Conjugate of indocyanine green and cholesterol, synthesis and application thereof

Abstract

The invention relates to the field of synthesis of organic fluorescent dyes, and discloses a conjugate of indocyanine green and cholesterol as well as synthesis and application of the conjugate. The conjugate is formed by connecting an indocyanine green derivative and a cholesterol derivative through an amido bond; the indocyanine green derivative is ICG-R-COOH; iCG is indocyanine green, R is-(CH2) nH. [I-], n is an integer of 1-6, or R is-(CH2) mSO3-, and m is 3 or 4; and the cholesterol derivative is cholesterole-NH2, beta-sitosterole-NH2, stigmasterole-NH2 or ergosterole-NH2. The invention also discloses a preparation method of the cholesterol derivative. The lipidosome constructed by the conjugate is high in stability, good in dispersity and strong in NIR-II fluorescence, the blood vessel and tumor imaging contrast ratio and definition under a 1300 nm near-infrared imaging window are superior to those of lipidosome constructed by commercial ICG, more excellent diagnosis and treatment performance is shown, and wide application prospects are achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of organic fluorescent dye synthesis, in particular to an indocyanine green and cholesterol conjugate, synthesis and application thereof. BACKGROUND

[0002] Near-infrared region two (NIR-II, 900-1880 nm) fluorescence imaging technology has higher spatial resolution and penetration depth, which is helpful for accurate positioning of small lesions, and has great advantages and application potential in the fields of biomedicine and surgical navigation. ICG, as the only near-infrared fluorescent dye approved by FDA for clinical application, is commonly used for fluorescence imaging of blood vessels, lymph nodes and tumors. However, because of the extremely short in vivo half-life of ICG (2-3 minutes in mice) and rapid clearance, as well as the poor in vivo loading stability of liposome nanoparticles constructed by ICG (rapid leakage of ICG), the fluorescence imaging of target sites is severely limited. In addition, the NIR-II fluorescence of ICG in liposome nanoparticles is weak, which further causes the NIR-II fluorescence imaging effect of the target site to be unsatisfactory. These factors result in the very limited application of ICG in clinical practice. SUMMARY

[0003] In view of the above defects or improvement needs of the prior art, the present application provides an indocyanine green and cholesterol conjugate, synthesis and application thereof, which aims to couple indocyanine green derivatives and cholesterol derivatives through amide bonds, thereby solving the problems of poor in vivo loading stability and weak NIR-II fluorescence of liposome nanoparticles constructed by ICG.

[0004] To achieve the above-mentioned purpose, in one aspect of the present application, an indocyanine green and cholesterol conjugate is provided, which is coupled by amide bonds from indocyanine green derivatives and cholesterol derivatives; The indocyanine green derivative is ICG-R-COOH; ICG is indocyanine green, R is -(CH2) n H·[I - ], n is an integer from 1 to 6, or R is -(CH2) m SO3 - , m is 3 or 4; The cholesterol derivative is cholestanol-NH2, β-sitosterol-NH2, stigmasterol-NH2 or ergosterol-NH2.

[0005] Preferably, the indocyanine green derivative is ICG-CH3·[I - ]-COOH, and the cholesterol derivative is cholestanol-NH2.

[0006] In another aspect of the present application, a method for synthesizing the indocyanine green-cholesterol conjugate is provided, comprising: dispersing 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, N,N-diisopropylethylamine, an indocyanine green derivative and a cholester derivative in a solvent, concentrating, re-dispersing and washing, then extracting, drying and concentrating, and performing chromatographic elution to obtain the indocyanine green-cholesterol conjugate.

[0007] Preferably, the dispersing solvent is one or more of DMF, DMSO, CH3OH, CHCl3, CH2Cl2, and EA. The volume of the dispersing solvent is 0.1-50 mL. The molar amount of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, the molar amount of N,N-diisopropylethylamine, the molar amount of the indocyanine green derivative, and the molar amount of the cholester derivative are (0.1-5):(0.1-10):1:(0.1-3). The reaction time is 0.5 h-12 h, preferably, the reaction time is 4 h.

[0008] Preferably, the re-dispersing solvent is any one or more of CH2Cl2, CHCl3, and EA, and the volume of the re-dispersing solvent is 1-100 mL. The washing solution is any one or more of NaHCO3, HCl, and NaCl. The drying solution is an organic layer, and the drying agent is any one or more of anhydrous magnesium sulfate and anhydrous sodium sulfate. The chromatographic elution solvent is any one or more of CH2Cl2 and MeOH.

