Conjugate of indocyanine green and multi-arm polyethylene glycol or pharmaceutically acceptable salt thereof as well as preparation method and application of conjugate

Abstract

The invention relates to the technical field of tumor fluorescence contrast agents, and particularly provides a conjugate of indocyanine green and multi-arm polyethylene glycol or pharmaceutically acceptable salt of the conjugate and a preparation method and application of the conjugate, the conjugate has the structural formula shown in the formula (I), the binding capacity of the conjugate and serum protein is remarkably reduced, and the conjugate can be applied to tumor fluorescence contrast agents. According to the present invention, the tumor targeting property is significantly improved, and the tumor is accumulated and detained in the tumor tissue, such that the targeted treatment or the high-resolution imaging is achieved so as to achieve the accurate tumor positioning and diagnosis, the high application value is provided, and the new method is developed for the tumor imaging and the treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of tumor fluorescent contrast agents, and in particular to a conjugate of indocyanine green and multi-arm polyethylene glycol or a pharmaceutically acceptable salt thereof, and a preparation method and use thereof. Background Art

[0002] Tumor near-infrared fluorescence imaging is a biological imaging technology based on near-infrared light. It uses the characteristics of near-infrared light that has a large penetration depth in tissues and little interaction with tissues, combined with targeted fluorescent probes to image tumors. This technology usually involves the use of specific fluorescent probes (such as indocyanine green ICG, etc.) to label tumor-related molecules or features, such as receptors or specific proteins on the surface of tumor cells, so that they emit fluorescent signals under near-infrared light excitation. Through the deep penetration ability of near-infrared light and the high sensitivity of fluorescent signals, tumor near-infrared fluorescence imaging can provide high-resolution, real-time tumor imaging, which is expected to be used for early diagnosis of tumors, surgical guidance, and monitoring of treatment effects.

[0003] Indocyanine green (ICG) is an FDA-approved near-infrared fluorescent probe that can be used clinically to guide certain tumor resection surgeries. It has good biocompatibility and fluorescence imaging properties in the body and can be used for angiography, tumor diagnosis, surgical guidance, etc. However, its half-life in the body is only 2 to 3 minutes. After injection, ICG immediately binds tightly to serum proteins, and then quickly passes through the liver and is excreted from the body by bile. Even though it can visualize blood vessels and lymph nodes, the rapid clearance of ICG limits its application in tumor resection. In addition, the contrast agent lacks targeting to tumors, resulting in low contrast between the tumor area and normal tissues. These factors make the use of ICG in tumor resection surgery very limited. 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 and a preparation method and use thereof.

[0005] To this end, the present application provides a conjugate of indocyanine green and multi-arm polyethylene glycol or a pharmaceutically acceptable salt thereof, wherein the conjugate has a structural formula shown in the following formula (I):

[0006]

[0007] Wherein, M is the branching center of the 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 are the same or different, and mk R1 are independently selected from H or any other substituent;

[0010] PEG is a polyethylene glycol group;

[0011] L1 are the same or different, L1 is a linker connecting the polyethylene glycol unit to the X group;

[0012] L2 are the same or different, and L2 is a linker connecting M to the X group.

[0013] In the present invention, the conjugate of indocyanine green and multi-arm polyethylene glycol is referred to as ICG-mArmPEG, wherein m is the number of arms of the multi-arm polyethylene glycol.

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

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

[0016] Furthermore, k is 1.

[0017] Furthermore, the conjugate has a structural formula as shown in the following formula (II), formula (III), formula (IV), formula (V), formula (VI) or formula (VII):

[0018]

[0019]

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

[0021] Further, L1 or L2 is selected from -AB-, wherein A is selected from one of the following groups: -CO-NH-, -NH-CO-NH-, -CO-NH-CO-, -O-, -S-, -SS-, an azide-alkynyl cycloaddition linking group, a tetrazine-trans-cyclooctene cycloaddition linking group, a maleimide-thiol addition linking group, an azide-dibenzocyclooctynyl cycloaddition linking group or a cyanobenzothiazolyl-aminothiol click reaction linking group; and 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-, wherein x, y, z, j, p, i, and u are independently selected from integers of 1 to 10;

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

[0023]

[0024] wherein x and p are independently selected from integers of 1-5.

[0025] Wherein, any end of the L1 or L2 group can be connected to X.

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

[0027] Further, 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, R8 are independently selected from unsubstituted or substituted C1-C5 alkyl, unsubstituted or substituted C2-C5 alkenyl, unsubstituted or substituted C1-C5 lactam group or unsubstituted or substituted C5-C25 heterocyclic compound;

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

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

[0031] In the present application, 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), lutetium (Lu). Heterocyclic compounds are organic compounds containing heterocyclic structures in molecules. The atoms constituting the ring contain at least one heteroatom in addition to carbon atoms. 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 spiro heterocycle formed by two alicyclic heterocycles, aromatic heterocycles or fused heterocyclic compounds sharing a carbon atom.

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

[0033] Further, 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 be 10, 20, 28, 30, 50, 56, 60, 110, 200, 220, 300, 500, 1000 or an integer between any two of the above. For example, n in formulas A1-A2 and A4-A6 can be approximately 28, 56, 110 or 220. n in formula A3 can be approximately 28 or 56.

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

[0043] Further, the coupling reaction functional group includes amino, sulfhydryl, azido, alkynyl, hydroxyl, carboxylate, carbonate, succinimide, maleimide, aldehyde, dibenzocyclooctyne, trans-cyclooctenyl, cyanobenzothiazolyl, aminothiol, tetrazine or sulfonic acid-succinimide ester. For example, the carboxylate can be succinimide carboxylate. The carbonate can be succinimide carbonate.

