtration. The near-infrared fluorescent probe Cy7.5-αCD was obtained by column chromatography.

[0112] Example 7

[0113] The preparation method of the near-infrared fluorescent probe Cy9-αCD is as follows:

[0114] (1) Mix 2.40 g of heptadenal diphenylamine hexafluorophosphate, 47.8 g of α-cyclodextrin, 70 mL of water, and 7 mL of DMSO at room temperature and stir for 30 minutes. Add 3.42 g of 1,2,3,3-tetramethyl-3H-indolium iodide and 6.71 g of anhydrous potassium acetate. After deoxygenation, heat the solution to 55 °C and stir for 2 days.

[0115] (2) After the solution cools to room temperature, dilute it with water and wash the aqueous phase with dichloromethane.

[0116] (3) The aqueous solution was evaporated to dryness using a rotary evaporator, the crude product was dissolved in an appropriate amount of ethanol, and some inorganic salts were removed by filtration. The near-infrared fluorescent probe Cy9-αCD was obtained by column chromatography.

[0117] Example 8

[0118] The preparation method of the near-infrared fluorescent probe Cy9-αCD-OMe is as follows:

[0119] (1) Mix 2.40 g of heptaenal diphenylamine hexafluorophosphate, 47.8 g of α-cyclodextrin, 70 mL of water, and 7 mL of DMSO at room temperature and stir for 30 minutes. Add 3.76 g of 5-methoxy-1,2,3,3-tetramethyl-3H-indolium iodide and 6.71 g of anhydrous potassium acetate. After deoxygenation, heat the solution to 55 °C and stir for 2 days.

[0120] (2) After the solution cools to room temperature, dilute it with water and wash the aqueous phase with dichloromethane.

[0121] (3) The aqueous solution was evaporated to dryness using a rotary evaporator, the crude product was dissolved in an appropriate amount of ethanol, and some inorganic salts were removed by filtration. The near-infrared fluorescent probe Cy9-αCD-OMe was obtained by column chromatography.

[0122] Example 9

[0123] The preparation method of the near-infrared fluorescent probe Cy9-αCD-NMe2 is as follows:

[0124] (1) Mix 2.40 g of heptadenal diphenylamine hexafluorophosphate, 47.8 g of α-cyclodextrin, 70 mL of water, and 7 mL of DMSO at room temperature and stir for 30 minutes. Add 3.91 g of 5-dimethylamino-1,2,3,3-tetramethyl-3H-indolium iodide and 6.71 g of anhydrous potassium acetate. After deoxygenation, heat the solution to 55 °C and stir for 2 days.

[0125] (2) After the solution cools to room temperature, dilute it with water and wash the aqueous phase with dichloromethane.

[0126] (3) The aqueous solution was evaporated to dryness using a rotary evaporator, the crude product was dissolved in an appropriate amount of ethanol, and some inorganic salts were removed by filtration. The near-infrared fluorescent probe Cy9-αCD-NMe2 was obtained by column chromatography.

[0127] Example 10

[0128] The preparation method of the near-infrared fluorescent probe Cy9.5-αCD includes the following steps:

[0129] (1) Mix 2.40 g of heptadenal diphenylamine hexafluorophosphate, 47.8 g of α-cyclodextrin, 70 mL of water, and 7 mL of DMSO at room temperature and stir for 30 minutes. Add 3.99 g of 1,1,2,3-tetramethyl-1H-benzo[e]indolium iodide and 6.71 g of anhydrous potassium acetate. After deoxygenation, heat the solution to 55 °C and stir for 2 days.

[0130] (2) After the solution cools to room temperature, dilute it with water and wash the aqueous phase with dichloromethane.

[0131] (3) The aqueous solution was evaporated to dryness using a rotary evaporator, the crude product was dissolved in an appropriate amount of ethanol, and some inorganic salts were removed by filtration. The near-infrared fluorescent probe Cy9.5-αCD was obtained by column chromatography.

[0132] Example 11

[0133] The preparation method of the near-infrared fluorescent probe Cy7-αCD-2COOH is as follows:

[0134] (1) Mix 0.30 g of (1E,3E)-5-oxopentan-1,3-diene-1-acetate, 1.51 g of 1-(5-carboxypentyl)-2,3,3-trimethyl-3H-indole-1-onium bromide, 20.4 g of α-cyclodextrin, 4.91 g of anhydrous potassium acetate, 30 mL of water, and 3 mL of DMSO at room temperature. After deoxygenation, heat the solution to 80 °C and stir for 2 days.

[0135] (2) After the solution cools to room temperature, dilute it with water and wash the aqueous phase with dichloromethane.

