A conjugate of indocyanine green and multi-arm polyethylene glycol or a pharmaceutically acceptable salt thereof, and a preparation method and use thereof

Abstract

The present application relates to the technical field of tumor fluorescence contrast agent, and specifically provides a kind of indocyanine green and multi-arm polyethylene glycol conjugate or its pharmaceutically acceptable salt and preparation method and purposes thereof, the conjugate has the structural formula shown in formula (I), the binding capacity of the conjugate with serum protein is significantly reduced, and the tumor targeting is significantly improved, accumulates and lingers in tumor tissue, to realize targeted treatment or high-resolution imaging, to realize more accurate tumor localization and diagnosis, with very high application value, and open up new method for tumor imaging and treatment development.

Description

Technical Field

[0001] This invention relates to the field of tumor fluorescence contrast agents, specifically to a conjugate of indocyanine green and multi-arm polyethylene glycol or a pharmaceutically acceptable salt thereof, as well as its preparation method and uses. Background Technology

[0002] Near-infrared fluorescence imaging (NIFI) of tumors is a bioimaging technique based on near-infrared light. It leverages the deep penetration of near-infrared light into tissues and its relatively low interaction with tissue, combined with targeted fluorescent probes to image tumors. This technique typically involves labeling tumor-related molecules or features, such as receptors or specific proteins on the surface of tumor cells, with specific fluorescent probes (e.g., indocyanine green, ICG), to emit fluorescent signals upon near-infrared light excitation. Through the deep penetration of near-infrared light and the high sensitivity of the fluorescence signal, NIFI can provide high-resolution, real-time tumor imaging, holding promise for early tumor diagnosis, surgical guidance, and monitoring of treatment efficacy.

[0003] Indocyanine green (ICG) is an FDA-approved near-infrared fluorescent probe for clinical use in guiding certain tumor resection procedures. It exhibits good biocompatibility and fluorescence imaging properties in vivo, making it suitable for angiography, tumor diagnosis, and surgical guidance. However, its half-life in vivo is only 2 to 3 minutes. After injection, ICG immediately binds tightly to serum proteins and is then rapidly excreted through the liver via bile. Even though it can visualize blood vessels and lymph nodes, the rapid clearance of ICG limits its application in tumor resection. Furthermore, this contrast agent lacks tumor targeting, resulting in low contrast between the tumor area and normal tissue. These factors severely limit the use of ICG in tumor resection surgery. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the defects of short half-life and lack of tumor targeting of ICG in the prior art, thereby providing a conjugate of indocyanine green and multi-arm polyethylene glycol or a pharmaceutically acceptable salt thereof, as well as its preparation method and use.

[0005] Therefore, this application provides a conjugate of indocyanine green and multi-arm polyethylene glycol or a pharmaceutically acceptable salt thereof, said conjugate having the structural formula shown in formula (I):

[0006]

[0007] Where M is the branching center of multi-arm polyethylene glycol, m is an integer between 3 and 8, k is an integer between 0 and m; q is an integer between 0 and 3; and k and q are not both 0.

[0008] X is

[0009] R1 may be the same or different, and mk R1s are independently selected from H or any other substituents;

[0010] PEG stands for polyethylene glycol group;

[0011] L1 may be the same or different, where L1 is the linker group connecting the polyethylene glycol unit to the X group;

[0012] L2 may be the same or different, where L2 is the linker group from which M is attached to the X group.

[0013] In this invention, the coupling compound of indocyanine green and multi-arm polyethylene glycol is abbreviated as ICG-mArmPEG. m represents the number of arms of the multi-arm polyethylene glycol.

[0014] Furthermore, M is selected from or Where m is an integer between 3 and 8. Furthermore, PEG has the following structure: Where n is the number of repeating units;

[0015] Optional, n is an integer between 1 and 1000.

[0016] Furthermore, k is 1.

[0017] Furthermore, the coupling has the structural formula shown in formulas (II), (III), (IV), (V), (VI), or (VII):

[0018]

[0019]

[0020] In equation (II), m is an integer between 3 and 8. In equations (II), (III), (IV), (V), (VI), or (VII), n1 is an integer between 10 and 1000. In equations (II), (III), (IV), and (V), n2 is an integer between 1 and 1000. R2 is independently selected from R1 or -L1-X, where R1 is as defined above, L is as defined above, and X is as defined above.

[0021] Furthermore, L1 or L2 is selected from -AB-, where A is selected from one of the following groups: -CO-NH-, -NH-CO-NH-, -CO-NH-CO-, -O-, -S-, -SS-, azido-ynyl cycloaddition linking group, tetrazine-trans-cyclooctene cycloaddition linking group, maleimide-mercaptoaddition linking group, azido-dibenzocyclooctynyl cycloaddition linking group, or cyanobenzothiazolyl-aminothiol click reaction linking group; B is selected from one of the following groups: -(CH2) x -、-O(CH2) y -、-CONH-(CH2) z -、-CONH-(CH2) j -NHC(O)O-, -CO-NH-(CH2) P -NH-CO-, -CONH-(CH2) i -C(O)-, -CONH-(CH2) u -O-, where x, y, z, j, p, i, u are independent integers selected from 1 to 10;

[0022] Preferably, L1 or L2 is selected from -(CH2). x -NH-CO-, -CO-NH-(CH2) P -NH-CO-、

[0023]

[0024] Where x and p are independent integers selected from 1 to 5.

[0025] X can be attached to either the L1 or L2 group.

[0026] Furthermore, the A group is linked to the X group; the B group is linked to PEG or M.

[0027] Furthermore, R1 is selected from H, halogen, -OH, -NH2, -COOH, -CN, -SH, -OCOOR3, -R4-COOR5, -COOR6, -NH-CO-R7-COOH, -NH-CO-R8, unsubstituted or substituted C1-C5 alkyl, unsubstituted or substituted C2-C5 alkenyl, unsubstituted or substituted C1-C5 alkoxy, unsubstituted or substituted C3-C5 cycloalkyl, unsubstituted or substituted C1-C5 aldehyde, unsubstituted or substituted C5-C20 heterocyclic compound, unsubstituted or substituted C1-C5 lactam, or unsubstituted or substituted C2-C10 amino acid residue;

[0028] R3, R4, R5, R6, R7, and R8 are independently selected from unsubstituted or substituted C1-C5 alkyl groups, unsubstituted or substituted C2-C5 alkenyl groups, unsubstituted or substituted C1-C5 lactam groups, or unsubstituted or substituted C5-C25 heterocyclic compounds.

[0029] "Substituted" means that the H atom on the group in R1, R3, R4, R5, R6, R7 or R8 is replaced by one or more of the following: an oxygen atom, -OH, halogen, -NH2, C1-C3 alkyl, C1-C3 carboxyl or salt thereof, or a C1-C3 alkyl group.

[0030] Preferably, R1 is selected from -H, -OH, C1-C5 alkyl, C1-C5 alkoxy, -NH2, -NH-CO-R7-COOH, hydroxyl-substituted C5-C20 oxygen-containing heterocyclic compounds, or -NH-CO-R8; R7 is selected from methyl-substituted C2-C3 alkenyl; and R8 is selected from C1-C3 carboxyl or rare earth metal salt-substituted C1-C3 alkyl-substituted C5-C10 nitrogen-containing heterocyclic compounds.