[0009] In another aspect of the present application, a liposome nanoparticle is provided, comprising the indocyanine green-cholesterol conjugate of any one of the above, a phospholipid, a PEGylated lipid, and cholester, and is assembled by a thin film ultrasonic method.

[0010] In another aspect of the present application, the liposome nanoparticle of the above is used for preparing a near-infrared imaging reagent, and the wavelength of the near-infrared is 850-1800 nm.

[0011] Preferably, the wavelength of the near-infrared is 1300 nm.

[0012] Preferably, the concentration of the indocyanine green-cholesterol conjugate in the liposome nanoparticle is 0.1-1.6%, and preferably, the concentration is 0.4%.

[0013] Preferably, when the near-infrared imaging reagent is a near-infrared in vivo imaging reagent, it is used for vascular imaging, lymph node imaging, tumor imaging, or metastatic tumor imaging.

[0014] In summary, compared with the prior art, the above-described technical solutions conceived by this invention mainly possess the following technical advantages: 1. This invention develops a class of indocyanine green and cholesterol conjugates. By regulating the hydrophilicity / hydrophobicity and structural symmetry of ICG, high-resolution fluorescence imaging of liposome nanoparticles constructed with this conjugate is achieved in the 850~1800 nm near-infrared imaging window.

[0015] 2. The liposome nanoparticles constructed in this invention have excellent stability and can be stably loaded in bovine serum albumin (BSA) or serum environments without leakage.

[0016] 3. The liposome nanoparticles constructed in this invention also possess good NIR-II fluorescence performance. Compared with commercial ICG, the liposome nanoparticles in this invention show better contrast and clarity in fluorescence imaging under the 1300 nm near-infrared imaging window, making them a very promising candidate for far-end near-infrared imaging. Attached Figure Description

[0017] Figure 1 The ICG-C-Chol in Embodiment 1 of this invention 1 H NMR spectrum.

[0018] Figure 2 The ICG-S-Chol in Embodiment 2 of the present invention 1 H NMR spectrum.

[0019] Figure 3 NIR-II fluorescence imaging tests were performed on ICG-C-Chol liposomes at different molar concentrations in Example 1 of this invention. Figure 3 The left image in the figure shows NIR-II fluorescence imaging of ICG-C-Chol liposomes at different molar concentrations in Example 1, and the right image shows the quantitative analysis data of the left image.

[0020] Figure 4 This is a TEM image of the ICG-C-Chol liposome (ICG-C-Chol LP) in Example 1 of the present invention.

[0021] Figure 5 This is a TEM image of the ICG liposomes (ICG LP) in Comparative Example 1 of the present invention.

[0022] Figure 6The images show the UV absorption spectra of ICG-C-Chol LP in Example 1 and ICG LP in Comparative Example 1 of this invention.

[0023] Figure 7 The fluorescence emission spectra of ICG-C-Chol LP in Example 1 and ICG LP in Comparative Example 1 are shown.

[0024] Figure 8 The results show the leakage of ICG in serum from ICG-C-Chol LP in Example 1 and ICG LP in Comparative Example 1.

[0025] Figure 9 The images show NIR-II fluorescence imaging of ICG-C-Chol LP in Example 1 and ICG LP in Comparative Example 1 in PBS, BSA and serum environments.

[0026] Figure 10 This is a vascular imaging image of ICG-C-Chol LP injected into a normal mouse via the tail vein in Example 1 of the present invention.

[0027] Figure 11 This is a vascular imaging image of ICG LP injected into a normal mouse via the tail vein in Comparative Application Example 1 of the present invention.

[0028] Figure 12 This is an NIR-II fluorescence imaging image of ICG-C-Chol LP injected subcutaneously into mice bearing bladder cancer via the tail vein in Example 1 of this invention.

[0029] Figure 13 The image shows an NIR-II fluorescence image of ICG LP injected subcutaneously into mice with bladder cancer via the tail vein in Comparative Application Example 1 of this invention. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0031] In the following embodiments, all instruments and other equipment used, unless otherwise specified, are conventional products that can be purchased through legitimate channels. Unless otherwise specified, all methods described are conventional methods, and all raw materials are available from publicly available commercial sources.