[0044] Furthermore, the indocyanine green derivative comprises one or more of ICG-NHS, ICG-COOH, indocyanine green maleimide, indocyanine green azide, indocyanine green alkynyl, indocyanine green thiol, indocyanine green amino, indocyanine green dibenzocyclooctyne, indocyanine green trans-cyclooctenyl, indocyanine green cyanobenzothiazole, indocyanine green aminothiol, indocyanine green tetrazine, and indocyanine green-sulfonic acid-succinimide ester.

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

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

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

[0048] The conjugate was prepared by amidation reaction between ICG-NHS and mArmPEG-NH2.

[0049] Further, the molar ratio of ICG-NHS to mArmPEG-NH2 is 1 to 8:1; and / or, the reaction is carried out at room temperature in the dark, and the reaction is stirred at 500 to 3000 rpm for 20 to 40 hours; and / or, after the reaction, it is purified by chromatography and the purified product is collected; and / or, mArmPEG-NH2 includes 4mArmPEG-NH2 or 8ArmPEG-NH2; and / or, after the amidation reaction, it also includes a step of modification with dimethylmaleic anhydride, FITC-NHS or gadoteric acid. Synthesis route see Figure 1 shown.

[0050] The step of modifying with gadoteric acid may be direct modification with gadoteric acid, or modification with DOTA-NHS and gadolinium salt in sequence, wherein the gadolinium salt may be conventional salts such as gadolinium chloride and gadolinium sulfate.

[0051] Furthermore, the preparation method includes the following steps: mixing ICG-NHS and 4ArmPEG-NH2 at a molar ratio of 1:1, stirring at 1000 rpm in the dark at room temperature for 24 hours, and after stirring, separating by liquid chromatography using water as an eluent, collecting the eluate, and obtaining a conjugate.

[0052] The present invention also provides the use of any of the conjugates or pharmaceutically acceptable salts thereof, or the conjugates or pharmaceutically acceptable salts thereof prepared by any of the above-mentioned preparation methods in the preparation of tumor contrast agents and / or drugs for preventing or treating tumors.

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

[0054] Furthermore, the tumor includes one or more of 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] The term "pharmaceutically acceptable salt" as used herein refers to a salt that retains the biological effectiveness of the free acid and free base of the specified compound and has no adverse effects in biology or other aspects. Pharmaceutically acceptable salts refer to the form of a base group in the parent compound converted into a salt. Pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of base groups such as amine (amino) groups. For example, 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 with the above acids after forming esters or amides and then with inorganic bases, such as sodium, potassium, calcium, aluminum or ammonium salts. Salts formed with acidic groups such as sulfonic acid groups and inorganic bases, for example, sodium, potassium, calcium, aluminum or ammonium salts.

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

[0057] 1. The study found that the conjugate formed by indocyanine green or single-arm polyethylene glycol and indocyanine green group decays rapidly, while the conjugate of indocyanine green and multi-arm polyethylene glycol or its pharmaceutically acceptable salt provided by the present invention has a structural formula shown in the following formula (I), comprising a multi-arm polyethylene glycol group and an indocyanine green group connected to at least one arm of the multi-arm polyethylene glycol group or to the branching center, the multi-arm polyethylene glycol group and the indocyanine green group are connected by a linker, the binding ability of the conjugate to serum proteins is significantly reduced, and it has significantly improved tumor targeting, accumulates and retains in tumor tissues, thereby achieving targeted treatment or high-resolution imaging to achieve more accurate tumor localization and diagnosis, and can be metabolized by the liver to achieve more accurate tumor localization and diagnosis, has high application value, and opens up new methods for tumor imaging and treatment development.

[0058] 2. The conjugate of indocyanine green and multi-arm polyethylene glycol provided by the present invention or a pharmaceutically acceptable salt thereof, wherein 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 0 at the same time, that is, the multi-arm polyethylene glycol has 3 to 8 arms, wherein at least one arm or branching center is connected to an indocyanine green group, especially when one arm of the multi-arm polyethylene glycol group is connected to an indocyanine green group (i.e., when k is 1), the tumor targeting can be further improved, the half-life can be prolonged, and the tumor localization and diagnosis can be better achieved.

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

[0060] 4. The preparation method of the conjugate of indocyanine green and multi-arm polyethylene glycol or its pharmaceutically acceptable salt provided by the present invention is simple in preparation method, has strong reaction controllability, can design reaction products according to needs, and has mild reaction conditions (room temperature), especially by using ICG-NHS and mArmPEG-NH2 for amidation reaction to obtain the conjugate, using mArmPEG-NH2 of different molecular weights as reactants, and after the reaction is completed, mArmPEG-NH- of various molecular weights is bonded to -C=O-, so as to achieve the purpose of completing the mArmPEG modification process of different molecular weights, wherein the NHS ester group has strong activity, and it is easy to undergo ester exchange with mArmPEG-NH2, remove one molecule of NHS, connect mArmPEG-NH- to -C=O-, and complete the process of ICG being modified by mArmPEG, thereby easily obtaining the medical imaging contrast agent. It has high selectivity and yield, and is easy to be converted for industrial production.

[0061] 5. The conjugate of indocyanine green and multi-arm polyethylene glycol or a pharmaceutically acceptable salt thereof provided by the present invention can further improve tumor targeting, prolong the half-life, and thus better achieve tumor localization and diagnosis by limiting the molecular weight of the multi-arm polyethylene glycol group to between 5000 and 40000, especially to between 20000 and 40000.