[0031] In this application, the halogen is fluorine, chlorine, bromine, or iodine. Rare earth metals are scandium (Sc), yttrium (Y), lanthanum (La), cerium (Ce), praseodymium (Pr), neodymium (Nd), promethium (Pm), samarium (Sm), europium (Eu), gadolinium (Gd), terbium (Tb), dysprosium (Dy), holmium (Ho), erbium (Er), thulium (Tm), ytterbium (Yb), and lutetium (Lu). Heterocyclic compounds are organic compounds containing heterocyclic structures in their molecules. In addition to carbon atoms, the atoms constituting the rings contain at least one heteroatom. The heteroatom can be a nitrogen atom, a sulfur atom, an oxygen atom, etc. It can be an alicyclic heterocycle, an aromatic heterocycle, or a fused heterocyclic compound composed of an alicyclic heterocycle, an aromatic heterocycle, and an aromatic ring, or a spirocyclic heterocycle formed by two alicyclic heterocycles, aromatic heterocycles, or fused heterocyclic compounds sharing a single carbon atom.

[0032] Furthermore, R1 is selected from -H, -OH, C1-C5 alkyl, C1-C5 alkoxy, -NH2, -NH-CO-R7-COOH, hydroxyl-substituted C5-C20 oxygen-containing heterocyclic compounds, or -NH-CO-R8; R7 is selected from methyl-substituted C2-C3 alkenyl; R8 is selected from C1-C3 carboxyl or rare earth metal salt-substituted C1-C3 alkyl-substituted C5-C10 nitrogen-containing heterocyclic compounds.

[0033] Furthermore, R1 is selected from -H, -OH, -CH3, -OCH3, -NH2, -NH-CO-C(CH3)=C(CH3)COOH, or

[0034] Furthermore, at least one of A1-A9:

[0035]

[0036]

[0037]

[0038]

[0039]

[0040] n is an integer between 10 and 1000.

[0041] For example, n can take the values ​​10, 20, 28, 30, 50, 56, 60, 110, 200, 220, 300, 500, 1000, or any integer between two of these values. For instance, n in equations A1-A2 and A4-A6 can be approximately 28, 56, 110, or 220. n in equation A3 can be approximately 28 or 56.

[0042] The present invention provides a method for preparing a coupling compound of indocyanine green and multi-arm polyethylene glycol or a pharmaceutically acceptable salt thereof, as described above, comprising a process of coupling multi-arm polyethylene glycol containing coupling reaction functional groups with an indocyanine green derivative.

[0043] Furthermore, the coupling reaction functional groups include amino, mercapto, azide, alkynyl, hydroxyl, carboxylic acid ester group, carbonate group, succinimide group, maleimide group, aldehyde group, dibenzocyclooctynyl, transcyclooctenyl, cyanobenzothiazolyl, aminothiol, tetraazinyl, or sulfonic acid-succinimide ester group. For example, the carboxylic acid ester group can be succinimide carboxylic acid ester. The carbonate group can be succinimide carbonate.

[0044] Furthermore, the indocyanine green derivative comprises one or more of the following: ICG-NHS, ICG-COOH, indocyanine green maleimide, indocyanine green azide, indocyanine green alkynyl, indocyanine green thiol, indocyanine green amino, indocyanine green dibenzocyclooctynyl, indocyanine green transcyclooctenyl, indocyanine green cyanobenzothiazole, indocyanine green aminothiol, indocyanine green tetraazinyl, and indocyanine green sulfonic acid succinimide ester.

[0045] Furthermore, the weight-average molecular weight of the multi-arm polyethylene glycol with the coupling reaction functional group is 5000-40000.

[0046] Furthermore, the weight-average molecular weight of multi-arm polyethylene glycol containing coupling reaction functional groups is 20,000-40,000.

[0047] Furthermore, the preparation method includes the following steps:

[0048] The coupling compound was prepared by amidation reaction of ICG-NHS and mArmPEG-NH2.

[0049] Furthermore, the molar ratio of ICG-NHS to mArmPEG-NH2 is 1–8:1; and / or, the reaction is carried out at room temperature in the dark, with stirring at 500–3000 rpm for 20–40 h; and / or, the reaction is purified by chromatographic separation, and the purified product is collected; and / or, mArmPEG-NH2 includes 4 mArmPEG-NH2 or 8 mArmPEG-NH2; and / or, the amidation reaction further includes a modification step with dimethylmaleic anhydride, FITC-NHS, or gadolinium acid. The synthetic route is shown below. Figure 1 As shown.

[0050] The modification of gadoteric acid can be performed by direct modification with gadoteric acid, or by sequential modification with DOTA-NHS and gadolinium salt. The gadolinium salt can be a conventional salt such as gadolinium chloride or gadolinium sulfate.

[0051] Furthermore, the preparation method includes the following steps: ICG-NHS and 4ArmPEG-NH2 are mixed at a molar ratio of 1:1, stirred at 1000 rpm in the dark at room temperature for 24 h, and after stirring is completed, the eluent is separated by liquid chromatography using water as the eluent, and the eluent is collected to obtain the conjugate.

[0052] The present invention also provides the use of any of the conjugates described above or a pharmaceutically acceptable salt thereof, or a conjugate or a pharmaceutically acceptable salt thereof prepared by any of the preparation methods described above, in the preparation of tumor contrast agents and / or medicaments for the prevention or treatment of tumors.

[0053] Furthermore, the tumor includes primary or metastatic tumors.

[0054] Furthermore, the tumor includes one or more of the following: bladder tumor, bone tumor, brain tumor, breast tumor, colorectal tumor, esophageal tumor, kidney tumor, lung tumor, ovarian tumor, pancreatic tumor, prostate tumor, stomach tumor, and liver tumor.

[0055] As used herein, the term "pharmaceutically acceptable salt" refers to a salt that retains the biological potency of the free acid and free base of a specified compound and has no adverse effects on biology or otherwise. A pharmaceutically acceptable salt is one in which the base groups of the parent compound are converted into salt form. Pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts with base groups such as amine (amino) groups. Examples include salts formed with inorganic acids such as phosphoric acid, sulfuric acid, and hydrochloric acid, or organic acids such as acetic acid, tartaric acid, citric acid, and malic acid, or acidic amino acids such as aspartic acid and glutamic acid, or salts formed by esterification or amide formation with the aforementioned acids followed by formation with an inorganic base, such as sodium, potassium, calcium, aluminum, or ammonium salts. Salts formed with acid groups such as sulfonic acid groups and inorganic bases, such as sodium, potassium, calcium, aluminum, or ammonium salts.

[0056] The technical solution of this invention has the following advantages:

[0057] 1. Studies have found that conjugates formed by indocyanine green or single-arm polyethylene glycol with an indocyanine green group decay rapidly. The present invention provides a conjugate of indocyanine green with multi-arm polyethylene glycol or a pharmaceutically acceptable salt thereof. This conjugate has the structural formula shown in formula (I), comprising a multi-arm polyethylene glycol group and an indocyanine green group attached to at least one arm or branching center of the multi-arm polyethylene glycol group. The multi-arm polyethylene glycol group and the indocyanine green group are connected by a linker. This conjugate exhibits significantly reduced binding affinity to serum proteins and significantly enhanced tumor targeting. It accumulates and remains in tumor tissue, thereby enabling targeted treatment or high-resolution imaging for more accurate tumor localization and diagnosis. Furthermore, it can be metabolized by the liver for even more accurate tumor localization and diagnosis, demonstrating high application value and opening up new methods for the development of tumor imaging and treatment.

[0058] 2. The indocyanine green and multi-arm polyethylene glycol conjugate or its pharmaceutically acceptable salt provided by the present invention, in formula (I), m is an integer between 3 and 8, k is an integer between 0 and m; q is an integer between 0 and 3; and k and q are not simultaneously 0, that is, the multi-arm polyethylene glycol has 3-8 arms, wherein at least one arm or branching center is connected to an indocyanine green group, especially controlling one arm of the multi-arm polyethylene glycol group to be connected to an indocyanine green group (i.e., when k is 1), which can further improve tumor targeting, prolong half-life, and thus better achieve tumor localization and diagnosis.