[0136] (3) The aqueous solution was evaporated to dryness using a rotary evaporator, the crude product was dissolved in an appropriate amount of ethanol, and some inorganic salts were removed by filtration. The near-infrared fluorescent probe Cy7-αCD-2COOH was obtained by column chromatography.

[0137] Example 12

[0138] The preparation method of the near-infrared fluorescent probe Cy7-αCD-2NHS ester is as follows:

[0139] (1) Mix 0.15 g Cy7-αCD-2COOH, 0.15 g 1-ethyl-(3-dimethylaminopropyl)carbodiimide, 0.12 g N-hydroxysuccinimide and 10 mL DMF at room temperature and stir for 5 hours.

[0140] (2) Add acetonitrile to precipitate the product, and filter to obtain the near-infrared fluorescent probe Cy7-αCD-2NHS ester.

[0141] Example 13

[0142] The preparation method of the near-infrared fluorescent probe Cy7-αCD-OMe-2COOH is as follows:

[0143] (1) Mix 0.30 g of (1E,3E)-5-oxopentan-1,3-diene-1-acetate, 1.77 g of 1-(5-carboxypentyl)-5-methoxy-2,3,3-trimethyl-3H-indole-1-onium bromide, 20.4 g of α-cyclodextrin, 4.91 g of anhydrous potassium acetate, 30 mL of water, and 3 mL of DMSO at room temperature. After deoxygenation, heat the solution to 70 °C and stir for 2 days.

[0144] (2) After the solution cools to room temperature, dilute it with water and wash the aqueous phase with dichloromethane.

[0145] (3) The aqueous solution was evaporated to dryness using a rotary evaporator, the crude product was dissolved in an appropriate amount of ethanol, and some inorganic salts were removed by filtration. The near-infrared fluorescent probe Cy7-αCD-OMe-2COOH was obtained by column chromatography.

[0146] Example 14

[0147] The preparation method of the near-infrared fluorescent probe Cy7-αCD-OMe-2NHS ester is as follows:

[0148] (1) Mix 0.15 g Cy7-αCD-2COOH, 0.15 g 1-ethyl-(3-dimethylaminopropyl)carbodiimide, 0.12 g N-hydroxysuccinimide and 10 mL DMF at room temperature and stir for 5 hours.

[0149] (2) Add acetonitrile to precipitate the product, and filter to obtain the near-infrared fluorescent probe Cy7-αCD-OMe-2NHS ester.

[0150] Example 15

[0151] The preparation methods of near-infrared fluorescent probes Cy7-αCD-OMe-COOH-1 and Cy7-αCD-OMe-COOH-2 are as follows:

[0152] (1) Mix 0.30 g of (1E,3E)-5-oxopentan-1,3-diene-1-acetate, 0.89 g of 1-(5-carboxypentyl)-2,3,3-trimethyl-5-methoxy-3H-indole-1-onium bromide, 0.78 g of 5-methoxy-1,2,3,3-tetramethyl-3H-indole-onium iodide, 20.4 g of α-cyclodextrin, 4.91 g of anhydrous potassium acetate, 30 mL of water, and 3 mL of DMSO at room temperature. After deoxygenation, heat the solution to 70 °C and stir for 2 days.

[0153] (2) After the solution cools to room temperature, dilute it with water and wash the aqueous phase with dichloromethane.

[0154] (3) The aqueous solution was evaporated to dryness using a rotary evaporator, the crude product was dissolved in an appropriate amount of ethanol, and some inorganic salts were removed by filtration. The near-infrared fluorescent probes Cy7-αCD-OMe-COOH-1 and Cy7-αCD-OMe-COOH-2 were obtained by preparative high performance liquid chromatography.

[0155] Example 16

[0156] The preparation method of the near-infrared fluorescent probe Cy7-αCD-NMe2-2COOH is as follows:

[0157] (1) Mix 0.30 g of (1E,3E)-5-oxopentan-1,3-diene-1-acetate, 1.69 g of 1-(5-carboxypentyl)-2,3,3-trimethyl5-dimethylamino-3H-indole-1-onium bromide, 20.4 g of α-cyclodextrin, 4.91 g of anhydrous potassium acetate, 30 mL of water, and 3 mL of DMSO at room temperature. After deoxygenation, heat the solution to 70 °C and stir for 2 days.

[0158] (2) After the solution cools to room temperature, dilute it with water and wash the aqueous phase with dichloromethane.

[0159] (3) The aqueous solution was evaporated to dryness using a rotary evaporator, the crude product was dissolved in an appropriate amount of ethanol, and some inorganic salts were removed by filtration. The near-infrared fluorescent probe Cy7-αCD-NMe2-2COOH was obtained by column chromatography.