[0032] The specific technical solution of the present invention is as follows: A conjugate of indocyanine green and cholesterol is synthesized by coupling an indocyanine green derivative and a cholesterol derivative via an amide bond. The indocyanine green derivative is ICG-R-COOH, where ICG is indocyanine green and R is -(CH2). n H·[I - ], n = 1 to 6, or R = -(CH2). m SO3 - m is an integer from 3 to 4; Chol derivatives are cholesterol-NH2 (Chol-NH2), β-sitosterol-NH2 (β-Siol-NH2), stigmasterol-NH2 (Stol-NH2) or ergosterol-NH2 (Erol-NH2).

[0033] The structural formula of the indocyanine green derivative is as follows: ; Preferably, the indocyanine green derivative is ICG-CH3·[I - ]-COOH.

[0034] The structural formulas of Chol derivatives are as follows: .

[0035] Preferably, the cholesterol derivative is Chol-NH2.

[0036] In this invention, the preferred embodiment yields the following coupling structure: .

[0037] This invention also provides a method for synthesizing an indocyanine green and cholesterol conjugate, comprising: 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate (HBTU), N,N-diisopropylethylamine (DIPEA), indocyanine green derivatives, and cholesterol derivatives were dispersed in a solvent, reacted, and concentrated. The mixture was then redispersed and washed, extracted, dried, and concentrated. Chromatographic elution yielded a solid product, which is the indocyanine green-cholesterol conjugate.

[0038] Furthermore, the dispersing solvent is any one or more of DMF, DMSO, CH3OH, CHCl3, CH2Cl2, and EA, and the volume of the dispersing solvent is 0.1~50 mL; the molar ratio of HBTU, DIPEA, ICG derivative, and Chol derivative is 0.1~5:0.1~10:1:0.1~3; the reaction is carried out by stirring, and the reaction time is 0.5 h~12 h, preferably 4 h.

[0039] Furthermore, the concentration method is any one or more of the following that can cause solvent evaporation: rotary evaporation, dialysis, distillation, crystallization, and nitrogen blowing; the redispersing solvent is any one or more of CH2Cl2, CHCl3, and EA, and the volume of the dispersing solvent is 1~100 mL; the washing solution is any one or more of NaHCO3, HCl, and NaCl.

[0040] Furthermore, the dried solution is an organic layer, and the drying agent is any one or more of anhydrous magnesium sulfate and anhydrous sodium sulfate; the chromatographic elution solvent is any one or more of CH2Cl2 and MeOH.

[0041] The present invention also provides a liposome nanoparticle constructed from the above-mentioned indocyanine green and cholesterol conjugate, comprising the above-mentioned indocyanine green and cholesterol conjugate, phospholipids, PEGylated lipids and cholesterol, and assembled by thin film sonication.

[0042] For example, 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC) as the matrix lipid, 1,2-distearate phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) as the functional lipid, and cholesterol (Chol) for regulating membrane fluidity were dissolved in chloroform in a molar ratio of 60:5:35. Then, an indocyanine green-cholesterol conjugate was added, and the mixture was rotary dried to form a membrane. After hydration with PBS, sonication, and extrusion through a polycarbonate membrane, liposomes with uniform particle size were obtained.

[0043] The present invention also provides an application of the above-mentioned liposome nanoparticles in a near-infrared imaging window, wherein the wavelength of the near-infrared is 850~1800 nm, preferably 1300 nm.

[0044] Furthermore, the molar concentration of the indocyanine green and cholesterol conjugate in the liposome nanoparticles is 0.1-1.6%, preferably 0.4%.

[0045] Furthermore, liposome nanoparticles were applied to fluorescence imaging within the 1300 nm near-infrared imaging window, exhibiting significantly superior imaging contrast and resolution compared to commercially available ICG-constructed liposome nanoparticles. Applications of liposome nanoparticles within the 1300 nm near-infrared imaging window include vascular imaging, lymph node imaging, tumor imaging, and metastatic tumor imaging. The liposome nanoparticles demonstrated stable in vitro and in vivo loading and strong NIR-II fluorescence performance.