[0059] 3. The indocyanine green and multi-arm polyethylene glycol conjugates or their pharmaceutically acceptable salts provided by the present invention, the conjugates with the structures shown in formula (II), (III), (IV), (V), (VI) or (VII) can further improve tumor targeting, prolong half-life, and thus better achieve tumor localization and diagnosis, especially the conjugate with the structure shown in formula (II).

[0060] 4. The method for preparing indocyanine green and multi-arm polyethylene glycol conjugates or their pharmaceutically acceptable salts provided by this invention is simple, highly controllable, allows for the design of reaction products as needed, and operates under mild (room temperature) reaction conditions. In particular, by using an amidation reaction between ICG-NHS and mAmPEG-NH2 to obtain the conjugate, different molecular weight mAmPEG-NH2 molecules are used as reactants. After the reaction, the mAmPEG-NH- groups of various molecular weights are bonded to -C=O-, achieving the purpose of mAmPEG modification at different molecular weights. The NHS ester group is highly reactive and readily undergoes transesterification with mAmPEG-NH2, removing one molecule of NHS and attaching mAmPEG-NH- to -C=O-, thus completing the ICG modification process by mAmPEG. This facilitates the preparation of this medical imaging contrast agent. It exhibits high selectivity and yield, and is easily adaptable for industrial production.

[0061] 5. The indocyanine green and multi-arm polyethylene glycol conjugate or its pharmaceutically acceptable salt provided by the present invention, by limiting the molecular weight of the multi-arm polyethylene glycol group to between 5,000 and 40,000, especially to between 20,000 and 40,000, can further improve tumor targeting, prolong the half-life, and thus better achieve tumor localization and diagnosis.

[0062] 6. The use of indocyanine green and multi-arm polyethylene glycol conjugates or pharmaceutically acceptable salts thereof provided by the present invention, which can be used as tumor-specific targeted contrast agents, expected to achieve precise localization of tumor boundaries, and can be used for preoperative, intraoperative or postoperative contrast imaging. Preoperative and postoperative contrast agents are mainly used for examination, and intraoperative contrast agents are used for image navigation, which has the advantages of improving surgical precision and examination accuracy. Attached Figure Description

[0063] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0064] Figure 1 The synthetic route for ICG-mArmPEG;

[0065] Figure 2The UV-Vis absorption spectra of ICG-NHS, ICG-4ArmPEG5K, ICG-4ArmPEG10K, ICG-8ArmPEG10K, ICG-4ArmPEG20K, ICG-8ArmPEG20K and ICG-4ArmPEG40K in Experimental Example 1 are shown.

[0066] Figure 3 The UV-Vis absorption spectrum of ICG4-4ArmPEG40K in Experimental Example 1 is shown below.

[0067] Figure 4 The fluorescence emission spectra (excitation wavelength: 713 nm) of ICG-NHS, ICG-4ArmPEG5K, ICG-4ArmPEG10K, ICG-8ArmPEG10K, ICG-4ArmPEG20K, ICG-8ArmPEG20K and ICG-4ArmPEG40K in Experimental Example 1 are shown.

[0068] Figure 5 The fluorescence emission spectra of ICG-4ArmPEG40K and ICG4-4ArmPEG40K in Experimental Example 1 (excitation wavelength: 713 nm);

[0069] Figure 6 The UV-Vis absorption and fluorescence emission spectra of ICG-4ArmPEG40K-FITC and ICG-4ArmPEG40K-DMA in Experimental Example 1 are shown (excitation wavelength: 713 nm).

[0070] Figure 7 The UV-Vis absorption spectrum and fluorescence emission spectrum (excitation wavelength: 713 nm) of ICG-4ArmPEG40K-DOTA-Gd in Experimental Example 1 are shown.

[0071] Figure 8 The results of ninhydrin detection for ICG-4ArmPEG40K and ICG-4ArmPEG40K-DMA in Experimental Example 1 are shown.

[0072] Figure 9 This is the ICG standard curve established in Experiment Example 1.

[0073] Figure 10 The image shows the agarose gel electrophoresis results from Experiment Example 2.

[0074] Figure 11In vivo imaging of tumor-bearing mice at different time points after tail vein injection of three types of ICG-mArmPEG (ICG-4ArmPEG20K, ICG-8ArmPEG20K, and ICG-4ArmPEG40K). BF is bright field image, and the rest are fluorescence images at various time points (808nm exciter; 900nm long-pass filter).

[0075] Figure 12 The graphs show the changes in fluorescence intensity of tumors and skin tissues over time, as well as the changes in fluorescence contrast (tumor fluorescence intensity / background tissue fluorescence intensity) over time, after tail vein injection of three types of ICG-mArmPEG (ICG-4ArmPEG20K, ICG-8ArmPEG20K and ICG-4ArmPEG40K) in tumor-bearing mice.

[0076] Figure 13 Two weeks (14 days) after tail vein injection of three types of ICG-mArmPEG (ICG-4ArmPEG20K, ICG-8ArmPEG20K and ICG-4ArmPEG40K), in vitro fluorescence imaging and fluorescence intensity analysis of various major organs or tissues (Heart; Liver; Spleen; Lung; Kidney; Tumor; Brain; Muscle).

[0077] Figure 14 In vivo fluorescence imaging of mice with orthotopic gliomas 48 hours after tail vein injection of ICG-4ArmPEG40K. Fluorescence is mainly concentrated in the brain region where the orthotopic glioma is located.

[0078] Figure 15 Fluorescence imaging of the brain of mice with orthotopic gliomas 48 hours after tail vein injection of ICG-4ArmPEG40K.

[0079] Figure 16 Fluorescence images of ICG4-4ArmPEG40K injected via the tail vein, curves showing the changes in fluorescence intensity of the tumor and skin tissue in tumor-bearing mice over time, and curves showing the changes in fluorescence contrast (tumor fluorescence intensity / background tissue fluorescence intensity) over time.

[0080] Figure 17 Fluorescence images of ICG-4ArmPEG40K-FITC injected via the tail vein, curves showing the changes in fluorescence intensity of the tumor and skin tissue over time in tumor-bearing mice, and curves showing the changes in fluorescence contrast (tumor fluorescence intensity / background tissue fluorescence intensity) over time.

[0081] Figure 18 The graph shows the changes in in vivo fluorescence imaging, fluorescence intensity and fluorescence contrast (tumor fluorescence intensity / background tissue fluorescence intensity) of tumor-bearing mice at different time points after tail vein injection of ICG-4ArmPEG40K-DOTA-Gd.

[0082] Figure 19 The image shows magnetic resonance (MRI) images of mice with orthotopic gliomas 48 hours after intravenous injection of gadopentetate and ICG-4ArmPEG40K-DOTA-Gd, respectively. Detailed Implementation

[0083] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0084] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0085] 1. ICG-NHS, full Chinese name is indocyanine green succinimide ester, CAS number is: 1622335-40-3.

[0086] 2. 4ArmPEG-NH2, whose full Chinese name is four-armed polyethylene glycol amino, has the following structural formula:

[0087]

[0088] 3. 8ArmPEG-NH2, whose full Chinese name is eight-arm polyethylene glycol amino, has the following structural formula:

[0089]

[0090] Where m is 8 and R is -NH2.