[0160] Example 17

[0161] The preparation method of the near-infrared fluorescent probe Cy7-αCD-NMe2-2NHS ester includes the following steps:

[0162] (1) Mix 0.15 g Cy7-αCD-2COOH, 0.15 g 1-ethyl-(3-dimethylaminopropyl)carbodiimide, 0.12 g N-hydroxysuccinimide and 10 mL DMF at room temperature and stir for 5 hours.

[0163] (2) Acetonitrile was added to precipitate the product, and the near-infrared fluorescent probe Cy7-αCD-NMe2-2NHS ester was obtained by filtration.

[0164] Example 18

[0165] The preparation method of the near-infrared fluorescent probe Cy7.5-αCD-2COOH is as follows:

[0166] (1) Mix 0.30 g of (1E,3E)-5-oxopentan-1,3-diene-1-acetate, 1.71 g of 3-(5-carboxypentyl)-1,1,2-trimethyl-1H-benzo[e]indole-3-onium bromide, 20.4 g of α-cyclodextrin, 4.91 g of anhydrous potassium acetate, 30 mL of water, and 3 mL of DMSO at room temperature. After deoxygenation, heat the solution to 70 °C and stir for 2 days.

[0167] (2) After the solution cools to room temperature, dilute it with water and wash the aqueous phase with dichloromethane.

[0168] (3) The aqueous solution was evaporated to dryness using a rotary evaporator, the crude product was dissolved in an appropriate amount of ethanol, and some inorganic salts were removed by filtration. The near-infrared fluorescent probe Cy7.5-αCD-2COOH was obtained by column chromatography.

[0169] Example 19

[0170] The preparation method of the near-infrared fluorescent probe Cy7.5-αCD-2NHS ester is as follows:

[0171] (1) Mix 0.15 g Cy7-αCD-2COOH, 0.15 g 1-ethyl-(3-dimethylaminopropyl)carbodiimide, 0.12 g N-hydroxysuccinimide and 10 mL DMF at room temperature and stir for 5 hours.

[0172] (2) Add acetonitrile to precipitate the product, and filter to obtain the near-infrared fluorescent probe Cy7.5-αCD-2NHS ester.

[0173] Example 20

[0174] The preparation method of the near-infrared fluorescent probe Cy9-αCD-2COOH is as follows:

[0175] (1) Mix 2.40 g of heptadenal diphenylamine hexafluorophosphate, 47.8 g of α-cyclodextrin, 70 mL of water, and 7 mL of DMSO at room temperature and stir for 30 minutes. Add 4.02 g of 1-(5-carboxypentyl)-2,3,3-trimethyl-3H-indole-1-onium bromide and 6.71 g of anhydrous potassium acetate. After deoxygenation, heat the solution to 55 °C and stir for 2 days.

[0176] (2) After the solution cools to room temperature, dilute it with water and wash the aqueous phase with dichloromethane.

[0177] (3) The aqueous solution was evaporated to dryness using a rotary evaporator, the crude product was dissolved in an appropriate amount of ethanol, and some inorganic salts were removed by filtration. The near-infrared fluorescent probe Cy9-αCD-2COOH was obtained by column chromatography.

[0178] Example 21

[0179] The preparation method of the near-infrared fluorescent probe Cy9-αCD-2NHS ester is as follows:

[0180] (1) Mix 0.15 g Cy9-αCD-2COOH, 0.15 g 1-ethyl-(3-dimethylaminopropyl)carbodiimide, 0.12 g N-hydroxysuccinimide and 10 mL DMF at room temperature and stir for 5 hours.

[0181] (2) Add acetonitrile to precipitate the product, and filter to obtain the near-infrared fluorescent probe Cy9-αCD-2NHS ester.

[0182] Example 22

[0183] The preparation method of the near-infrared fluorescent probe Cy9-αCD-OMe-2COOH is as follows:

[0184] (1) Mix 2.40 g of heptadenal diphenylamine hexafluorophosphate, 47.8 g of α-cyclodextrin, 70 mL of water, and 7 mL of DMSO at room temperature and stir for 30 minutes. Add 4.36 g of 1-(5-carboxypentyl)-5-methoxy-2,3,3-trimethyl-3H-indole-1-onium bromide and 6.71 g of anhydrous potassium acetate. After deoxygenation, heat the solution to 55 °C and stir for 2 days.

[0185] (2) After the solution cools to room temperature, dilute it with water and wash the aqueous phase with dichloromethane.

[0186] (3) The aqueous solution was evaporated to dryness using a rotary evaporator, the crude product was dissolved in an appropriate amount of ethanol, and some inorganic salts were removed by filtration. The near-infrared fluorescent probe Cy9-αCD-OMe-2COOH was obtained by column chromatography.