[0046] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0047] Example 1: Synthesis of indocyanine green and cholesterol conjugates and preparation of liposomes Synthesis of the indocyanine green-cholesterol conjugate (ICG-C-Chol): HBTU, DIPEA, ICG-CH3·[I - ICG-C-Chol was dispersed in 10 mL of DMF at a molar ratio of 1.5:3:1.2:1 and stirred for 4 h. The mixture was then rotary evaporated, redispersed in CH2Cl2, and washed with NaHCO3 and HCl. Extraction with CH2Cl2 followed by drying with anhydrous magnesium sulfate was then performed. Finally, purification was achieved by elution with a mixed solvent of CH2Cl2 and MeOH using a silica gel column. The resulting product was ICG-C-Chol. The ICG-C-Chol synthesized in this example... 1 H NMR such as Figure 1 As shown, ICG-C-Chol was successfully synthesized.

[0048] Preparation of ICG-C-Chol LP: DPPC, DSPE-PEG2000, and Chol were dissolved in chloroform at a molar ratio of 60:5:35. Then, different molar amounts of ICG-C-Chol (0.1%, 0.2%, 0.4%, 0.8%, and 1.6% of the total lipids) were added. The mixture was rotary evaporated and dried to form a film. After hydration with PBS and sonication, the film was extruded through a polycarbonate membrane to obtain ICG-C-Chol liposomes with uniform particle size. Preferably, the liposomes were prepared with 0.4% ICG-C-Chol, i.e., ICG-C-Chol LP, for subsequent applications. NIR-II imaging of the ICG-C-Chol liposomes with different molar concentrations prepared in this example is shown below. Figure 3 As shown, the TEM image of the prepared ICG-C-Chol LP is as follows. Figure 4 As shown.

[0049] Example 2: Synthesis of indocyanine green and cholesterol conjugates and preparation of liposomes Synthesis of the indocyanine green-cholesterol conjugate (ICG-S-Chol): HBTU, DIPEA, ICG-(CH2)4SO3 - -COOH and Chol-NH2 were dispersed in 10 mL of DMF at a molar ratio of 1.5:3:1.2:1 and stirred for 4 h. The mixture was then rotary evaporated, redispersed in CH2Cl2, and washed with NaHCO3 and HCl. Extraction with CH2Cl2 followed by drying with anhydrous magnesium sulfate was then performed. Finally, purification was achieved by elution with a mixed solvent of CH2Cl2 and MeOH using a silica gel column. The resulting product was ICG-S-Chol. The ICG-S-Chol synthesized in this example... 1 H NMR such as Figure 2As shown, ICG-S-Chol was successfully synthesized.

[0050] Preparation of ICG-S-Chol LP: DPPC, DSPE-PEG2000, and Chol were dissolved in chloroform at a molar ratio of 60:5:35. Then, 0.4% ICG-S-Chol was added, and the mixture was dried by rotary evaporation to form a film. After hydration with PBS and sonication, the film was extruded through a polycarbonate membrane to obtain ICG-S-Chol liposomes with uniform particle size, namely ICG-C-Chol LP.

[0051] It should be noted that other indocyanine green derivatives, ICG-(CH), n ·[I - ]-COOH, n=2~6, or ICG-(CH2)3SO3 - -COOH can be obtained through commercial channels or simple synthesis. Other cholesterol derivatives, such as β-sitosterol-NH2, stigmasterol-NH2, or ergosterol-NH2, can also be obtained through commercial channels or simple synthesis. The indocyanine green and cholesterol conjugates constructed from the above-mentioned indocyanine green and cholesterol derivatives exhibit similar properties to the preferred molecule ICG-C-Chol because they share the same regulatory mechanism on the hydrophilicity / hydrophobicity and structural symmetry of ICG.

[0052] Application Example 1: The application of the liposome nanoparticles described in Example 1 in the 1300 nm near-infrared imaging window includes: NIR-II in vivo fluorescence imaging 1: After injecting normal mice via the tail vein with the liposome nanoparticles (ICG = 0.2 mM, 0.1 mL) described above, imaging was performed using a NIR-II in vivo imaging system. Imaging conditions: 808 nm laser light source, 75 mW / cm² laser power. 2 The exposure time was 800 ms, and the filter was a 1300 nm long-pass filter.