[0091] 4. 2ArmPEG-NH2, whose full Chinese name is two-arm polyethylene glycol amino, has the following structural formula:

[0092]

[0093] Example 1

[0094] This embodiment provides a method for preparing a coupling compound of indocyanine green and multi-arm polyethylene glycol, the process route of which is shown below. Figure 1 As shown, it includes the following steps:

[0095] ICG-NHS was weighed and added to dimethyl sulfoxide (DMSO) to prepare a 0.24 mmol / L ICG-NHS solution; 4ArmPEG-NH2 with a weight average molecular weight of 40,000 was weighed and added to DMSO to prepare a 0.24 mmol / L 4ArmPEG-NH2 solution; the two solutions were mixed in equal volumes and stirred at 1000 rpm in the dark at room temperature for 24 h. After stirring, the solution was eluted by liquid chromatography using water as the eluent, and the eluent was collected to obtain a medical imaging contrast agent, designated ICG-4ArmPEG40K, which was stored at -20℃. Alternatively, the collected eluent could be freeze-dried to obtain a lyophilized powder.

[0096] The structural formula of the prepared conjugate ICG-4ArmPEG40K is as follows:

[0097]

[0098] Examples 2-4

[0099] Examples 2-4 provide a series of methods for preparing coupling compounds. The only difference from Example 1 is that 4ArmPEG-NH2 with a weight average molecular weight of 5000, 10000 and 20000 respectively is used instead of 4ArmPEG-NH2 with a weight average molecular weight of 40000 in Example 1. The products obtained are denoted as ICG-4ArmPEG5K, ICG-4ArmPEG10K and ICG-4ArmPEG20K respectively.

[0100] Examples 5-6

[0101] Examples 5 and 6 provide two methods for preparing the conjugates. The only difference from Example 1 is that 8ArmPEG-NH2 with a weight average molecular weight of 10,000 and 20,000 respectively is used instead of 4ArmPEG-NH2 with a weight average molecular weight of 40,000 in Example 1. The products obtained are denoted as ICG-8ArmPEG10K and ICG-8ArmPEG20K respectively.

[0102] The structural formulas of the coupling compounds ICG-8ArmPEG10K and ICG-8ArmPEG20K are as follows:

[0103]

[0104] Example 7

[0105] Example 7 provides a method for preparing a coupling compound, which differs from Example 1 only in that 2ArmPEG-NH2 with a weight average molecular weight of 40,000 in the same molar amount is used instead of 4ArmPEG-NH2 with a weight average molecular weight of 40,000 in Example 1. The resulting coupling compound is denoted as ICG-2ArmPEG40K.

[0106] The structural formula of the coupling compound ICG-2ArmPEG40K is as follows:

[0107]

[0108] Example 8

[0109] This embodiment provides a method for preparing a conjugate. The only difference from Example 1 is that ICG-NHS is weighed and added to dimethyl sulfoxide (DMSO) to prepare a 1 mmol / L ICG-NHS solution. The 1 mmol / L ICG-NHS solution is used instead of the 0.24 mmol / L ICG-NHS solution and mixed with the 0.24 mmol / L 4ArmPEG-NH2 solution in the same volume. The reaction, reaction conditions and subsequent treatment are the same as in Example 1. The obtained product is denoted as ICG4-4ArmPEG40K.

[0110] The structural formula of the coupling compound ICG4-4ArmPEG40K is as follows:

[0111]

[0112] Example 9

[0113] This embodiment provides a method for preparing a coupling compound. Compared with Example 1, this method allows for the modification of the R1 group of the original coupling compound through a chemical reaction after the coupling compound is formed in Example 1, thereby obtaining coupling compounds containing different R1 groups. The method is as follows: The lyophilized powder obtained by freeze-drying the coupling compound ICG-4ArmPEG40K obtained in Example 1 is dissolved in DMSO to form a 0.24 mmol / L ICG-4ArmPEG40K solution. Dimethylmaleic anhydride (DMA, CAS: 766-39-2) is dissolved in DMSO to form a 0.72 mmol / L solution, and mixed with an equal volume of the ICG-4ArmPEG40K solution. The mixture is stirred at 1000 rpm at room temperature in the dark for 24 h. After stirring, the mixture is separated by liquid chromatography using water as the eluent, and the eluent is collected to obtain the dimethylmaleic anhydride-modified coupling compound, denoted as ICG-4ArmPEG40K-DMA, which is stored at -20°C.

[0114] The structural formula of the conjugate ICG-4ArmPEG40K-DMA is as follows:

[0115]

[0116] Example 10

[0117] This embodiment provides a method for preparing a conjugate through R-group modification. ICG-NHS is weighed and added to DMSO to prepare a 0.24 mmol / L ICG-NHS solution; 4ArmPEG-NH2 is weighed and added to DMSO to prepare a 0.24 mmol / L 4ArmPEG-NH2 solution; the two solutions are mixed in equal volumes and stirred at 1000 rpm at room temperature in the dark for 24 h.

[0118] After stirring, FITC-NHS (5-carboxyfluorescein succinimide ester, CAS: 92557-80-7) was weighed and added to DMSO to prepare a 0.72 mmol / L FITC-NHS solution. The FITC-NHS solution was slowly added dropwise to the reaction solution, with the volume of FITC-NHS solution added being the same as the volume of 4ArmPEG-NH2 solution added in the reaction. The mixture was stirred at 1000 rpm at room temperature in the dark for 24 h. After stirring, the mixture was eluted by liquid chromatography using water as the eluent, and the eluent was collected to obtain the medical imaging contrast agent ICG-4ArmPEG40K-FITC, which was stored at -20℃. Alternatively, the collected eluent could be freeze-dried to obtain a lyophilized powder.

[0119] The structural formula of the conjugate ICG-4ArmPEG40K-FITC is as follows:

[0120]

[0121] Example 11

[0122] This embodiment provides a method for preparing gadolinium salt of a coupling compound. Compared with Example 1, in this method, after the coupling compound is formed in Example 1, the R1 group of the original coupling compound is modified by a modified ligand compound, and then the gadolinium salt of the coupling compound with NMR contrast properties is obtained through coordination complexation of gadolinium-ligand compound.

[0123] The specific method is as follows: The lyophilized powder obtained by freeze-drying the coupling compound ICG-4ArmPEG40K obtained in Example 1 was dissolved in water to form a 0.24 mmol / L solution. The NHS-modified ligand compound DOTA (DOTA-NHS, CAS: 170908-81-3) was dissolved in DMSO to form a 3.84 mmol / L solution, and mixed with an equal volume of the ICG-4ArmPEG40K solution. The mixture was stirred at 1000 rpm in the dark for 24 hours. After stirring, the solution was dialyzed in water using a 3500 Da dialysis bag, and then freeze-dried to obtain the lyophilized powder of ICG-4ArmPEG40K-DOTA. The ICG-4ArmPEG40K-DOTA powder was dissolved in water to form a 0.24 mmol / L solution. Gadolinium chloride (GdCl3, CAS: 10138-52-0) was dissolved in water to form a 4.8 mmol / L solution, and the pH of the solution was adjusted to 5-6. An equal volume of ICG-4ArmPEG40K-DOTA solution was mixed with gadolinium chloride solution and stirred at 1000 rpm in the dark for 24 hours. After stirring, the solution was dialyzed in water using a 3500 Da dialysis bag to obtain the medical imaging contrast agent ICG-4ArmPEG40K-DOTA-Gd, which was stored at -20°C. Alternatively, the collected eluent could be freeze-dried to obtain a lyophilized powder.