[0187] Example 23

[0188] The preparation method of the near-infrared fluorescent probe Cy9-αCD-OMe-2NHS ester is as follows:

[0189] (1) Mix 0.15 g Cy9-αCD-OMe-2COOH, 0.15 g 1-ethyl-(3-dimethylaminopropyl)carbodiimide, 0.12 g N-hydroxysuccinimide and 10 mL DMF at room temperature and stir for 5 hours.

[0190] (2) Acetonitrile was added to precipitate the product, and the near-infrared fluorescent probe Cy9-αCD-OMe-2NHS ester was obtained by filtration.

[0191] Example 24

[0192] The preparation method of the near-infrared fluorescent probe Cy7-αCD-2Alkyne is as follows:

[0193] (1) Mix 0.30 g of (1E,3E)-5-oxopram-1,3-diene-1-acetate, 1.19 g of 1-propynyl-2,3,3-trimethyl-3H-indole-1-onium bromide, 20.4 g of α-cyclodextrin, 4.91 g of anhydrous potassium acetate, 30 mL of water, and 3 mL of DMSO at room temperature. After deoxygenation, heat the solution to 80 °C and stir for 2 days.

[0194] (2) After the solution cools to room temperature, dilute it with water and wash the aqueous phase with dichloromethane.

[0195] (3) The near-infrared fluorescent probe Cy7-αCD-2Alkyne was obtained by rotary evaporation of the aqueous solution.

[0196] Example 25

[0197] The preparation method of the near-infrared fluorescent probe Cy7-αCD-OMe-2Alkyne is as follows:

[0198] (1) Mix 0.30 g of (1E,3E)-5-oxopentan-1,3-diene-1-acetate, 1.31 g of 1-propynyl-2,3,3-trimethyl-5-methoxy-3H-indole-1-onium bromide, 20.4 g of α-cyclodextrin, 4.91 g of anhydrous potassium acetate, 30 mL of water, and 3 mL of DMSO at room temperature. After deoxygenation, heat the solution to 80 °C and stir for 2 days.

[0199] (2) After the solution cools to room temperature, dilute it with water and wash the aqueous phase with dichloromethane.

[0200] (3) The aqueous solution was evaporated to dryness using a rotary evaporator, the crude product was dissolved in an appropriate amount of ethanol, and some inorganic salts were removed by filtration. The near-infrared fluorescent probe Cy7-αCD-OMe-2Alkyne was obtained by column chromatography.

[0201] Example 26

[0202] The preparation methods of the near-infrared fluorescent probes Cy7-αCD-OMe-2Alkyne-1 and Cy7-αCD-NMe2-2Alkyne-2 are as follows:

[0203] (1) Mix 0.30 g of (1E,3E)-5-oxopentane-1,3-diene-1-acetate, 0.66 g of 1-propynyl-2,3,3-trimethyl-5-methoxy-3H-indole-1-onium bromide, 0.78 g of 5-methoxy-1,2,3,3-tetramethyl-3H-indole-1-iodide, 20.4 g of α-cyclodextrin, 4.91 g of anhydrous potassium acetate, 30 mL of water, and 3 mL of DMSO at room temperature. After deoxygenation, heat the solution to 80 °C and stir for 2 days.

[0204] (2) After the solution cools to room temperature, dilute it with water and wash the aqueous phase with dichloromethane.

[0205] (3) The aqueous solution was evaporated to dryness using a rotary evaporator, the crude product was dissolved in an appropriate amount of ethanol, and some inorganic salts were removed by filtration. The near-infrared fluorescent probes Cy7-αCD-OMe-2Alkyne-1 and Cy7-αCD-OMe-2Alkyne-2 were obtained by preparative high performance liquid chromatography.

[0206] Example 27

[0207] The preparation method of the near-infrared fluorescent probe Cy7-αCD-NMe2-2Alkyne includes the following steps:

[0208] (1) Mix 0.30 g of (1E,3E)-5-oxopentan-1,3-diene-1-acetate, 1.37 g of 1-propynyl-2,3,3-trimethyl-5-dimethylamino-3H-indole-1-onium bromide, 20.4 g of α-cyclodextrin, 4.91 g of anhydrous potassium acetate, 30 mL of water, and 3 mL of DMSO at room temperature. After deoxygenation, heat the solution to 80 °C and stir for 2 days.

[0209] (2) After the solution cools to room temperature, dilute it with water and wash the aqueous phase with dichloromethane.

[0210] (3) The aqueous solution was evaporated to dryness using a rotary evaporator, the crude product was dissolved in an appropriate amount of ethanol, and some inorganic salts were removed by filtration. The near-infrared fluorescent probe Cy7-αCD-NMe2-2Alkyne was obtained by column chromatography.