[0053] NIR-II in vivo fluorescence imaging 2: After subcutaneous injection of the liposome nanoparticles (ICG = 0.2 mM, 0.1 mL) described above into mice bearing bladder cancer via the tail vein, imaging was performed using a NIR-II in vivo imaging system. Imaging conditions: 808 nm laser light source, 75 mW / cm² laser power. 2 The exposure time was 150 ms, and the filter was a 1300 nm long-pass filter.

[0054] Comparative Example 1: Synthesis and liposome preparation of indocyanine green (commercial ICG) This embodiment prepares ICG liposomes according to the preparation method and parameters of ICG-C-Chol liposomes described above, the difference being that the indocyanine green used was commercially available and not coupled with cholesterol derivatives. The TEM image of the ICGLP prepared in this comparative example is shown below. Figure 5 As shown.

[0055] Comparative Application Example 1: Application of Comparative Example 1 within the 1300 nm near-infrared imaging window, including: NIR-II in vivo fluorescence imaging 1: Normal mice were injected with ICG LP (ICG = 0.2 mM, 0.1 mL) via the tail vein, and then imaged using a NIR-II in vivo imaging system. Imaging conditions: 808 nm laser source, 75 mW / cm² laser power. 2 The exposure time was 800ms, and the filter was a 1300 nm long-pass filter.

[0056] NIR-II in vivo fluorescence imaging 2: Mice with subcutaneous bladder cancer were injected subcutaneously via the tail vein with ICG LP (ICG = 0.2 mM, 0.1 mL), and then imaged using a NIR-II in vivo imaging system. Imaging conditions: 808 nm laser source, 75 mW / cm² laser power. 2 The exposure time was 150 ms, and the filter was a 1300 nm long-pass filter.

[0057] The in vitro fluorescence properties of the liposome nanoparticles prepared in the above embodiments and comparative examples were studied, including: UV-Vis absorption spectroscopy: The liposome nanoparticles prepared in Example 1 and Comparative Example 1 were diluted to 5 μM with PBS and then measured in a UV-Vis absorption spectrometer.

[0058] Fluorescence spectroscopy: The liposome nanoparticles prepared in Example 1 and Comparative Example 1 were diluted to 5 μM with PBS and then measured in a fluorescence spectrometer with 740 nm as the excitation light.

[0059] Serum loading stability: The liposome nanoparticles prepared in Example 1 and Comparative Example 1 were co-incubated with serum, and then the free ICG-C-Chol in the serum was quantitatively analyzed by fluorescence spectroscopy.

[0060] In vitro NIR-II fluorescence imaging: The liposome nanoparticles prepared in Example 1 and Comparative Example 1 were diluted to 1 μM with PBS and then measured in an NIR-II in vivo imaging device. Imaging conditions: Laser source: 808 nm; Laser power: 75 mW / cm². 2The exposure time was 800 ms, and the filter was a 1300 nm long-pass filter.

[0061] See Figure 3 NIR-II fluorescence imaging results showed that liposomes containing different molar concentrations of ICG-C-Chol could all exhibit a certain fluorescence intensity, and the fluorescence was strongest when the molar concentration of ICG-C-Chol was 0.4%. Therefore, this concentration was set as the molar concentration for subsequent experiments.

[0062] See Figure 4 and Figure 5 TEM results showed that the liposome particles constructed in Example 1 and Comparative Example 1 were spherical in shape, uniform in size, and ranged from 30 to 70 nm.

[0063] See Figure 6 The ultraviolet absorption spectra of ICG-C-Chol LP in Example 1 and ICG LP in Comparative Example 1 were measured. ICG-C-Chol showed better dispersibility in liposomes than ICG, and its emission peak red-shifted, indicating that the interaction between ICG-C-Chol and phospholipids in liposomes was stronger than that between ICG and liposomes.

[0064] See Figure 7 The fluorescence emission spectra of ICG-C-Chol LP in Example 1 and ICG LP in Comparative Example 1 were measured. The fluorescence intensity of ICG-C-Chol in liposomes was higher than that of ICG, and the emission peak was red-shifted, indicating that the interaction between ICG-C-Chol and phospholipids in liposomes was stronger than that of ICG.

[0065] See Figure 8 In serum, ICG leaks rapidly from liposomes, while ICG-C-Chol is relatively stable.

[0066] See Figure 9 In PBS, the fluorescence intensity of ICG-C-Chol LP was much higher than that of ICG LP, with a significant enhancement in fluorescence. Furthermore, the NIR-II fluorescence of ICG-C-Chol LP showed little change in BSA and serum, indicating that it was relatively stable.