[0062] 6. The use of the conjugate of indocyanine green and multi-arm polyethylene glycol or its pharmaceutically acceptable salt provided by the present invention. These conjugates or their pharmaceutically acceptable salts can be used as tumor-specific targeted contrast agents, which are expected to achieve the effect of accurately positioning the tumor boundary and can be used for preoperative, intraoperative or postoperative angiography. They are mainly used for examination before and after surgery and for image navigation during surgery, and have the advantages of improving surgical precision and examination accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

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

[0065] Figure 2are the UV-visible absorption spectra of ICG-NHS, ICG-4ArmPEG5K, ICG-4ArmPEG10K, ICG-8ArmPEG10K, ICG-4ArmPEG20K, ICG-8ArmPEG20K and ICG-4ArmPEG40K in Experimental Example 1;

[0066] Figure 3 is the UV-visible absorption spectrum of ICG4-4ArmPEG40K in Experimental Example 1,

[0067] Figure 4 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;

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

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

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

[0071] Figure 8 These are the ninhydrin test results of ICG-4ArmPEG40K and ICG-4ArmPEG40K-DMA in Experimental Example 1.

[0072] Fig. 9 This is the ICG standard curve established in Experimental Example 1.

[0073] Fig.10 This is the result of agarose gel electrophoresis in Experimental Example 2.

[0074] Fig.11Live 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 the bright field of view, and the rest are fluorescence imaging images at various time points (808nm excitation; 900nm long-wave filter).

[0075] Fig.12 This is a graph showing the change in fluorescence intensity of the tumor (Tumor) and background tissue (Skin) of tumor-bearing mice over time after tail vein injection of three types of ICG-mArmPEG (ICG-4ArmPEG20K, ICG-8ArmPEG20K and ICG-4ArmPEG40K), as well as a graph showing the change in fluorescence contrast (tumor fluorescence intensity / background tissue fluorescence intensity) over time.

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

[0077] Fig.14 This is an in vivo fluorescence imaging of a mouse with orthotopic glioma 48 hours after tail vein injection of ICG-4ArmPEG40K. The fluorescence is mainly concentrated in the brain area with orthotopic glial tumors.

[0078] Fig.15 Fluorescence imaging of the brain of an orthotopic glioma-bearing mouse dissected 48 hours after tail vein injection of ICG-4ArmPEG40K.

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

[0080] Fig.17 It is the fluorescence image of ICG-4ArmPEG40K-FITC injected into the tail vein, the fluorescence intensity of the tumor (Tumor) and background tissue (Skin) of the tumor-bearing mouse changing with time, and the fluorescence contrast (tumor fluorescence intensity / background tissue fluorescence intensity) changing with time.

[0081] Fig.18 This is a graph showing the in vivo fluorescence imaging of tumor-bearing mice at different time points after tail vein injection of ICG-4ArmPEG40K-DOTA-Gd, as well as the changes in fluorescence intensity of tumor and background tissues and fluorescence contrast (tumor fluorescence intensity / background tissue fluorescence intensity) over time.

[0082] Fig.19 Magnetic resonance imaging (MRI) images of mice bearing orthotopic glioma 48 hours after injection of gadopentetate and ICG-4ArmPEG40K-DOTA-Gd via the tail vein. DETAILED DESCRIPTION

[0083] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0084] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

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

[0086] 2. 4ArmPEG-NH2, the full name of which is four-arm polyethylene glycol amino, the structural formula of 4ArmPEG-NH2 is as follows:

[0087]

[0088] 3. 8ArmPEG-NH2, the full name in Chinese is eight-arm polyethylene glycol amino, the structural formula of 8ArmPEG-NH2 is as follows:

[0089]

[0090] Wherein, m is 8 and R is -NH2.

[0091] 4. 2ArmPEG-NH2, the full name of which is two-arm polyethylene glycol amino, the structural formula of 2ArmPEG-NH2 is as follows:

[0092]

[0093] Example 1

[0094] This embodiment provides a method for preparing a conjugate of indocyanine green and multi-arm polyethylene glycol, and its process route is shown in Figure 1 As shown, the following steps are included:

[0095] Weigh ICG-NHS and add dimethyl sulfoxide (DMSO) to prepare a 0.24mmol / L ICG-NHS solution; weigh 4ArmPEG-NH2 with a weight average molecular weight of 40000 and add DMSO to prepare a 0.24mmol / L 4ArmPEG-NH2 solution; mix the above two solutions in equal volumes and stir at 1000rpm in the dark at room temperature for 24h. After stirring, use water as the eluent for liquid chromatography elution and separation, collect the eluate, and obtain a medical imaging contrast agent, which is recorded as ICG-4ArmPEG40K and stored at -20℃. The collected eluate can also be freeze-dried to obtain a lyophilized powder.

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

[0097]

[0098] Embodiments 2 to 4

[0099] Examples 2 to 4 provide a series of methods for preparing conjugates, which differ from Example 1 only in that the same molar amounts of 4ArmPEG-NH2 with weight average molecular weights of 5000, 10000 and 20000 are used in sequence to replace the 4ArmPEG-NH2 with a weight average molecular weight of 40000 in Example 1. The obtained products are respectively recorded as ICG-4ArmPEG5K, ICG-4ArmPEG10K and ICG-4ArmPEG20K.

[0100] Embodiment 5-6

[0101] Examples 5 to 6 provide two methods for preparing conjugates, which differ from Example 1 only in that the same molar amounts of 8ArmPEG-NH2 with weight average molecular weights of 10,000 and 20,000 are used, respectively, to replace the 4ArmPEG-NH2 with a weight average molecular weight of 40,000 in Example 1. The obtained products are respectively designated as ICG-8ArmPEG10K and ICG-8ArmPEG20K.