[0124] The structural formula of ICG-4ArmPEG40K-DOTA-Gd is as follows:

[0125]

[0126] Characterization in Experiment Example 1

[0127] 1. The spectroscopic properties (including absorption and emission spectroscopic properties) of the conjugates prepared in the above embodiments were tested. The specific procedures are as follows:

[0128] (1) ICG powder and lyophilized coupling powders of ICG-4ArmPEG5K, ICG-4ArmPEG10K, ICG-8ArmPEG10K, ICG-4ArmPEG20K, ICG-8ArmPEG20K, and ICG-4ArmPEG40K were respectively added to water to prepare solutions with a concentration of 38 μmol / L. The UV-Vis absorption spectra of ICG, ICG-4ArmPEG5K, ICG-4ArmPEG10K, ICG-8ArmPEG10K, ICG-4ArmPEG20K, ICG-8ArmPEG20K, and ICG-4ArmPEG40K were measured using a UV-2550 spectrometer in the spectral range of 500 nm-900 nm. See [link to relevant documentation]. Figure 2 As shown. By Figure 2It is known that ICG-4ArmPEG5K, ICG-4ArmPEG10K, ICG-8ArmPEG10K, ICG-4ArmPEG20K, ICG-8ArmPEG20K and ICG-4ArmPEG40K have spectral absorption capabilities that are basically consistent with those of ICG.

[0129] (2) ICG4-4ArmPEG40K lyophilized powder was added to water to prepare solutions with a concentration of 15 μmol / L. The UV-Vis absorption spectrum of ICG4-4ArmPEG40K was measured using a UV-2550 spectrometer in the spectral range of 500 nm-900 nm. Figure 3 As shown in the red spectrum, in aqueous solution, the main absorption peak of ICG4-4ArmPEG40K blue-shifts from 790 nm to 700 nm, indicating that ICG4-4ArmPEG40K forms H aggregates. ICG is hydrophobic, and PEG is hydrophilic; therefore, ICG molecules tend to aggregate in aqueous solution. Thus, when a PEG molecule couples with more ICG, ICG4-4ArmPEG40K forms H aggregates. The aqueous solution of ICG4-4ArmPEG40K was diluted with an equal volume of DMSO, and the UV-Vis absorption spectrum of ICG4-4ArmPEG40K was retested. Figure 3 As shown in the black spectrum, the main absorption peak of ICG4-4ArmPEG40K recovered from 700nm to 790nm, indicating that the H aggregates of ICG can be redispersed in an equal volume of DMSO.

[0130] (3) ICG powder and lyophilized conjugates of ICG-4ArmPEG5K, ICG-4ArmPEG10K, ICG-8ArmPEG10K, ICG-4ArmPEG20K, ICG-8ArmPEG20K, and ICG-4ArmPEG40K were respectively added to water to prepare solutions with a concentration of 38 μmol / L. A Spectrofluorometer FS5 was used with an excitation wavelength of 713 nm to measure the fluorescence emission spectra of ICG, ICG-4ArmPEG5K, ICG-4ArmPEG10K, ICG-8ArmPEG10K, ICG-4ArmPEG20K, ICG-8ArmPEG20K, and ICG-4ArmPEG40K. The test results are as follows: Figure 4 As shown. By Figure 4It is known that ICG-4ArmPEG5K, ICG-4ArmPEG10K, ICG-8ArmPEG10K, ICG-4ArmPEG20K, ICG-8ArmPEG20K and ICG-4ArmPEG40K have good near-infrared fluorescence properties comparable to ICG.

[0131] (4) ICG4-4ArmPEG40K lyophilized powder and ICG-4ArmPEG40K lyophilized powder were respectively added to water to prepare solutions with a concentration of 15 μmol / L. The fluorescence emission spectra of ICG-4ArmPEG40K and ICG4-4ArmPEG40K were measured using a Spectrofluorometer FS5 with an excitation wavelength of 713 nm. The results are as follows: Figure 5 As shown, the fluorescence signal of ICG4-4ArmPEG40K is significantly weaker than that of ICG-4ArmPEG40K. This is because the four ICG molecules on the ICG4-4ArmPEG40K molecule have a more pronounced aggregation effect, which leads to fluorescence aggregation-induced quenching.

[0132] (5) ICG-4ArmPEG40K-DMA and ICG-4ArmPEG40K-FITC lyophilized powders were respectively added to water to prepare solutions with a concentration of 13 μmol / L. The UV-Vis absorption spectra of ICG-4ArmPEG40K-FITC were measured in the spectral range of 350 nm-900 nm, and the UV-Vis absorption spectra of ICG-4ArmPEG40K-DMA were measured in the spectral range of 500 nm-900 nm using a UV-2550 spectrometer. See [link to relevant documentation] Figure 6 As shown. By Figure 6 It can be seen that ICG-4ArmPEG40K-DMA and ICG-4ArmPEG40K-FITC have spectral absorption capabilities basically consistent with ICG, while ICG-4ArmPEG40K-FITC exhibits spectral absorption similar to FITC at 490 nm. Using a Spectrofluorometer FS5 with an excitation wavelength of 713 nm, the fluorescence emission spectra of ICG-4ArmPEG40K-DMA and ICG-4ArmPEG40K-FITC were measured. The test results are shown in the figure. Figure 6 It can be seen that ICG-4ArmPEG40K-DMA and ICG-4ArmPEG40K-FITC have good near-infrared fluorescence properties comparable to ICG.

[0133] (6) A 22 μmol / L solution was prepared by adding ICG-4ArmPEG40K-DOTA-Gd lyophilized powder to water. The UV-Vis absorption spectrum of ICG-4ArmPEG40K-DOTA-Gd was measured using a UV-2550 spectrometer in the spectral range of 500 nm-900 nm. See [link to relevant documentation] Figure 7 As shown. By Figure 7 It can be seen that ICG-4ArmPEG40K-DOTA-Gd has a spectral absorption capability that is basically the same as that of ICG. Using a Spectrofluorometer FS5 fluorescence spectrometer with an excitation light set to 713 nm, the fluorescence emission spectrum of ICG-4ArmPEG40K-DOTA-Gd was measured. The results are shown in the figure; ICG-4ArmPEG40K-DOTA-Gd still exhibits near-infrared fluorescence properties.

[0134] 2. Ninhydrin detection

[0135] The R group of the coupling compound ICG-4ArmPEG40K is -NH2. Modification of the R group reduces the concentration of -NH2. This experiment uses ninhydrin, which reacts with -NH2 and produces a color change, to verify whether the R group of the coupling compound has been successfully modified.

[0136] ICG-4ArmPEG40K lyophilized powder and ICG-4ArmPEG40K-DMA lyophilized powder were dissolved in water to prepare ICG-4ArmPEG40K solution and ICG-4ArmPEG40K-DMA solution with concentrations of 0.5 mmol / L, respectively. 20 μL of each solution was mixed with 10 μL of 200 mg / mL ninhydrin solution and 10 μL of phosphate buffer, respectively, and incubated at 90 °C for 15 minutes. The color changes of the solutions were then observed.

[0137] 20 μL of deionized water, 10 μL of 200 mg / mL ninhydrin solution, and 10 μL of phosphate buffer were mixed and incubated at 90 °C for 15 minutes as a negative control for this experiment.

[0138] 20 μL of an aqueous solution containing 20 μg / mL leucine, 10 μL of a 200 mg / mL ninhydrin solution, and 10 μL of phosphate buffer were mixed and incubated at 90°C for 15 minutes as a positive control for this experiment.

[0139] Experimental results are as follows Figure 8As shown, after co-incubation of deionized water and ninhydrin, the solution remained colorless; after co-incubation of leucine and ninhydrin, the solution turned purple; after reaction of ICG-4ArmPEG40K-DMA with ninhydrin, the solution was green; while ICG-4ArmPEG40K remained blue. This indicates that ICG-4ArmPEG40K contains -NH2, while the R-group-modified ICG-4ArmPEG40K-DMA does not. This demonstrates that the R-group of ICG-4ArmPEG40K-DMA was successfully modified.