[0211] Example 28

[0212] The preparation method of the near-infrared fluorescent probe Cy7.5-αCD-2Alkyne includes the following steps:

[0213] (1) Mix 0.30 g of (1E,3E)-5-oxopentan-1,3-diene-1-acetate, 1.40 g of 1,1,2-trimethyl-3-(prop-2-yn-1-yl)-1H-benzo[e]indole-3-onium bromide, 20.4 g of α-cyclodextrin, 4.91 g of anhydrous potassium acetate, 30 mL of water, and 3 mL of DMSO at room temperature. After deoxygenation, heat the solution to 80 °C and stir for 2 days.

[0214] (2) After the solution cools to room temperature, dilute it with water and wash the aqueous phase with dichloromethane.

[0215] (3) The aqueous solution was evaporated to dryness using a rotary evaporator, the crude product was dissolved in an appropriate amount of ethanol, and some inorganic salts were removed by filtration. The near-infrared fluorescent probe Cy7.5-αCD-2Alkyne was obtained by column chromatography.

[0216] Example 29

[0217] The preparation method of the near-infrared fluorescent probe Cy9-αCD-2Alkyne includes the following steps:

[0218] (1) Mix 2.40 g of heptadenal diphenylamine hexafluorophosphate, 47.8 g of α-cyclodextrin, 70 mL of water, and 7 mL of DMSO at room temperature and stir for 30 minutes. Add 3.16 g of 1-propynyl-2,3,3-trimethyl-3H-indole-1-onium bromide and 6.71 g of anhydrous potassium acetate. After deoxygenation, heat the solution to 55 °C and stir for 2 days.

[0219] (2) After the solution cools to room temperature, dilute it with water and wash the aqueous phase with dichloromethane.

[0220] (3) The aqueous solution was evaporated to dryness using a rotary evaporator, the crude product was dissolved in an appropriate amount of ethanol, and some inorganic salts were removed by filtration. The near-infrared fluorescent probe Cy9-αCD-2Alkyne was obtained by column chromatography.

[0221] Example 30

[0222] The preparation method of the near-infrared fluorescent probe Cy9-αCD-OMe-2Alkyne includes the following steps:

[0223] (1) Mix 2.40 g of heptaenal diphenylamine hexafluorophosphate, 47.8 g of α-cyclodextrin, 70 mL of water, and 7 mL of DMSO at room temperature and stir for 30 minutes. Add 3.50 g of 5-methoxy-2,3,3-trimethyl-1-(prop-2-yn-1-yl)-3H-indol-1-onium bromide and 6.71 g of anhydrous potassium acetate. After deoxygenation, heat the solution to 55 °C and stir for 2 days.

[0224] (2) After the solution cools to room temperature, dilute it with water and wash the aqueous phase with dichloromethane.

[0225] (3) The aqueous solution was evaporated to dryness using a rotary evaporator, the crude product was dissolved in an appropriate amount of ethanol, and some inorganic salts were removed by filtration. The near-infrared fluorescent probe Cy9-αCD-OMe-2Alkyne was obtained by column chromatography.

[0226] Application Example 1

[0227] Application of the near-infrared fluorescent probe prepared in Example 1 in fluorescence imaging of the ureter in mice.

[0228] 100 μL of the Cy9-αCD fluorescent probe prepared in Example 1 was injected into BABL / c mice via tail vein injection after anesthesia and disinfection. Under anesthesia, the mouse skin, peritoneum, and intestines were dissected sequentially to expose the kidneys and ureters. Imaging was performed under 860 nm laser irradiation and 1100 nm, 1200 nm, and 1300 nm wavelength filters to observe the excretion of the probe by the ureter.

[0229] Using 860nm laser excitation, the imaging of the intravenously injected Cy9-αCD probe was demonstrated under different wavelength filters, as shown in Figure 8. Under the 1100nm wavelength filter, the probe could be clearly seen being excreted from the kidney to the bladder with urine. Furthermore, as the filter wavelength was increased to 1200nm and 1300nm, the fluorescence imaging resolution increased accordingly, the ureter imaging became clearer, and the tissue autofluorescence signal decreased, further highlighting the ureteral morphology. After homogenizing the ureteral signals at each wavelength, as shown in Figure 9, the tissue background signal at 3mm using the 1300nm long-pass filter was reduced by 3.8 times compared to the 1100nm condition, thus demonstrating excellent signal-to-noise ratio (SNR).

[0230] The near-infrared fluorescent probe prepared in Example 1 can be rapidly excreted by the kidneys. With the peristalsis of the ureter, the probe in the kidneys is excreted into the bladder along with the urine, as shown in Figure 10. Within 2 minutes after injection, the near-infrared fluorescent probe prepared in Example 1, under 860nm laser excitation and a 1200nm long-pass filter, can be clearly visualized in the kidneys, ureters, and bladder, forming a sharp contrast with the background tissue signal. At this time, the SNR reaches 7.5, and the SNR peaks at 14 at 18 minutes. As time progresses, most of the probe is rapidly excreted through the urine; probe excretion is still visible in the ureter at 30 minutes, at which point the SNR is 11.3.