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

[0103]

[0104] Example 7

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

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

[0107]

[0108] Example 8

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

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

[0111]

[0112] Example 9

[0113] This embodiment provides a method for preparing a conjugate. Compared with Example 1, this method can modify the R1 group of the original conjugate by chemical reaction after the conjugate is formed in Example 1, thereby obtaining a conjugate containing different R1 groups. The method is as follows: the lyophilized powder obtained by freeze-drying the conjugate ICG-4ArmPEG40K obtained in Example 1 is dissolved in DMSO to form a 0.24mmol / L ICG-4ArmPEG40K solution. Dimethylmaleic anhydride (DMA, CAS: 766-39-2) is dissolved in DMSO to form a 0.72mmol / L solution, and mixed with the ICG-4ArmPEG40K solution in equal volumes, and stirred at room temperature for 24h at 1000rpm in the dark. After stirring, water is used as the eluent for liquid chromatography separation, and the eluate is collected to obtain a conjugate modified with dimethylmaleic anhydride, which is recorded as ICG-4ArmPEG40K-DMA and 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 coupling, weighing ICG-NHS and adding DMSO to prepare a 0.24 mmol / L ICG-NHS solution; weighing 4ArmPEG-NH2 and adding 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 hours.

[0118] After stirring, weigh FITC-NHS (5-carboxyfluorescein succinimidyl ester, CAS: 92557-80-7) and add DMSO to prepare a 0.72mmol / L FITC-NHS solution, slowly drop the FITC-NHS solution into the reaction solution, the added volume of the FITC-NHS solution is the same as the volume of the 4ArmPEG-NH2 solution added in the reaction, and stir at room temperature at 1000rpm in the dark for 24h. After stirring, use water as the eluent for liquid chromatography elution and separation, collect the eluate, and obtain the medical imaging contrast agent ICG-4ArmPEG40K-FITC, which is stored at -20°C. The collected eluate can also be freeze-dried to obtain a lyophilized powder.

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

[0120]

[0121] Embodiment 11

[0122] This embodiment provides a method for preparing a gadolinium salt of a conjugate. Compared with Example 1, after the conjugate is formed in Example 1, the present method modifies the R1 group of the original conjugate by a modified ligand compound, and then obtains a gadolinium salt of the conjugate having nuclear magnetic resonance contrast properties through coordination complexation of the gadolinium-ligand compound.

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

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

[0125]

[0126] Experimental Example 1 Characterization

[0127] 1. Test the spectral properties (including absorption spectral properties and emission spectral properties) of the conjugates prepared in the above embodiments. The specific operation is as follows:

[0128] (1) ICG powder and ICG-4ArmPEG5K, ICG-4ArmPEG10K, ICG-8ArmPEG10K, ICG-4ArmPEG20K, ICG-8ArmPEG20K and ICG-4ArmPEG40K conjugate lyophilized powder were added to water to prepare a solution with a concentration of 38 μmol / L. The UV-visible absorption spectra of ICG, ICG-4ArmPEG5K, ICG-4ArmPEG10K, ICG-8ArmPEG10K, ICG-4ArmPEG20K, ICG-8ArmPEG20K and ICG-4ArmPEG40K were measured in the spectral range of 500nm-900nm using a UV-2550 spectrometer. Figure 2 As shown. Figure 2It can be seen that ICG-4ArmPEG5K, ICG-4ArmPEG10K, ICG-8ArmPEG10K, ICG-4ArmPEG20K, ICG-8ArmPEG20K and ICG-4ArmPEG40K have spectral absorption capabilities that are basically consistent with that of ICG.

[0129] (2) ICG4-4ArmPEG40K lyophilized powder was added to water to prepare a solution with a concentration of 15 μmol / L, and the UV-visible absorption spectrum of ICG4-4ArmPEG40K was measured in the spectral range of 500nm-900nm using a UV-2550 spectrometer. Figure 3 As shown in the red spectrum, in aqueous solution, the main absorption peak of ICG4-4ArmPEG40K blue-shifts from 790nm to 700nm, which indicates that ICG4-4ArmPEG40K forms H aggregates. ICG is hydrophobic and PEG is hydrophilic, so in aqueous solution, ICG molecules tend to aggregate with each other. Therefore, when a PEG molecule couples more ICG, ICG4-4ArmPEG40K forms H aggregates. After diluting the above ICG4-4ArmPEG40K aqueous solution with an equal volume of DMSO, the UV-visible absorption spectrum of ICG4-4ArmPEG40K was retested. Figure 3 As shown in the black spectrum in , 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 powder of ICG-4ArmPEG5K, ICG-4ArmPEG10K, ICG-8ArmPEG10K, ICG-4ArmPEG20K, ICG-8ArmPEG20K and ICG-4ArmPEG40K conjugates were added to water to prepare a solution with a concentration of 38 μmol / L. The fluorescence emission spectra of ICG, ICG-4ArmPEG5K, ICG-4ArmPEG10K, ICG-8ArmPEG10K, ICG-4ArmPEG20K, ICG-8ArmPEG20K and ICG-4ArmPEG40K were tested using a fluorescence spectrometer FS5 with the excitation light set to 713 nm. The test results are as follows: Figure 4 As shown. Figure 4It can be seen 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 added to water to prepare a solution with a concentration of 15 μmol / L. The fluorescence emission spectra of ICG-4ArmPEG40K and ICG4-4ArmPEG40K were measured using a fluorescence spectrometer FS5 with the excitation light set to 713 nm. The results are shown in Figure 2. Figure 5 As shown, the fluorescence signal of ICG4-4ArmPEG40K is significantly weaker than that of ICG-4ArmPEG40K. This is because the four ICGs on the ICG4-4ArmPEG40K molecule have a more obvious aggregation effect, resulting in fluorescence aggregation-induced quenching.

