WO2025190388A2 - Sonde fluorescente proche infrarouge, son procédé de préparation et son utilisation - Google Patents

Sonde fluorescente proche infrarouge, son procédé de préparation et son utilisation

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Publication number
WO2025190388A2
WO2025190388A2 PCT/CN2025/082583 CN2025082583W WO2025190388A2 WO 2025190388 A2 WO2025190388 A2 WO 2025190388A2 CN 2025082583 W CN2025082583 W CN 2025082583W WO 2025190388 A2 WO2025190388 A2 WO 2025190388A2
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Prior art keywords
imaging
arm
nir
tumor
fluorescent probe
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English (en)
Chinese (zh)
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WO2025190388A3 (fr
Inventor
戴宏杰
刘浩然
唐梅杰
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Shenzhen Institute of Research and Innovation HKU
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Shenzhen Institute of Research and Innovation HKU
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0054—Macromolecular compounds, i.e. oligomers, polymers, dendrimers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0052—Small organic molecules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0056—Peptides, proteins, polyamino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0058—Antibodies

Definitions

  • 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.
  • NIR fluorescent dyes/probes in fluorescence-guided surgery (FGS) is rapidly developing, primarily to enhance visualization of tumor tissue during surgery for precise resection.
  • NIR fluorescent dyes such as Indocyanine Green (ICG)
  • ICG Indocyanine Green
  • ICG Indocyanine Green
  • ICG 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.
  • 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.
  • 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.
  • NIR-II 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.
  • 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.
  • 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.
  • the multi-arm polymer is multi-arm polyethylene glycol, dextran or a dendrimer.
  • the NIR fluorescent molecules are cyanine dyes, electron donor-electron acceptor-electron donor (donor-acceptor-donor, D-A-D) dyes, boron-dipyrromethene (BODIPY) dyes, rhodamine dyes, metal-macrocycles complexes and gold cluster dyes.
  • 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, Zwitterionic cyanine dyes (ZW800, ZW800-1), Dylight 755, Dylight 800, Cy-7-NHS dyes, Alexa Fluor 750, Alexa Fluor 790, Alexa Fluor 800, NovaFluor Yell NovaFluor Yellow 700, NovaFluor Yellow 730, NovaFluor Yellow 755, NovaFluor Red 75, Brilliant Ultra Violet 737, Brilliant Ultra Violet 805, Brilliant Violet 711, Brilliant Violet 786, PE-Cyanine7, Qdot 705 Probe
  • the electron donor-electron acceptor-electron donor (donor-acceptor-donor, D-A-D) dyes include but are not limited to CH1055, Fluorene-based D-A-D 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.
  • 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.
  • 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.
  • the metal-macrocycles complexes dyes include but are not limited to Pd-1, Pd-2, Pd-3, Pt-1, F-Yb, etc.
  • the gold cluster dyes include but are not limited to Au 25 , Au 28 , Au 7 Cd 1 , AuNCs, and the like.
  • 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 multi-arm polyethylene glycol is 8-arm-PEG-NH 2 , 8-arm-PEG-SH, 8-arm-PEG-alkyne, 8-arm-PEG-N 3 or 8-arm-PEG-COOH.
  • the dextran is selected from one or more of dextran methoxypolyethylene glycol, dextran ester, diethylaminoethyl dextran, dextran polyethylene glycol, Dextran-NH 2 , Dextran-NHS, and DEAE-Dextran;
  • the structure of the near-infrared fluorescent probe is shown in Figure 1, which is based on n-arm-PEG as the core, and n-arm-PEG is coupled with m NIR fluorescent molecules through its n arms, wherein n can be 2, 4, 6, 8, 10, and m is the number of NIR fluorescent molecules on each n-arm-PEG, and m ⁇ n.
  • the present invention also provides a method for preparing a near-infrared fluorescent probe, comprising the following steps:
  • the solvent in step (1) is selected from DMSO, pure water or buffer solution.
  • step (2) Furthermore, the pH value in step (2) is 5-9.
  • reaction time of step (2) is 1-3 hours.
  • the catalyst or coupling agent in step (2) is EDC or copper ions.
  • step (2) Furthermore, the reaction in step (2) is carried out in the dark at room temperature.
  • step (3) according to the molecular weight of the multi-arm polymer, an ultrafiltration centrifuge tube of corresponding molecular weight is used for centrifugation.
  • the present invention also provides the use of the near-infrared fluorescent probe in preparing a fluorescent imaging agent.
  • the fluorescent imaging agent is used for imaging the ureters, lymph nodes, lymphatic vessels, gastrointestinal tract, blood vessels, and nerves of animals and humans.
  • 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.
  • the present invention also provides a method for fluorescence imaging of animal or human blood vessels, which comprises intravenously injecting a near-infrared fluorescent probe with a molecular weight of 30kDa-200kDa in the present invention, and then imaging the blood vessels in the body with a NIR-I or NIR-II fluorescence endoscope, or imaging the blood vessels on the surface of the whole body with an in vitro imaging device in the NIR-I or NIR-II region.
  • This method is used to develop blood vessels during surgery, thereby avoiding damage to the blood vessels.
  • 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 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 present invention also provides a method for fluorescence imaging of the ureter of an animal or human body, the method comprising injecting a near-infrared fluorescent probe of the present invention having a molecular weight of less than 30 kDa into the ureter through a vein, tissue, or subcutaneous injection, followed by excretion into the ureter through the kidneys, and imaging the ureter within 2 minutes to 5 hours using a fluorescence endoscope in the NIR-I or NIR-II region or a surgical robot fluorescence imaging system.
  • imaging can be performed using an in vitro imaging device in the NIR-I or NIR-II region after the ureter is exposed by opening the abdominal cavity.