[0231] To test the photostability of the Cy9-αCD probe, an 860nm laser (35mW cm⁻¹) was used. -2 Cy9-αCD samples dissolved in 10% FBS, deionized water, and PBS were continuously irradiated. Figure 11A shows the fluorescence imaging images of the samples after irradiation with an 860nm laser for 10 seconds to 60 minutes. As shown in Figure 11B, the fluorescence intensity curve shows that the fluorescence intensity of the samples decreased by only 0.25% after irradiation for 60 minutes, proving that Cy9-αCD has good photostability.

[0232] Application Example 2

[0233] Application of the near-infrared fluorescent probe Cy9-αCD prepared in Example 1 in fluorescence imaging of lymph nodes and lymphatic vessels in mice.

[0234] The near-infrared fluorescent probe prepared in Example 1 was used for in vivo lymph node and lymphatic vessel fluorescence imaging at different time points after subcutaneous injection, under conditions of 860nm laser excitation and a 1200nm long-pass filter. As shown in Figure 12, significant lymph node and lymphatic vessel signals were observed 10 minutes after subcutaneous injection, with the strongest lymph node signal at 20 minutes. Subsequently, the probe was rapidly excreted through the kidneys in the urine, and almost all of the probe had been excreted from the mouse body by 120 minutes.

[0235] Application Example 3

[0236] Application of the near-infrared fluorescent probe Cy7.5-αCD prepared in Example 2 in fluorescence imaging of ureter, lymph node and lymphatic vessel in mice.

[0237] 100 μL of the fluorescent probe prepared in Example 2 was injected into BABL / c mice via intratumoral injection and tail vein injection after anesthesia and disinfection. Under anesthesia, the mice's skin, peritoneum, and intestines were dissected sequentially to expose their kidneys and ureters. Imaging was performed under 808 nm laser irradiation and long-pass filters at 1000 nm, 1100 nm, 1200 nm, and 1300 nm.

[0238] As shown in Figure 13, the Cy7.5-αCD probe was injected into the 4T1 tumor of BABL / c mice via intratumoral injection. Under different wavelength filters, as the wavelength of the filter increased, the fluorescence imaging resolution of lymph nodes and lymphatic vessels increased, the imaging became clearer, and the tissue autofluorescence signal decreased.

[0239] As shown in Figure 14, the probe can clearly visualize the lymph nodes and lymphatic vessels next to the tumor 5 minutes after injection into the tumor, and obvious kidney signals can be observed at the same time, indicating that the probe can also be rapidly excreted from the kidneys in urine.

[0240] Mice were dissected under anesthesia within 2 minutes to expose their kidneys, ureters, and bladder. Cy7.5-αCD fluorescent probes were excited using an 808nm laser. Imaging of the intravenously injected Cy7.5-αCD probes was demonstrated under different wavelength filters. The probe is rapidly excreted by the kidneys; with ureteral peristalsis, the probe in the kidneys is excreted into the bladder with urine, as shown in Figure 15. Within 2 minutes of injection, the kidneys, ureters, and bladder were clearly visualized, forming a sharp contrast with the background tissue signal. Furthermore, with increasing filter wavelength, the fluorescence imaging resolution increased, the ureter imaging became clearer, and the tissue autofluorescence signal decreased, further highlighting the ureteral morphology. After homogenizing the ureteral signal at various wavelengths, as shown in Figure 16, the tissue background signal at 2.5mm using a 1500nm long-pass filter was reduced by 6.9 times compared to a 900nm long-pass filter. At this point, the ureteral morphology could be clearly observed under conditions of lower background signal.

[0241] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A near-infrared fluorescent probe based on cyclodextrin, characterized in that, The near-infrared fluorescent probe is a conjugate in which fluorescent molecules are coated with cyclodextrin; the fluorescent molecules are cyanine dyes or electron donor-electron acceptor-electron donor dyes.

2. The near-infrared fluorescent probe based on cyclodextrin according to claim 1, characterized in that, The cyanine dyes are any one or more of Cyanine7, Cyanine7.5, Cyanine9, Cyanine9.5, Cyanine11, Cyanine11.5, Cyanine13, Cyanine13.5, Cyanine 15, Cyanine15.5, Flab 7, FD-1080, IR-1048, IR-26, IR-1061, and IR-1051.