[0132] (5) ICG-4ArmPEG40K-DMA and ICG-4ArmPEG40K-FITC lyophilized powders were added to water to prepare a solution with a concentration of 13 μmol / L. The UV-visible absorption spectra of ICG-4ArmPEG40K-FITC in the spectral range of 350nm-900nm and ICG-4ArmPEG40K-DMA in the spectral range of 500nm-900nm were measured using a UV-2550 spectrometer. Figure 6 As shown. Figure 6 It can be seen that ICG-4ArmPEG40K-DMA and ICG-4ArmPEG40K-FITC have spectral absorption capabilities that are basically consistent with ICG, and ICG-4ArmPEG40K-FITC has spectral absorption of FITC at 490nm. Using the fluorescence spectrometer Spectrofluorometer FS5, set the excitation light to 713nm, and test the fluorescence emission spectra of ICG-4ArmPEG40K-DMA and ICG-4ArmPEG40K-FITC respectively. 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) The ICG-4ArmPEG40K-DOTA-Gd lyophilized powder was added to water to prepare a solution with a concentration of 22 μmol / L. The UV-visible absorption spectrum of ICG-4ArmPEG40K-DOTA-Gd was measured in the spectral range of 500 nm-900 nm using a UV-2550 spectrometer. Figure 7 As shown. Figure 7 It can be seen that ICG-4ArmPEG40K-DOTA-Gd has a spectral absorption capacity that is basically consistent with that of ICG. Using a fluorescence spectrometer Spectrofluorometer FS5, set the excitation light to 713nm, and test the fluorescence emission spectrum of ICG-4ArmPEG40K-DOTA-Gd. The results are shown in the figure, and ICG-4ArmPEG40K-DOTA-Gd still has the property of near-infrared fluorescence.

[0134] 2. Ninhydrin detection

[0135] The R group of the conjugate ICG-4ArmPEG40K is -NH2. After the R group of the conjugate is modified, the -NH2 concentration will decrease. In this experiment, ninhydrin, which can react with -NH2 and develop color, is used to verify whether the R group of the conjugate has been successfully modified.

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

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

[0139] The experimental results are as follows Figure 8As shown, after incubation with ninhydrin, deionized water did not change color and became colorless; after incubation with leucine, the solution became purple; after the reaction of ICG-4ArmPEG40K-DMA with ninhydrin, the solution color was green; ICG-4ArmPEG40K was blue, which means that there is -NH2 in ICG-4ArmPEG40K, while ICG-4ArmPEG40K-DMA after R group modification does not contain -NH2. This shows that the R group of ICG-4ArmPEG40K-DMA has been successfully modified.

[0140] 3. Establishment of ICG standard curve

[0141] In order to ensure the consistency of ICG concentration in ICG-mArmPEG solution in animal experiments, the ultraviolet absorption of ICG solutions of different concentrations was tested by ultraviolet spectrophotometry to fit a standard curve.

[0142] Weigh ICG powder and add it to a mixed solution of DMSO and water in a volume ratio of 1:1 to prepare a 0.05mmol / L solution, which was diluted 2, 4, 8, 16 and 32 times to obtain 0.025mmol / L, 0.0125mmol / L, 0.00625mmol / L, 0.00313mmol / L and 0.00156mmol / L solutions, and then use a UV-2550 spectrometer to measure the UV-visible absorption spectrum in the spectral range of 500nm-900nm, and then take the maximum absorption value at 789nm to obtain the ICG concentration standard curve by fitting, see Fig. 9 The concentration of ICG is calculated as follows: concentration (mmol / L) = (Abs + 0.008) / 46.6, where Abs is the absorbance value. The standard curve has a good linear relationship. The absorbance of ICG-mArmPEG injection is tested by UV spectrophotometry and then inserted into the standard curve to obtain the concentration of ICG in ICG-mArmPEG injection.

[0143] Experimental Example 2

[0144] The prepared ICG-mArmPEG was tested for serum protein binding by 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 phosphate buffered saline (PBS) supplemented with 10% (v / v) fetal bovine serum (FBS) to prepare 0.02 mM test samples, and incubated at 37°C for 30 minutes. 5 μL Coomassie Brilliant Blue (CBB) 250 staining solution was added for mixed staining, and CBB 250 staining solution contained 10% (v / v) CBB and 90% (v / v) PBS.

[0146] Control group (including ICG group and ICG-4ArmPEG40K group): ICG-NHS or ICG-4ArmPEG40K lyophilized powder was added to 20 μL PBS buffer without FBS to prepare 0.02 mmol / L test samples, and incubated at 37°C for 30 minutes. 5 μL CBB 250 staining solution was added for mixed staining.

[0147] Staining agent control group (CBB group): 20 μL of phosphate buffer without FBS was incubated at 37°C for 30 minutes. 5 μL of CBB 250 staining solution was added for mixed 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 added for mixed staining.

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

[0150] Depend on Fig.10 It can be seen that in agarose gel electrophoresis, after co-incubation with FBS, ICG-NHS moved completely in the direction of FBS, while the movement of ICG-4ArmPEG40K was basically unaffected by FBS. Free ICG-NHS can strongly bind to serum proteins; in contrast, the binding ability of PEG-modified ICG-mArmPEG to serum proteins is significantly reduced.