  • 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 about 10 KD;
  • 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 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.
  • 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.
  • 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.
  • the present invention also provides a kit for fluorescence imaging, which comprises the above-mentioned near-infrared fluorescent probe.
  • 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).
  • n-Arm-PEG-NIR Dye/targeting ligand a near-infrared fluorescent probe for tumor-specific targeted imaging
  • the fluorescent probe is a conjugate of a NIR fluorescent molecule, a multi-arm polymer, and a specific targeting molecule (targeting ligand).
  • the multi-arm polymer is multi-arm polyethylene glycol, dextran or a dendrimer.
  • the near-infrared fluorescent probe for tumor-specific targeted imaging is formed by coupling a near-infrared fluorescent probe with a specific targeting molecule.
  • the specific targeting molecule is selected from any one of peptide, aptamers, affibodies, and antibodies.
  • the specific targeting molecules are directed against PSMA, ⁇ v ⁇ 3integrin, ⁇ 4 ⁇ 1integrin , ⁇ 6 ⁇ 1integrin , 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, PDGFR ⁇ , VEGFR, CAIX, LMP, LMP1, PD-L1, PD-1, PSMA, TNF ⁇ , IL-1, IL-6, IL-12, IL-13, IL-17A, IL-23, MCP-1, IGF-1, IFN-
  • the specific targeting molecule is any one of peptides, nucleic acid aptamers, affinity bodies, and antibodies targeting PSMA, ⁇ v ⁇ 3integrin, HER2, EGFR, VEGFR1, VEGFR2, VEGFR3, TROP-2, Nectin-4, CD8, CD4, CD105, CD133, PD-L1 or PD-1 targets.
  • the specific targeting molecule is a small molecule vitamin B12, vitamin H, thiamine, riboflavin, adenosine, N-acetylglucosamine, folic acid, HS-PEG-Folic acid (PEG molecular weight is 1-20kDa), methotrexate, mannose, carbohydrates, hyaluronic acid, fructose, cRGD and any one of Bevacizumab, Cetuximab, Atezolizumab, CD105 monoclonal antibody, Andecaliximab monoclonal antibody, and TRC105 antibody.
  • the structure of the near-infrared fluorescent probe for tumor-specific targeted imaging is shown in Figure 2, which is based on 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 ⁇ n-m.
  • 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;
  • the specific targeting molecule is a folic acid molecule, RGD polypeptide or PSMA polypeptide.
  • the present invention also provides a method for preparing a near-infrared fluorescent probe for tumor-specific targeted imaging, comprising the following steps:
  • the reactants are centrifuged and washed to obtain the tumor-specific targeted imaging near-infrared fluorescent probe.
  • the solvent in step (1) is selected from DMSO, pure water or buffer solution.
  • step (2) Furthermore, the pH value in step (2) is 5-9.
  • reaction time of step (2) is 1-3 hours.
  • the catalyst or coupling agent in step (2) is EDC or copper ions.
  • step (2) Furthermore, the reaction in step (2) is carried out in the dark at room temperature.
  • step (3) according to the molecular weight of the multi-arm polymer, an ultrafiltration centrifuge tube of corresponding molecular weight is used for centrifugation.
  • step (4) Furthermore, the pH value in step (4) is 5-9.
  • step (4) is carried out in the dark at room temperature, and the reaction time is 1-3 hours.
  • the linking molecule in step (4) is SMCC, SPDP or SPDB.
  • step (5) according to the molecular weight of the multi-arm polymer, an ultrafiltration centrifuge tube of corresponding molecular weight is used for centrifugation.
  • step (6) Furthermore, the pH value in step (6) is 5-9.
  • step (6) is carried out in the dark at room temperature, and the reaction time is 1-3 hours.
  • the catalyst and coupling agent in step (6) are EDC or copper ions.
  • step (7) according to the molecular weight of the multi-arm polymer, centrifugation is performed using an ultrafiltration centrifuge tube of corresponding molecular weight.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the tumor is imaged using a fluorescence endoscope or surgical robot in the NIR-I or NIR-II range.
  • 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.
  • 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.
  • 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.
  • 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.
  • the nerves are imaged using fluorescence in the NIR-I or NIR-II region to provide surgical navigation to avoid nerve damage.
  • the present invention has the following beneficial effects:
  • 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.
  • 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.
  • 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.
  • FIG1 is a schematic structural diagram of the near-infrared fluorescent probe of the present invention.
  • FIG2 is a schematic diagram of the structure of the near-infrared fluorescent probe for tumor-specific targeted imaging of the present invention.
  • FIG3A and FIG3B are the absorption spectrum and emission spectrum of the near-infrared fluorescent probe prepared in Example 1 of the present invention, respectively.
  • FIG4A and FIG4B are the absorption spectrum and emission spectrum of the near-infrared fluorescent probe prepared in Example 2 of the present invention, respectively.
  • FIG5A and FIG5B are respectively 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.
  • FIG6A and FIG6B are respectively 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.
  • FIG7A and FIG7B are respectively 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.
  • FIG8 is a fluorescence imaging of the ureter of a mouse in vivo using the near-infrared fluorescent probe prepared in Example 1 of the present invention.
  • FIG9 is a fluorescence imaging of the lymph nodes and lymphatic vessels in mice using the near-infrared fluorescent probe prepared in Example 1 of the present invention.
  • FIG10 is a fluorescence imaging of the ureter of a mouse in vivo using the near-infrared fluorescent probe prepared in Example 2 of the present invention.
  • FIG11 is a fluorescence imaging of the lymph nodes and lymphatic vessels in mice using the near-infrared fluorescent probe prepared in Example 2 of the present invention.