3. The near-infrared fluorescent probe based on cyclodextrin according to claim 1, characterized in that, The electron donor-electron acceptor-electron donor dyes are any one or more of the following: CH1055, Fluorene-based DAD dyes, IR-FGP, IR-FEP, IR-FEPC, IR-FTAP, CH4T, IR-pFE, IRT, IR-BGP6, IR-BEMC6P, IR-1048-MZ, IR-FE, IR-FTP, IR-BBE, IR-BBEP, IR-FTX, IR-FTXP, IR-FX, IR-FXP, SXH1100, Q-FITBBTTFI, IR-FT, IR-FP8P, 2TT-oC6B, 2TT-oC26B, TT3-oCB, and HL3.

4. The near-infrared fluorescent probe based on cyclodextrin according to claim 1, characterized in that, The fluorescent molecule includes, but is not limited to, any one or more of the following structures: Among them, R1, R2, R3, R4, R5, R6, R7, R8, R 26 R 27 R 28 R 29 Each group is independently selected from hydrogen, amino, hydroxy, dimethylamino, sulfonic acid, methoxy, methylthio, fluorine, chlorine, bromine, iodine, ester or carboxyl, and one or more of substituted or unsubstituted straight-chain or branched alkyl, triphenylamine, straight-chain or branched alkoxy, or substituted or unsubstituted aryl groups. R9, R 10 R 11 R 12 Each group is independently selected from one or more of the following groups: substituted or unsubstituted straight-chain or branched alkyl, alkoxy, phenyl, benzyl, sulfonic acid, carboxyl, alkynyl, mercapto, and N-hydroxysuccinimide ester groups. R 15 R 16 R 17 R 18 R 19 R 20 R 21 R 22 R 23 R 24 R 25 Each of the following is independently selected from one or more of hydrogen, substituted or unsubstituted straight-chain or branched alkyl groups, fluorine, chlorine, bromine, and iodine; R 13 R 14 Each group is independently selected from one or more of the following groups: amino, hydroxy, dimethylamino, sulfonic acid, methoxy, methylthio, fluorine, chlorine, bromine, iodine, ester, carboxyl, alkynyl, mercapto, and N-hydroxysuccinimide ester. L and L', as linking groups, are independently selected from one or more of the following: substituted or unsubstituted straight-chain or branched alkyl groups, straight-chain or branched alkoxy groups, phenyl groups, and benzyl groups. X - As anionic groups, they are independently selected from chloride ions, bromide ions, iodide ions, fluoride ions, and acetate ions, respectively.

5. The near-infrared fluorescent probe based on cyclodextrin according to claim 4, characterized in that, The cyclodextrin-based near-infrared fluorescent probe includes, but is not limited to, any one or more of the following structural formulas: in, It is a cyclodextrin.

6. The near-infrared fluorescent probe based on cyclodextrin according to claim 1, characterized in that, The cyclodextrin-based near-infrared fluorescent probe includes, but is not limited to, any one or more of the structural formulas: in, It is a cyclodextrin.

7. The near-infrared fluorescent probe based on cyclodextrin according to claim 1, characterized in that, The molecular weight of the cyclodextrin is between 500 Da and 5000 Da.

8. The near-infrared fluorescent probe based on cyclodextrin according to claim 1, characterized in that, The cyclodextrin is selected from one or more of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, hydroxypropyl-α-cyclodextrin (HP-α-cyclodextrin), hydroxypropyl-β-cyclodextrin (HP-β-cyclodextrin), hydroxypropyl-γ-cyclodextrin (HP-γ-cyclodextrin), methyl-α-cyclodextrin (Mα-cyclodextrin), methyl-β-cyclodextrin (Mβ-cyclodextrin), and methyl-γ-cyclodextrin (Mγ-cyclodextrin).

9. A method for preparing a near-infrared fluorescent probe based on cyclodextrin according to any one of claims 1-8, characterized in that, Includes the following steps: (1) Dissolve (1E,3E)-5-oxopram-1,3-diene-1-acetate, pentaenal diphenylamine hexafluorophosphate, heptaenal diphenylamine hexafluorophosphate or nonadienal diphenylamine hexafluorophosphate in solvent one to obtain solution one; (2) Dissolve cyclodextrin, indolon halide and potassium acetate in the first solution, remove oxygen and heat and stir at 25℃-110℃ for 40-50 hours to obtain the reaction solution; (3) The reaction solution was diluted with water, washed with dichloromethane, and the aqueous solution was purified by dialysis and column chromatography to obtain a near-infrared fluorescent probe based on cyclodextrin. The solvent is water, or a mixture of water and a water-miscible organic solvent, wherein the organic solvent includes, but is not limited to, acetonitrile, N,N-dimethylformamide, N,N-dimethylacetamide, acetone, methanol, ethanol, isopropanol, DMSO and other organic solvents.

10. The method for preparing a near-infrared fluorescent probe based on cyclodextrin according to claim 9, characterized in that, In step (3), the column chromatography includes, but is not limited to, normal phase column chromatography, reversed phase column chromatography, ion exchange chromatography, size exclusion chromatography, hydrophilic interaction chromatography, and preparative high performance liquid chromatography.