[0151] Experimental Example 3

[0152] To test the tumor imaging effect of ICG-mArmPEG, the FOBI in vivo imaging system was used to observe the distribution and metabolism of the fluorescent contrast agent in vivo. The specific steps are as follows:

[0153] ICG-4ArmPEG20K, ICG-8ArmPEG20K and ICG-4ArmPEG40K lyophilized powders were weighed respectively and added with water to prepare ICG-4ArmPEG20K solution, ICG-8ArmPEG20K solution and ICG-4ArmPEG40K solution with the ICG concentration of 0.02 mmol / L.

[0154] Nine 4T1 tumor-bearing Balb / c mice with hair removed from the imaging area were divided into three groups, with three mice in each group. The mice were anesthetized by the animal anesthesia system SWD, and the three groups of mice were injected with ICG-4ArmPEG20K solution, ICG-8ArmPEG20K solution and ICG-4ArmPEG40K solution through the tail vein, with an injection volume of 125μL / mouse. Then, the mice were imaged with fluorescence at different time points. Imaging conditions: The tumor-bearing mice were excited by 808nm laser and photographed with FOBI near-infrared camera through 900nm long-wave filter FELH0900.

[0155] The results are as follows Fig.11 As shown in the figure, after three types of ICG-mArmPEG (ICG-4ArmPEG20K, ICG-8ArmPEG20K and ICG-4ArmPEG40K) were injected through the tail vein, ICG-mArmPEG was observed to be mainly concentrated in the liver tissue area at 2 minutes. After 12 hours of injection, the fluorescence signal intensity of the liver began to gradually decrease, while the fluorescence intensity of the tumor area gradually increased until it reached a peak at 48h, and then began to gradually decrease until 14 days. There was still a detectable fluorescence signal in the tumor area, while the fluorescence signals in other tissue areas were completely undetectable. This shows that ICG-mArmPEG can specifically accumulate and remain in the tumor site for a long time, so that ICG-mArmPEG has the ability to target tumors and achieve fluorescence imaging of tumor sites.

[0156] After the tail vein injection of three ICG-mArmPEG (ICG-4ArmPEG20K, ICG-8ArmPEG20K and ICG-4ArmPEG40K), the changes in the fluorescence intensity of the tumor and the normal skin tissue around the tumor were analyzed over time. The fluorescence intensity ratio and the retention rate of the tumor part aggregation of each mouse were calculated according to the following formula: fluorescence intensity ratio = fluorescence intensity of the tumor part / fluorescence intensity of the skin tissue, and the average and SD of the fluorescence intensity ratio were calculated; the retention rate of the tumor part aggregation = fluorescence intensity of the tumor part at each time point / fluorescence intensity of the tumor part at the highest time, and the average and SD of the retention rate were calculated.

[0157] The results are as follows Fig.12As shown in Tables 1-4, after the tail vein injection of three ICG-mArmPEG (ICG-4ArmPEG20K, ICG-8ArmPEG20K and ICG-4ArmPEG40K), the fluorescence of the mouse tumor site gradually increased, reaching the highest value at 48h after injection, and the fluorescence of the tumor site always maintained a high intensity during the 14-day imaging process. In addition, by comparing the fluorescence intensity of the tumor and its surrounding normal skin tissue, it can be found that during the 14-day imaging process, the fluorescence intensity of the tumor area remained more than 5 times the fluorescence intensity of the surrounding skin for most of the time. This shows that ICG-mArmPEG can not only illuminate the tumor area, but also distinguish the tumor site from the surrounding normal tissue boundary. Moreover, it was unexpectedly found that ICG-4ArmPEG20K and ICG-4ArmPEG40K are more effective in distinguishing the tumor site from the surrounding normal tissue boundary than ICG-8ArmPEG20K, and the retention rate of fluorescence intensity is higher, with a significantly extended half-life, especially ICG-4ArmPEG40K.

[0158] Table 1 Fluorescence intensity of 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 of 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 in tumor site (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] Experimental Example 4

[0168] The mice injected with three ICG-mArmPEG (ICG-4ArmPEG20K, ICG-8ArmPEG20K and ICG-4ArmPEG40K) by tail vein were dissected 14 days after the experiment in Example 3, and the heart, liver, spleen, lung, kidney, tumor, brain and muscle were taken for near-infrared fluorescence in vitro imaging, and refer to Fig.13 .

[0169] The results are as follows Fig.13As shown, from the organ fluorescence imaging images 14 days after tail vein injection of three ICG-mArmPEG (ICG-4ArmPEG20K, ICG-8ArmPEG20K and ICG-4ArmPEG40K), obvious fluorescence signals can be observed in the tumor and liver, while the fluorescence signals of other organ tissues are lower, which further indicates that ICG-4ArmPEG can selectively illuminate the tumor site and maintain a strong fluorescence signal in the tumor area for a 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 orthotopic brain gliomas in mice.

[0172] Weigh the ICG-4ArmPEG40K lyophilized 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 selected and injected with ICG-4ArmPEG40K solution through the tail vein with an injection volume of 125 μL / mouse. 48 hours after the injection, the mice were anesthetized by the animal anesthesia system SWD and subjected to fluorescence imaging by the FOBI in vivo imaging system.

[0174] The results are as follows Fig.14 As shown in the figure, ICG-4ArmPEG40K mainly accumulates in the brain area. This result shows that ICG-4ArmPEG40K has a targeting effect on in situ brain glioma. Although the tumor is difficult to observe in the naked eye, the tumor-bearing site can still be identified under the fluorescent field using ICG-4ArmPEG40K.