  • FIG12 shows tumor-targeted imaging of a near-infrared fluorescent probe for tumor-specific targeted imaging prepared in Example 3-4 of the present invention in mice.
  • FIG13 shows tumor-targeted imaging of a near-infrared fluorescent probe for tumor-specific targeted imaging prepared in Example 5 of the present invention in mice.
  • the “near infrared” in this invention refers to infrared light (NIR), which is an electromagnetic wave between visible light (VIS) and mid-infrared light (MIR).
  • NIR infrared light
  • MIR mid-infrared light
  • the near infrared region is customarily divided into two regions: near infrared region 1 (750-900nm) and near infrared region 2 (1000-1700nm).
  • 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.
  • the "fluorescent probe” in the present invention means a moiety that can be detected by colorimetry or fluorescence measurement.
  • a method for preparing a near-infrared fluorescent probe comprises the following steps:
  • Figure 3A shows the absorption spectra of IRDye800CW and IRDye800CW-8-Arm-PEG (10 kDa) collected after filtration using an ultrafiltration tube with a 3 kDa molecular weight cutoff. The absorption peak is located at 775 nm. Because IRDye800CW has a molecular weight of only 0.96 kDa, the dye not conjugated to 8-Arm-PEG is filtered out, while the dye collected in the ultrafiltration tube is successfully conjugated IRDye800CW-8-Arm-PEG, with three NIR fluorescent molecules per 8-Arm-PEG. Its emission spectrum under 808 nm laser excitation is shown in Figure 3B at 890 nm.
  • a method for preparing a near-infrared fluorescent probe :
  • Example 1 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.
  • Figure 4A shows the absorption spectra of ICG-NHS and ICG-NHS-8-Arm-PEG (10 kDa) collected after filtration using a 3 kDa molecular weight cutoff ultrafiltration tube. The absorption peak is located at 789 nm. Because the molecular weight of ICG-NHS is only 0.82 kDa, the dye that is not coupled to 8-Arm-PEG is filtered out, while the dye collected in the ultrafiltration tube is successfully coupled ICG-NHS-8-Arm-PEG, with the number of NIR fluorescent molecules on each 8-arm-PEG being 3.
  • the emission spectrum of ICG-NHS-8-Arm-PEG under 808 nm laser excitation is shown in Figure 4B, with an emission peak at 890 nm.
  • a method for preparing a near-infrared fluorescent probe for tumor-specific targeted imaging comprises the following steps:
  • a method for preparing a near-infrared fluorescent probe for tumor-specific targeted imaging comprises the following steps:
  • 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.
  • Figures 7A and 7B are the absorption and emission spectra of IR-12-NHS-8-Arm-PEG-FA, respectively.
  • Figure 7A shows the absorption spectra 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 10 kDa. The absorption peak is located at 790 nm. Because the molecular weight of IR-12-NHS is only 1.7 kDa, the dye that is not coupled to 8-Arm-PEG is filtered out.
  • the dye collected in the ultrafiltration tube after two filtrations 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 per 8-arm-PEG is 1.8 and 3 respectively.
  • Figure 7B shows the emission spectrum of the IR-12-NHS-8-Arm-PEG-FA probe under 808 nm laser excitation. The probe has a clear fluorescence signal.
  • Example 1 Take 100 ⁇ L 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.
  • 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 (I.T.), and subcutaneously (S.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.
  • A intravenously
  • I.T. intratumorally
  • S.C. subcutaneously
  • 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, further highlighting the ureteral morphology.
  • the near-infrared fluorescent probe prepared in Example 1 was used for fluorescence imaging of lymph nodes and lymphatic vessels in vivo at different time points after tumor injection (A) and subcutaneous injection (B), using 808 nm laser excitation.
  • (C) shows the imaging of the fluorescent probe after subcutaneous injection using different wavelength filters. As the filter wavelength increases, the fluorescence imaging resolution of the lymph nodes and lymphatic vessels increases, the images become clearer, and the tissue autofluorescence signal decreases.
  • the probe was injected into BABL/c mice via tail vein injection, intratumoral injection, or subcutaneous injection into the mouse tail.
  • 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.
  • 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, and the kidneys, ureters, and bladders were exposed. 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 (i.v.), intratumorally (i.t.), and subcutaneously (s.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.
  • the near-infrared fluorescent probe prepared in Example 2 was used for fluorescence imaging of lymph nodes and lymphatic vessels in vivo at different time points after tumor injection (A) and subcutaneous injection (B), using 808 nm laser excitation.
  • (C) shows the imaging of the fluorescent probe after subcutaneous injection using different wavelength filters. As the filter wavelength increases, the fluorescence imaging resolution of the lymph nodes and lymphatic vessels increases, resulting in clearer images and a decrease in tissue autofluorescence signal.
  • IRDye800CW-8-Arm-PEG-FA and 820NHS-8-Arm-PEG-FA were imaged using different long-pass filters (1000 nm, 1100 nm, 1200 nm, and 1300 nm), showing that both probes exhibited distinct fluorescence signals.
  • Figure 12B 48 hours after tail vein injection, under the same conditions of 808 nm laser excitation and matching different long-pass filters (1000 nm, 1100 nm, 1200 nm, and 1300 nm), both IRDye800CW-8-Arm-PEG-FA and 820NHS-8-Arm-PEG-FA were well targeted to the tumor. As the filter wavelength was increased, the resolution of the tumor boundary increased, imaging became clearer, and tissue autofluorescence signal decreased.
  • IR-12-NHS-8-Arm-PEG-FA fluorescent probe prepared in Example 5 100 ⁇ L 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.
  • IR-12-NHS-8-Arm-PEG-FA was well targeted to the tumor. As the filter wavelength was extended, the resolution of the tumor boundary increased, the imaging became clearer, and the tissue autofluorescence signal decreased.