11. The method for preparing a near-infrared fluorescent probe based on cyclodextrin according to claim 9, characterized in that, The indolon halides include one or more of indolon iodides, indolon bromides, or indolon chlorides.

12. The application of a cyclodextrin-based near-infrared fluorescent probe according to any one of claims 1-8 in imaging of the ureter, lymph nodes, lymphatic vessels, gastrointestinal tract, blood vessels, and nerves in animals and humans.

13. A near-infrared fluorescent probe for tumor-targeted imaging, characterized in that, The conjugate of a cyclodextrin-based near-infrared fluorescent probe and a specific target molecule according to any one of claims 1-8, wherein the cyclodextrin-based near-infrared fluorescent probe comprises, but is not limited to, one or more of the following structures:

14. The near-infrared fluorescent probe for tumor-targeted imaging according to claim 13, characterized in that, The specific target molecule is any one of peptides, nucleic acid aptamers, affinity molecules, and antibodies.

15. The near-infrared fluorescent probe for tumor-targeted imaging according to claim 14, characterized in that, The specific target molecules are PSMA, αvβ3integrin, α4β1integrin, α6β1integrin, HER1-4, SSTR1-5, GnRH-R, VIP, NTSR1, CCK2R, EphA2, TD05, TE02, AS1411, TLS11a, Sgc8, 41t, TE17, KDED 2a-3, KCHA10, Sgd5, TTA1, MUC-1, A32, S11e, S6, J3, F3B, A10, VEGF165, and Folate. Receptor, HER2, HER3, Trop-2, Nectin-4, PDGFRβ, VEGFR, CAIX, LMP, LMP1, PD-L1, PD-1, 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, ALK, KRAS, FGFR2, ICAM -1. Any one of the following: ARTC2, CAIX, CapG, CD11b, CD20, CD33, CD38, CD4, CD44, CD45, CD47, CD52, CD7, CD8, CD105, CD133, CEA, c-Met, CTLA-4, CXCL11, CXCL12, CXCR2, CXCR4, CXCR7, Fibronectin, BCMA, HGF, Ly-6C / Ly-6G, MHC-II, MMR, Viral GPCR, peptides, nucleic acid aptamers, affinity molecules, or antibodies targeting US28.

16. The near-infrared fluorescent probe for tumor-targeted imaging according to claim 13, characterized in that, The specific target molecule is any one of vitamin B12, vitamin H, thiamine, riboflavin, adenosine, N-acetylglucosamine, folic acid, methotrexate, mannose, carbohydrates, hyaluronic acid, fructose, cRGD, Bevacizumab, or Avastin, Cetuximab, or Erbitux, Herceptin, trastuzumab, Sacituzumab, Ramucirumab, Enfortumab, Datopotamab, Atezolizumab, CD105 monoclonal antibody, Amivantamab-VMJM, or Rybrevant, Ivonescimab, or TRC105 antibody.

17. A method for preparing a near-infrared fluorescent probe for tumor-targeting imaging, wherein the near-infrared fluorescent probe for tumor-targeting imaging is the near-infrared fluorescent probe for tumor-targeting imaging according to claim 13, characterized in that, The preparation method of the tumor-targeting imaging near-infrared fluorescent probe includes the following steps: (1) Dissolve fluorescent molecules with carboxyl or alkyne groups in a solvent to obtain a fluorescent molecule solution; (2) Add the fluorescent molecule solution and cyclodextrin to the buffer solution, adjust the pH value, and react at room temperature in the dark for 2-4 hours; (3) Centrifuge and wash the reactants; (4) Resuspend the washed reactants in a buffer solution, add EDC / NHS or copper ions and specific targeting molecules with amino or azide, and react for 1-3 hours in the dark at room temperature. (5) Centrifuge and wash the reactants to obtain the tumor-targeting imaging near-infrared fluorescent probe.

18. The preparation method according to claim 17, characterized in that, In step (1), the solvent includes, but is not limited to, DMSO, pure water, physiological saline, or PBS buffer solution.

19. The preparation method according to claim 17, characterized in that, In step (2), the pH value is 5-9.

20. The preparation method according to claim 17, characterized in that, In step (3), a catalyst or coupling agent is also added.

21. The use of the near-infrared fluorescent probe for tumor-targeting imaging according to any one of claims 13-16 in the preparation of tumor tracers, tumor boundary determination imaging agents, tumor resection surgical navigation imaging agents, or tumor metastatic lymph node imaging agents.

22. The application according to claim 21, wherein the tumor includes one or more of the following: urogenital tumors, gastrointestinal tumors, skin cancer, head and neck cancer, breast cancer, and brain cancer.

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