[0175] Further anatomical imaging of the brain, such as Fig.15 As shown, the tumor area of ​​the brain tissue has strong fluorescence, while the surrounding normal tissue area has no obvious fluorescence, and the contrast (multiple of the fluorescence intensity of the tumor area and the normal tissue area) is 3.2. This result illustrates the accumulation of ICG-4ArmPEG40K in the tumor area, showing its potential for 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 water was added to prepare an ICG4-4ArmPEG40K solution with an ICG concentration of 0.02mmol / L. Three 4T1 tumor-bearing Balb / c mice with hair removed from the imaging area were anesthetized by the animal anesthesia system SWD, and the ICG4-4ArmPEG40K solution containing 0.02mmol / L ICG was injected into the tail vein with an injection volume of 125μL / mouse. Then, the mice were subjected to fluorescence imaging at different time points. The imaging conditions were consistent with those in Experimental Example 3.

[0178] The results are as follows Fig.16 As shown in Table 5, within two minutes of the injection of ICG4-4ArmPEG40K, the fluorescence signal was mainly concentrated in the liver area, and the fluorescence signal in the tumor area was interfered by the strong fluorescence signal of the liver in a short time. The tumor site can be distinguished according to the fluorescence from 12 hours to 48 hours, but the fluorescence intensity of the tumor area weakens over time. After one week, the fluorescence in the tumor area was weak. Compared with the mice injected with ICG-4ArmPEG40K in Experimental Example 3, the fluorescence signal of ICG4-4ArmPEG40K coupled with 4 ICGs was significantly weaker, which may be due to the rapid liver metabolism of ICG4-4ArmPEG40K and the fluorescence aggregation-induced quenching effect of ICG4-4ArmPEG40K, which caused its fluorescence to be weaker than ICG-4ArmPEG40K.

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

[0180]

[0181]

[0182] Experimental Example 7

[0183] To test the tumor imaging effect of ICG-4ArmPEG40K-FITC, the FOBI in vivo imaging system was used to observe the distribution and metabolism of this fluorescent contrast agent in vivo. The specific steps are as follows:

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

[0185] Three 4T1 tumor-bearing Balb / c mice with hair removed from the imaging area were anesthetized by the animal anesthesia system SWD, and the mice were injected with ICG-4ArmPEG40K-FITC solution through the tail vein of the injection solution, with an injection volume of 125μL / mouse. Then, the mice were imaged with fluorescence at different time points. Imaging conditions: The tumor-bearing mice were excited by 808nm laser and photographed with a FOBI near-infrared camera through a 900nm long-wave filter FELH0900.

[0186] The results are as follows Fig.17 As shown in the figure, after ICG-4ArmPEG40K-FITC was injected through the tail vein, the fluorescence of the mouse tumor site gradually increased, reaching the highest value at 12 hours after injection, and in the subsequent imaging process, the fluorescence of the tumor site always maintained a high intensity. After stabilization, the fluorescence intensity of the tumor area remained more than 4 times that of the surrounding skin.

[0187] Experimental Example 8

[0188] Test the fluorescence imaging effect of the gadolinium-containing contrast agent ICG-4ArmPEG40K-DOTA-Gd on tumors. Take the ICG-4ArmPEG40K-DOTA-Gd lyophilized powder and add water to make a 0.02mmol / L solution. Three 4T1 tumor-bearing Balb / c mice with hair removed from the imaging area were anesthetized by the animal anesthesia system SWD, and 125μL of the prepared ICG-4ArmPEG40K-DOTA-Gd solution was injected into the tail vein, and the mice were subjected to fluorescence imaging at different time points. The imaging conditions were consistent with those in Experimental Example 3.

[0189] The results are as follows Fig.18 As shown, similar to ICG-4ArmPEG40K without DOTA-Gd modification, within two minutes of injection, the fluorescence signal was mainly concentrated in the liver area, and the fluorescence in the mouse tumor area reached a peak within one to two days, and then slowly decreased. Within 1-14 days, the fluorescence contrast between the tumor and the surrounding normal tissue was always above 4.

[0190] Experimental Example 9

[0191] The gadolinium-containing contrast agent ICG-4ArmPEG40K-DOTA-Gd was tested for its MRI effect on orthotopic brain gliomas in mice. The specific steps are as follows: dissolve gadopentetate (DOTA-Gd, CAS: 86050-77-3) in normal saline to prepare a gadopentetate solution containing 0.3 mmol / L of gadolinium. Take the lyophilized powder of ICG-4ArmPEG40K-DOTA-Gd and add water to prepare an ICG-4ArmPEG40K-DOTA-Gd solution containing the same 0.3 mmol / L of gadolinium. Take two mice with orthotopic brain gliomas (nude mouse C6 brain glioma model), and inject 125 μL of gadopentetate solution and ICG-4ArmPEG40K-DOTA-Gd solution through the tail vein, respectively. 48 hours after the injection, the mice were subjected to MRI imaging.

[0192] The results are as follows Fig.19 As shown, the arrow indicates the location of the tumor. Compared with gadopentetate, the tumor boundary obtained by ICG-4ArmPEG40K-DOTA-Gd is clearer, indicating that it has a better tumor imaging effect. ICG-4ArmPEG40K-DOTA-Gd can be used for MRI imaging to determine the location and size of the tumor.