[0140] 3. Establishment of the ICG standard curve

[0141] To ensure consistent ICG concentration in ICG-mArmPEG solutions during animal experiments, the UV absorption of ICG solutions at different concentrations was measured using UV spectrophotometry to fit a standard curve.

[0142] ICG powder was weighed and added to a 1:1 mixture of DMSO and water to prepare a 0.05 mmol / L solution. This solution was then diluted 2, 4, 8, 16, and 32 times to obtain solutions of 0.025 mmol / L, 0.0125 mmol / L, 0.00625 mmol / L, 0.00313 mmol / L, and 0.00156 mmol / L, respectively. The UV-Vis absorption spectra were then measured using a UV-2550 spectrometer in the spectral range of 500 nm–900 nm. The maximum absorbance was then recorded at 789 nm, and a standard curve for ICG concentration was obtained by fitting the data. (See figure). Figure 9 The concentration of ICG is calculated using the formula: Concentration (mmol / L) = (Abs + 0.008) / 46.6, where Abs is the absorbance value. This standard curve shows good linearity. The concentration of ICG in the ICG-mArmPEG injection can be obtained by measuring the absorbance of the ICG-mArmPEG injection solution using ultraviolet spectrophotometry and then substituting it into the standard curve.

[0143] Experiment Example 2

[0144] The serum protein binding of the prepared ICG-mArmPEG was tested using agarose gel electrophoresis. The specific method is as follows:

[0145] Experimental groups (including ICG+FBS+CBB group and ICG-4ArmPEG40K+FBS+CBB group): ICG-NHS powder or ICG-4ArmPEG40K lyophilized powder was added to 20 μL of phosphate-buffered saline (PBS) supplemented with 10% (v / v) fetal bovine serum (FBS) to prepare 0.02 mM test samples, which were incubated at 37°C for 30 minutes. 5 μL of Coomassie Brilliant Blue (CBB) 250 staining solution (containing 10% (v / v) CBB and 90% (v / v) PBS) was added for staining.

[0146] Control groups (including ICG group and ICG-4ArmPEG40K group): ICG-NHS or ICG-4ArmPEG40K lyophilized powder was added to 20 μL of FBS-free PBS buffer to prepare 0.02 mmol / L test samples, and incubated at 37°C for 30 minutes. 5 μL of CBB 250 staining solution was then added for staining.

[0147] Staining control group (CBB group): Incubate 20 μL of FBS-free phosphate buffer at 37°C for 30 minutes. Add 5 μL of CBB 250 staining solution and mix for staining.

[0148] Negative control group (FBS+CBB group): 20 μL of phosphate buffer supplemented with 10% (v / v) fetal bovine serum (FBS) was incubated at 37°C for 30 minutes. 5 μL of CBB 250 staining solution was then added for staining.

[0149] All these samples were analyzed using 2% agarose gel electrophoresis. Fluorescence images were captured at 730 nm / 825 nm using an FOBI imaging system.

[0150] Depend on Figure 10 It was observed that in agarose gel electrophoresis, after co-incubation with FBS, ICG-NHS migrated entirely along the direction of FBS, while the migration of ICG-4ArmPEG40K was largely unaffected by FBS. Free ICG-NHS could bind strongly to serum proteins; in contrast, the binding ability of PEG-modified ICG-mArmPEG to serum proteins was significantly reduced.

[0151] Experimental Example 3

[0152] The tumor imaging effect of ICG-mArmPEG was tested, and the distribution and metabolism of this fluorescent contrast agent in vivo were observed using the FOBI in vivo imaging system. The specific steps are as follows:

[0153] Weigh out the lyophilized powders of ICG-4ArmPEG20K, ICG-8ArmPEG20K and ICG-4ArmPEG40K respectively, add water to prepare ICG-4ArmPEG20K solution, ICG-8ArmPEG20K solution and ICG-4ArmPEG40K solution with an ICG concentration of 0.02 mmol / L.

[0154] Nine Balb / c 4T1 tumor-bearing mice (with hair removed from the imaging area) were randomly divided into three groups of three. Mice were anesthetized using the SWD animal anesthesia system. The three groups of mice were injected intravenously with ICG-4ArmPEG20K solution, ICG-8ArmPEG20K solution, and ICG-4ArmPEG40K solution, respectively, at a volume of 125 μL per mouse. Fluorescence imaging was then performed on the mice at different time points. Imaging conditions: The tumor-bearing mice were photographed using a FOBI near-infrared camera with an 808 nm laser excitation and a 900 nm long-pass filter (FELH0900).

[0155] The results are as follows Figure 11 As shown, after tail vein injection of three types of ICG-mArmPEG (ICG-4ArmPEG20K, ICG-8ArmPEG20K, and ICG-4ArmPEG40K), ICG-mArmPEG was observed to mainly accumulate in the liver tissue area at 2 minutes. Twelve hours after injection, the fluorescence signal intensity in the liver gradually decreased, while the fluorescence intensity in the tumor area gradually increased, reaching a peak at 48 hours, and then gradually decreased. At 14 days, the tumor area still showed detectable fluorescence, while fluorescence signals in other tissue areas were completely undetectable. This indicates that ICG-mArmPEG can specifically accumulate and remain at the tumor site for a long time, enabling it to target the tumor and achieve fluorescence imaging at the tumor site.

[0156] Following tail vein injection of three types of ICG-mArmPEG (ICG-4ArmPEG20K, ICG-8ArmPEG20K, and ICG-4ArmPEG40K), the changes in fluorescence intensity over time in tumors and surrounding normal skin tissue were analyzed. The fluorescence intensity ratio and retention rate of tumor aggregates for each mouse were calculated using the following formulas: Fluorescence intensity ratio = Fluorescence intensity of tumor portion / Fluorescence intensity of skin tissue; the average fluorescence intensity ratio and SD were calculated. The retention rate of tumor aggregates = Fluorescence intensity of tumor portion at each time point / Fluorescence intensity of tumor portion at the highest time point; the average retention rate and SD were calculated.

[0157] The results are as follows Figure 12As shown in Tables 1-4, after tail vein injection of three types of ICG-mArmPEG (ICG-4ArmPEG20K, ICG-8ArmPEG20K, and ICG-4ArmPEG40K), the fluorescence of the tumor site in mice gradually increased, reaching its peak at 48 hours post-injection. Furthermore, during the 14-day imaging process, the fluorescence intensity of the tumor site remained consistently high. In addition, comparing the fluorescence intensity of the tumor and the surrounding normal skin tissue revealed that, for most of the 14-day imaging process, the fluorescence intensity of the tumor area remained more than five times that of the surrounding skin. This indicates that ICG-mArmPEG can not only illuminate the tumor area but also distinguish the boundary between the tumor site and the surrounding normal tissue. Unexpectedly, it was found that ICG-4ArmPEG20K and ICG-4ArmPEG40K, compared to ICG-8ArmPEG20K, not only demonstrated a more significant effect in distinguishing the boundary between the tumor site and the surrounding normal tissue but also exhibited a higher retention rate of fluorescence intensity and a significantly prolonged half-life, especially ICG-4ArmPEG40K.