[0067] The results of Application Example 1 and Comparative Application Example 1 in the 1300 nm near-infrared imaging window are as follows: See Figure 10 and Figure 11 Mouse vascular imaging results showed that ICG-C-Chol LP had better contrast and clarity than ICG LP in NIR-II vascular imaging.

[0068] See Figure 12 and Figure 13Imaging results in mice with bladder tumors showed that, compared with ICG LP, ICG-C-Chol LP could achieve NIR-II fluorescence imaging in mice with bladder tumors, and the contrast and clarity were superior to those of ICG LP.

[0069] Experimental results show that liposomes constructed from indocyanine green and cholesterol conjugates are stably loaded without leakage in BSA or serum environments, exhibit good dispersibility, strong NIR-II fluorescence, and can achieve high-resolution vascular imaging and tumor imaging in the 1300 nm near-infrared imaging window.

[0070] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of protection of this invention and its equivalents, this invention also intends to include these modifications and variations. The above-described embodiments are merely preferred embodiments given to fully illustrate this invention, and their scope of protection is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on this invention are all within the scope of protection of this invention.

Claims

1. A conjugate of indocyanine green and cholesterol, characterized in that, It is composed of indocyanine green derivatives and cholesterol derivatives coupled via amide bonds; The indocyanine green derivative is ICG-R-COOH; ICG stands for indocyanine green, and R stands for -(CH2). n H·[I - ], where n is an integer from 1 to 6, or R is -(CH2). m SO3 - m is 3 or 4; The cholesterol derivative is cholesterol-NH2, β-sitosterol-NH2, stigmasterol-NH2, or ergosterol-NH2.

2. The indocyanine green and cholesterol conjugate as described in claim 1, characterized in that, The indocyanine green derivative is ICG-CH3·[I - ]-COOH, wherein the cholesterol derivative is cholesterol-NH2.

3. A method for synthesizing the indocyanine green and cholesterol conjugate as described in any one of claims 1-2, characterized in that, include: 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate, N,N-diisopropylethylamine, indocyanine green derivative, and cholesterol derivative were dispersed in a solvent and reacted. The mixture was then concentrated, redispersed, washed, extracted, dried, and concentrated, followed by chromatographic elution to obtain the indocyanine green-cholesterol conjugate.

4. The method for synthesizing the indocyanine green and cholesterol conjugate as described in claim 3, characterized in that, The dispersing solvent is one or more selected from DMF, DMSO, CH3OH, CHCl3, CH2Cl2, and EA; The volume of the dispersing solvent is 0.1~50 mL; The molar amounts of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate, N,N-diisopropylethylamine, indocyanine green derivative, and cholesterol derivative are (0.1~5):(0.1~10):1:(0.1~3). The reaction time is 0.5 h to 12 h, preferably 4 h.

5. The method for synthesizing the indocyanine green and cholesterol conjugate as described in claim 3, characterized in that, The redispersible solvent is any one or more of CH2Cl2, CHCl3, and EA, and the dispersing solvent volume of the redispersible solvent is 1~100 mL. The washing solution is any one or more of NaHCO3, HCl, and NaCl; The dried solution is an organic layer, and the desiccant is any one or more of anhydrous magnesium sulfate and anhydrous sodium sulfate. The solvent used for chromatographic elution is any one or more of CH2Cl2 and MeOH.

6. A liposome nanoparticle, characterized in that, The product comprises a conjugate of indocyanine green and cholesterol as described in any one of claims 1-2, phospholipids, PEGylated lipids, and cholesterol, assembled by thin-film sonication.

7. The application of the liposome nanoparticles as described in claim 6 in the preparation of near-infrared imaging reagents, wherein the near-infrared wavelength is 850~1800 nm.

8. An application as described in claim 6, wherein the near-infrared wavelength is 1300 nm.

9. An application as described in claim 6, characterized in that, The molar concentration of the indocyanine green-cholesterol conjugate in the liposome nanoparticles is 0.1-1.6%; preferably, the concentration is 0.4%.

10. The application as described in claim 6, characterized in that, When the near-infrared imaging reagent is a near-infrared in vivo imaging reagent, it is used for vascular imaging, lymph node imaging, tumor imaging, or metastatic tumor imaging.

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