[0193] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A conjugate of indocyanine green and multi-arm polyethylene glycol or a pharmaceutically acceptable salt thereof, characterized in that: The conjugate has the structural formula shown in the following formula (I): Wherein, M is the branching center of the 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 are the same or different, and mk R1 are independently selected from H or any other substituent; PEG is a polyethylene glycol group; L1 are the same or different, L1 is a linker connecting the polyethylene glycol unit to the X group; L2 are the same or different, and L2 is a linker connecting M to the X group.

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

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

4. The conjugate or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: k is 1.

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

6. The conjugate or pharmaceutically acceptable salt thereof according to claim 1 or 5, characterized in that: L1 or L2 is selected from -AB-, wherein A is selected from one of the following groups: -CO-NH-, -NH-CO-NH-, -CO-NH-CO-, -O-, -S-, -SS-, an azido-alkynyl cycloaddition linking group, a tetrazine-trans-cyclooctene cycloaddition linking group, a maleimide-thiol addition linking group, an azido-dibenzocyclooctynyl cycloaddition linking group or a 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-, wherein x, y, z, j, p, i, and u are independently selected from integers of 1 to 10; Preferably, L1 or L2 is selected from -(CH2) x -NH-CO-, -CO-NH-(CH2) P -NH-CO-, wherein x and p are independently selected from integers of 1-5.

7. The conjugate or a pharmaceutically acceptable salt thereof according to claim 1 or 5, characterized in that: 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; R3, R4, R5, R6, R7, R8 are independently selected from unsubstituted or substituted C1-C5 alkyl, unsubstituted or substituted C2-C5 alkenyl, unsubstituted or substituted C1-C5 lactam group or unsubstituted or substituted C5-C25 heterocyclic compound; "Substituted" means that the H atom in the group in R1, R3, R4, R5, R6, R7 or R8 is substituted by one or more of oxygen atom, -OH, halogen, -NH2, C1-C3 alkyl, C1-C3 carboxyl or a salt thereof; Preferably, R1 is selected from -H, -OH, C1-C5 alkyl, C1-C5 alkoxy, -NH2, -NH-CO-R7-COOH, a C5-C20 oxygen-containing heterocyclic compound substituted with a hydroxyl group, or -NH-CO-R8; R7 is selected from a C2-C3 alkenyl group substituted with a methyl group; R8 is selected from a C5-C10 nitrogen-containing heterocyclic compound substituted with a C1-C3 carboxyl group or a rare earth metal salt thereof.

8. The conjugate or a pharmaceutically acceptable salt thereof according to claim 6, characterized in that: R1 is selected from -H, -OH, -CH3, -OCH3, -NH2, -NH-CO-C(CH3)=C(CH3)COOH, or 9. The conjugate or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The conjugate comprises at least one of the following A1-A9: A1； n is an integer between 10 and 1000.

10. A method for preparing the conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 9, characterized in that: Including, the process of coupling multi-arm polyethylene glycol containing coupling reaction functional groups with indocyanine green derivatives; Optionally, the coupling reaction functional group includes an amino group, a carboxyl group, a sulfhydryl group, an azide group, an alkynyl group, a hydroxyl group, a carboxylate group, a carbonate group, a succinimide group, a maleimide group, an aldehyde group, a dibenzocyclooctyne group, a trans-cyclooctenyl group, a cyanobenzothiazolyl group, an aminothiol group, a tetrazine group, or a sulfonic acid succinimide ester group; Optionally, the indocyanine green derivative comprises one or more of ICG-NHS, ICG-COOH, indocyanine green maleimide, indocyanine green azide, indocyanine green alkynyl, indocyanine green thiol, indocyanine green amino, indocyanine green dibenzocyclooctyne, indocyanine green trans-cyclooctenyl, indocyanine green cyanobenzothiazole, indocyanine green aminothiol, indocyanine green tetrazine, and indocyanine green-sulfonic acid-succinimide ester.

11. The method for preparing the conjugate or a pharmaceutically acceptable salt thereof according to claim 10, characterized in that: The weight average molecular weight of the multi-arm polyethylene glycol containing coupling reaction functional groups is 5000-40000, preferably 20000-40000.

12. The method for preparing the conjugate or a pharmaceutically acceptable salt thereof according to claim 10, characterized in that: The preparation method comprises the following steps: The conjugate was prepared by amidation reaction between ICG-NHS and mArmPEG-NH2.

13. The method for preparing the conjugate or a pharmaceutically acceptable salt thereof according to claim 12, characterized in that: The molar ratio of ICG-NHS to mArmPEG-NH2 is 1 to 8:1; and / or, the reaction is carried out at room temperature in the dark, and the reaction is stirred at 500 to 3000 rpm for 20 to 40 hours; and / or, after the reaction, it is purified by chromatography and the purified product is collected; and / or, mArmPEG-NH2 includes 4mArmPEG-NH2 or 8ArmPEG-NH2; and / or, after the amidation reaction, the step of modification with dimethylmaleic anhydride, FITC-NHS or gadoteric acid is also included.

14. The method for preparing the conjugate or a pharmaceutically acceptable salt thereof according to claim 12, characterized in that: The preparation method comprises the following steps: mixing ICG-NHS and 4ArmPEG-NH2 in a molar ratio of 1:1, stirring at 1000 rpm in a dark room for 24 hours, and after stirring, separating by liquid chromatography using water as an eluent, collecting the eluate, and obtaining a conjugate.

15. Use of the conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 9 or the conjugate or a pharmaceutically acceptable salt thereof obtained by the preparation method according to any one of claims 10 to 14 in the preparation of a tumor contrast agent and / or a drug for preventing or treating tumors; Optionally, the tumor comprises a primary or metastatic tumor.

16. The use according to claim 15, characterized in that The tumor includes one or more of 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.

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