[0158] Table 1. Fluorescence intensity at tumor sites (mean ± SD)

[0159] time ICG-4ArmPEG20K ICG-8ArmPEG20K ICG-4ArmPEG40K 1d 31608±1464 16910±2809 39362±4247 2d 33659±3723 15594±2576 40750±2666 4d 28547±2068 11818±1852 36222±2213 7d 23196±5164 7386±1563 33509±6057 14d 14516±4282 3396±539 17596±165

[0160] Table 2. Fluorescence intensity of skin tissue (mean ± SD)

[0161]

[0162]

[0163] Table 3. Fluorescence intensity ratio between tumor and skin tissue (mean ± SD)

[0164] time ICG-4ArmPEG20K ICG-8ArmPEG20K ICG-4ArmPEG40K 1d 4.38±0.46 4.91±1.16 5.05±0.49 2d 6.16±0.92 5.83±1.44 6.58±1.29 4d 5.39±1.00 5.34±1.05 6.37±0.92 7d 6.66±2.01 5.47±1.52 9.00±1.87 14d 5.04±1.74 3.92±0.40 8.58±0.42

[0165] Table 4. Retention rate of fluorescence intensity at tumor sites (mean ± SD)

[0166] time ICG-4ArmPEG20K ICG-8ArmPEG20K ICG-4ArmPEG40K 2d 99％±3.1％ 92％±2.3％ 99％±1.3％ 4d 85％±4.5％ 70％±2.0％ 88％±1.8％ 7d 68％±8.4％ 44％±4.5％ 81％±8.6％ 11d 48％±11％ 25％±3.9％ 54％±3.7％ 14d 43％±8.5％ 20％±2.4％ 45％±2.9％

[0167] Experiment Example 4

[0168] Mice from Experiment 3 that were injected intravenously with three types of ICG-mArmPEG (ICG-4ArmPEG20K, ICG-8ArmPEG20K, and ICG-4ArmPEG40K) for 14 days were dissected. Organ tissues including heart, liver, spleen, lung, kidney, tumor, brain, and muscle were collected for near-infrared fluorescence imaging in vitro. (See also...) Figure 13 .

[0169] The results are as follows Figure 13As shown, organ fluorescence imaging 14 days after tail vein injection of three types of ICG-mArmPEG (ICG-4ArmPEG20K, ICG-8ArmPEG20K and ICG-4ArmPEG40K) reveals that tumors and the liver show significant fluorescence signals, while other organs and tissues show lower fluorescence signals. This further indicates that ICG-4ArmPEG can selectively illuminate tumor sites and maintain a strong fluorescence signal in the tumor area for a relatively long period of time.

[0170] Experimental Example 5

[0171] To further test the tumor imaging effect of ICG-mArmPEG and evaluate its feasibility in tumor resection surgery, we tested its distribution in mouse orthotopic gliomas.

[0172] Weigh out the lyophilized ICG-4ArmPEG40K powder and add water to prepare an ICG-4ArmPEG40K solution with an ICG concentration of 0.02 mmol / L.

[0173] Three mice with orthotopic glioma (nude mouse C6 glioma model) were injected with ICG-4ArmPEG40K solution via tail vein, with an injection volume of 125 μL per mouse. Forty-eight hours after injection, the mice were anesthetized by the animal anesthesia system SWD, and fluorescence imaging was performed using the FOBI in vivo imaging system.

[0174] The results are as follows Figure 14 As shown, ICG-4ArmPEG40K mainly accumulates in brain regions. This result demonstrates that ICG-4ArmPEG40K has a targeting effect on orthotopic gliomas. Although the tumor is difficult to observe with the naked eye, the tumor-bearing site can still be identified using ICG-4ArmPEG40K under fluorescence imaging.

[0175] Further anatomical imaging of the brain, such as Figure 15 As shown, the tumor region of the brain tissue exhibits strong fluorescence, while the surrounding normal tissue region shows no significant fluorescence, with a contrast ratio (the multiple of fluorescence intensity between the tumor region and the normal tissue region) of 3.2. This result demonstrates the accumulation of ICG-4ArmPEG40K in the tumor region, showcasing its potential application in fluorescence-guided tumor resection surgery.

[0176] Experimental Example 6

[0177] The tumor imaging effect of PEG conjugates coupled with different numbers of ICG was tested. ICG4-4ArmPEG40K lyophilized powder was weighed and diluted with water to prepare an ICG4-4ArmPEG40K solution with an ICG concentration of 0.02 mmol / L. Three Balb / c 4T1 tumor-bearing mice (with hair removed from the imaging area) were anesthetized using the SWD animal anesthesia system. The mice were then injected via tail vein with an ICG4-4ArmPEG40K solution containing 0.02 mmol / L ICG at a volume of 125 μL per mouse. Fluorescence imaging was then performed on the mice at different time points. The imaging conditions were the same as in Experiment 3.

[0178] The results are as follows Figure 16 As shown in Table 5, within two minutes of ICG4-4ArmPEG40K injection, the fluorescence signal was mainly concentrated in the liver region. The fluorescence signal in the tumor region was interfered with by the strong fluorescence signal in the liver for a short period of time. From 12 to 48 hours, the tumor site could be distinguished by fluorescence, but the fluorescence intensity in the tumor region decreased over time. One week later, the fluorescence in the tumor region was weaker. Compared with the mice injected with ICG-4ArmPEG40K in Experiment 3, the fluorescence signal of ICG4-4ArmPEG40K coupled with 4 ICGs was significantly weaker. This may be because the rapid liver metabolism of ICG4-4ArmPEG40K and the fluorescence aggregation-induced quenching effect of ICG4-4ArmPEG40K result in weaker fluorescence than that of ICG-4ArmPEG40K.

[0179] Table 5. Test results of ICG4-4ArmPEG40K (mean ± SD)

[0180]

[0181]

[0182] Experimental Example 7

[0183] The tumor contrast enhancement effect of ICG-4ArmPEG40K-FITC was tested, and the distribution and metabolism of this fluorescent contrast agent in vivo were observed using the FOBI in vivo imaging system. The specific steps are as follows:

[0184] Weigh out the lyophilized ICG-4ArmPEG40K-FITC powder and add water to prepare an ICG-4ArmPEG40K-FITC solution with an ICG concentration of 0.02 mmol / L.

[0185] Three Balb / c 4T1 tumor-bearing mice (with hair removed from the imaging area) were anesthetized using the SWD animal anesthesia system. Each mouse received a 125 μL injection of ICG-4ArmPEG40K-FITC solution via the tail vein. Fluorescence imaging was then performed on the mice at different time points. Imaging conditions: The tumor-bearing mice were photographed using an FOBI near-infrared camera with an 808 nm laser excitation and a 900 nm long-pass filter (FELH0900).

[0186] The results are as follows Figure 17 As shown, after tail vein injection of ICG-4ArmPEG40K-FITC, the fluorescence of the tumor site in mice gradually increased, reaching its peak at 12 hours post-injection. Furthermore, the fluorescence intensity at the tumor site remained high throughout subsequent imaging. After stabilization, the fluorescence intensity of the tumor area remained more than four times that of the surrounding skin.

[0187] Experimental Example 8

[0188] The fluorescence imaging effect of the gadolinium-containing contrast agent ICG-4ArmPEG40K-DOTA-Gd on tumors was tested. A 0.02 mmol / L solution of lyophilized ICG-4ArmPEG40K-DOTA-Gd powder was prepared by dissolving water in water. Three Balb / c 4T1 tumor-bearing mice (with hair removed from the imaging area) were anesthetized using the SWD animal anesthesia system, and 125 μL of the prepared ICG-4ArmPEG40K-DOTA-Gd solution was injected via the tail vein. Fluorescence imaging was performed on the mice at different time points. The imaging conditions were the same as in Experiment 3.

[0189] The results are as follows Figure 18 As shown, similar to ICG-4ArmPEG40K without DOTA-Gd modification, the fluorescence signal was mainly concentrated in the liver area within two minutes of injection, and then reached a peak in the tumor area of ​​mice within one to two days, followed by a slow decrease. Moreover, the fluorescence contrast between the tumor and the surrounding normal tissue remained above 4 for 1-14 days.