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Abstract

La présente invention concerne une sonde fluorescente proche infrarouge, se rapportant au domaine technique des colorants fonctionnels biomédicaux. Dans la sonde fluorescente, des polymères à bras multiples ayant différentes masses moléculaires sont conjugués à des molécules fluorescentes proche infrarouge ou infrarouge ; ainsi, la sonde présente une meilleure solubilité dans l'eau, une meilleure histocompatibilité, une sécurité améliorée, une durée de vie de fluorescence plus longue, et une photostabilité supérieure tout en permettent d'obtenir une distribution et une réponse rapides dans l'uretère, les vaisseaux lymphatiques et les ganglions lymphatiques dans le corps, et peut être utilisée pour l'imagerie en NIR-I (850-1000 nm) et NIR-II (1000-1700 nm). La sonde fluorescente peut en outre marquer diverses molécules de ciblage de tumeur pour obtenir une sonde fluorescente proche infrarouge de ciblage, ce qui permet d'obtenir une imagerie par fluorescence ciblée précise de différents types de tumeurs telles que des tumeurs génito-urinaires, des cancers de la tête et du cou, un cancer du sein, un cancer du col de l'utérus et un cancer de l'ovaire, ainsi que des ganglions lymphatiques métastatiques dans les fenêtres NIR-I et NIR-II, pouvant en outre être utilisée dans une navigation d'imagerie par fluorescence peropératoire clinique.
PCT/CN2025/082583 2024-03-15 2025-03-14 Sonde fluorescente proche infrarouge, son procédé de préparation et son utilisation Pending WO2025190388A2 (fr)

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