[0190] Experimental Example 9

[0191] The effect of gadolinium-containing contrast agent ICG-4ArmPEG40K-DOTA-Gd on MRI imaging of orthotopic gliomas in mice was tested. The specific steps were as follows: Gadolinium (DOTA-Gd, CAS: 86050-77-3) was dissolved in physiological saline to prepare a gadolinium-containing solution of 0.3 mmol / L. ICG-4ArmPEG40K-DOTA-Gd lyophilized powder was also dissolved in water to prepare another ICG-4ArmPEG40K-DOTA-Gd solution containing 0.3 mmol / L. Two mice with orthotopic gliomas (nude mouse C6 glioma model) were injected with 125 μL of gadolinium-containing solution and ICG-4ArmPEG40K-DOTA-Gd solution, respectively, via tail vein. MRI imaging was performed on the mice 48 hours after injection.

[0192] The results are as follows Figure 19 As shown, the arrows indicate the tumor location. Compared to gadopentetate, ICG-4ArmPEG40K-DOTA-Gd produces clearer tumor boundaries, indicating better tumor imaging performance. ICG-4ArmPEG40K-DOTA-Gd can be used for MRI imaging to determine the location and size of tumors.

[0193] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A conjugate of indocyanine green and a multi-arm polyethylene glycol or a pharmaceutically acceptable salt thereof, characterized by, The coupling has the structural formula shown in formula (I): Formula (I); Where M is the branching center of multi-arm polyethylene glycol, m is an integer between 3 and 8, k is an integer between 0 and m; q is an integer between 0 and 3; and k and q are not both 0. X is ; R1 may be the same or different, and mk R1s are independently selected from -H, -OH, -CH3, -OCH3, -NH2, -NH-CO-C(CH3)=C(CH3)COOH, , or ; PEG stands for polyethylene glycol group; L1 may be the same or different, where L1 is the linker group connecting the polyethylene glycol unit to the X group; L2 may be the same or different, where L2 is the linker group from which M is attached to the X group.

2. The conjugate according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, M is selected from , , , , or , where m is an integer between 3 and 8.

3. The conjugate according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, PEG has the following structure: where n is the number of repeating units; Optional, n is an integer between 1 and 1000.

4. The conjugate or pharmaceutically acceptable salt thereof according to claim 1, characterized by, k is 1.

5. The conjugate or pharmaceutically acceptable salt thereof according to claim 1, characterized by, The coupling has a structural formula as shown in formula (II), formula (III), formula (IV), formula (V), formula (VI), or formula (VII): Formula (II); Formula (III); Formula (IV); Formula (V); Formula (VI); Equation (VII); In equation (II), m is an integer between 3 and 8; in equations (II), (III), (IV), (V), (VI), or (VII), n1 is an integer between 10 and 1000; in equations (II), (III), (IV), and (V), n2 is an integer between 1 and 1000; R2 is independently selected from R1 or -L1-X, where R1, L1, and X are as defined in claim 1.

6. The conjugate according to claim 1 or 5, or a pharmaceutically acceptable salt thereof, characterized in that, L1 or L2 is selected from -AB-, where A is selected from one of the following groups: -CO-NH-, -NH-CO-NH-, -CO-NH-CO-, -O-, -S-, -SS-, azido-alkynyl cycloaddition linker, tetrazine-trans-cyclooctene cycloaddition linker, maleimide-mercapto-addition linker, azido-dibenzocyclooctynyl cycloaddition linker, or cyanobenzothiazolyl-aminothiol click reaction linker; B is selected from one of the following groups: -(CH2) x -、-O(CH2) y -、-CONH-(CH2) z -、-CONH-(CH2) j -NHC(O)O-, -CO-NH-(CH2) P -NH-CO-, -CONH-(CH2) i -C(O)-, -CONH-(CH2) u -O-, where x, y, z, j, p, i, u are independent integers selected from 1 to 10; L1 or L2 is selected from -(CH2) x -NH-CO-, -CO-NH-(CH2) P -NH-CO-、 , , , , ; where x and p are independent integers selected from 1 to 5.

7. The conjugate or pharmaceutically acceptable salt thereof according to claim 1, wherein The coupling agent comprises at least one of the following A1-A9: A1； A2； A3； A4； A5； A6； A7； A8； A9； n is an integer between 10 and 1000.

8. A method of preparing the conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, characterized by, This includes the process of coupling multi-arm polyethylene glycol containing coupling reaction functional groups with indocyanine green derivatives; The coupling reaction functional groups include amino, carboxyl, mercapto, azide, alkynyl, hydroxyl, carboxylic acid ester, carbonate, succinimide, maleimide, aldehyde, dibenzocyclooctynyl, transcyclooctenyl, cyanobenzothiazolyl, aminothiol, tetraazinyl, or sulfosuccinimide ester. The indocyanine green derivative comprises one or more of the following: ICG-NHS, ICG-COOH, indocyanine green maleimide, indocyanine green azide, indocyanine green alkynyl, indocyanine green thiol, indocyanine green amino, indocyanine green dibenzocyclooctynyl, indocyanine green transcyclooctenyl, indocyanine green cyanobenzothiazole, indocyanine green aminothiol, indocyanine green tetraazinyl, and indocyanine green sulfonic acid succinimide ester.

9. A method of preparing the conjugate or pharmaceutically acceptable salt thereof according to claim 8, characterized by, The weight-average molecular weight of the multi-arm polyethylene glycol containing coupling reaction functional groups is 5000-40000.

10. A method of preparing the conjugate or pharmaceutically acceptable salt thereof according to claim 9, wherein, The weight-average molecular weight of the multi-arm polyethylene glycol containing coupling reaction functional groups is 20,000-40,000.

11. A method of preparing the conjugate or pharmaceutically acceptable salt thereof according to claim 8, wherein, The preparation method includes the following steps: The coupling compound was prepared by amidation reaction of ICG-NHS and mArmPEG-NH2.

12. A method of preparing the conjugate or pharmaceutically acceptable salt thereof according to claim 11, wherein, The molar ratio of ICG-NHS to mArmPEG-NH2 is 1~8:1; and / or, the reaction is carried out at room temperature in the dark, with stirring at 500~3000 rpm for 20~40 h; and / or, the reaction is purified by chromatographic separation and the purified product is collected; and / or, mArmPEG-NH2 includes 4ArmPEG-NH2 or 8ArmPEG-NH2; and / or, the amidation reaction is followed by a modification step with dimethylmaleic anhydride, FITC-NHS or gadolinium acid.

13. A method of preparing the conjugate or pharmaceutically acceptable salt thereof according to claim 11, wherein, The preparation method includes the following steps: ICG-NHS and 4ArmPEG-NH2 are mixed at a molar ratio of 1:1, stirred at 1000 rpm in the dark at room temperature for 24 h, and after stirring is completed, the eluent is separated by liquid chromatography using water as the eluent, and the eluent is collected to obtain the conjugate.

14. Use of the conjugate of any one of claims 1-7 or a pharmaceutically acceptable salt thereof, or the conjugate of any one of claims 8-13 or a pharmaceutically acceptable salt thereof, in the preparation of tumor contrast agents and / or medicaments for the prevention or treatment of tumors; The tumors include primary or metastatic tumors.

15. Use according to claim 14, characterized in that, The tumors include one or more of the following: bladder tumors, bone tumors, brain tumors, breast tumors, colorectal tumors, esophageal tumors, kidney tumors, lung tumors, ovarian tumors, pancreatic tumors, prostate tumors, stomach tumors, and liver tumors.

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