Sphingosine-modified fluorescent dye conjugate and synthesis thereof, liposome and application thereof

Abstract

The invention relates to the field of synthesis of organic fluorescent dyes, and discloses a sphingosine-modified fluorescent dye conjugate and synthesis thereof, a liposome and an application of the liposome. The conjugate is formed by simply coupling a fluorescent dye and sphingosine through an amido bond; the fluorescent dye comprises an indocyanine green derivative, a cyanine dye, a near-infrared dye or an FD dye; the indocyanine green derivative is ICG-R-COOH, R is-(CH2) nH. [I-], n is equal to 1-3, or R is-(CH2) 4SO3-; the structural formulas of the other dyes are formulas (1) to (6); and the sphingosine is D-sphingosine or D-dihydrosphingosine. The lipid nanoparticles constructed by the conjugate have the characteristics of strong NIR-II fluorescence, good in-vivo / in-vitro stability and large penetration depth. The liposome particles can act with serum protein in vivo, so that the NIR-II fluorescence intensity is further improved, and fluorescence imaging under a far-end near-infrared in-vivo imaging window is realized.

Description

Technical Field

[0001] This invention relates to the field of organic fluorescent dye synthesis, and more particularly to a sphingosine-modified fluorescent dye conjugate and its synthesis, liposomes and their applications. Background Technology

[0002] Fluorescence imaging is widely used to guide clinical surgical procedures, especially near-infrared II (NIR-II, 900-1880 nm) fluorescence imaging technology, which boasts high penetration depth and resolution, and has significant application potential in clinical surgery. However, existing NIR-II fluorescent dyes are scarce and suffer from poor pharmacokinetics and insufficient NIR-II imaging performance. For example, ICG is the only clinically applicable near-infrared fluorescent dye, but its clinical application faces significant challenges, such as an extremely short in vivo half-life (2-3 minutes in mice), weak NIR-II fluorescence due to molecular aggregation, and photobleaching caused by poor molecular stability. Furthermore, lipid nanoparticles constructed from ICG exhibit extremely poor in vivo stability, leading to rapid leakage of ICG and severely affecting its NIR-II fluorescence imaging performance. These factors significantly limit the clinical application of fluorescent dyes. Summary of the Invention

[0003] To address the above-mentioned deficiencies or improvement needs of existing technologies, this invention provides a sphingosine-modified fluorescent dye conjugate and its synthesis, liposomes and their applications. By providing a fluorescent dye conjugate with two NIR-II fluorescence enhancements, the constructed liposome nanoparticles achieve long-cycle, high-contrast and high-resolution fluorescence imaging in the near-infrared imaging window.

[0004] To achieve the above objectives, in one aspect of the present invention, a sphingosine-modified fluorescent dye conjugate is provided, which is formed by simple coupling of a fluorescent dye and sphingosine via an amide bond; The fluorescent dyes include indocyanine green derivatives, anthocyanin dyes, near-infrared dyes, or FD dyes. The indocyanine green derivative is ICG-R-COOH, where ICG stands for indocyanine green and R stands for -(CH2). n H·[I - ], n=1~3, or R is -(CH2)4SO3 - The structural formula of the cyanine dye is one of formulas (1) to (3), the structural formula of the near-infrared dye is formula (4) or (5), and the structural formula of the FD dye is formula (6). The sphingosine is D-sphingosine or D-dihydrosphingosine; , , .

[0005] Preferably, the fluorescent dye is ICG-CH3·[I - ]-COOH, wherein the sphingosine is D-sphingosine.

[0006] In another aspect of the present invention, a method for synthesizing a sphingosine-modified fluorescent dye conjugate as described above is provided, comprising: dispersing 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate, N,N-diisopropylethylamine, a fluorescent dye, and sphingosine in a solvent and reacting the mixture; concentrating the mixture; redispersing and washing the mixture; then extracting, drying, and concentrating the mixture; and finally eluting the mixture by chromatography to obtain the sphingosine-modified fluorescent dye conjugate.

[0007] In another aspect of the present invention, a liposome nanoparticle is provided, comprising the sphingosine-modified fluorescent dye conjugate, phospholipid, PEGylated lipid and cholesterol as described in any one of the above, assembled by thin-film sonication.

[0008] In another aspect of the invention, an application of liposome nanoparticles as described above in near-infrared imaging is provided, wherein the near-infrared wavelength is 850~1800 nm.

[0009] Preferably, the wavelength of the near-infrared light is 1300~1500 nm.

[0010] Preferably, when the near-infrared imaging reagent is a near-infrared in vivo imaging reagent, the liposome nanoparticles can assemble with proteins in in vivo serum to form secondary NIR-II fluorescence enhancement.

[0011] Preferably, when the near-infrared imaging reagent is a near-infrared in vivo imaging reagent, the pharmacokinetic half-life of the sphingosine-modified fluorescent dye conjugate is extended to 60-120 min, and the imaging time is 0.1-96 h, preferably 12-96 h.

[0012] Preferably, the molar concentration of the sphingosine-containing fluorescent dye conjugate in the liposome nanoparticles is 0.1~1.6%; more preferably, the molar 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 couples fluorescent dyes with sphingosine to design and synthesize a class of sphingosine-modified fluorescent dye conjugates. By controlling the hydrophilicity / hydrophobicity and structural symmetry of the fluorescent dyes, the conjugates achieve the effect of two-fold NIR-II fluorescence enhancement.

[0015] 2. The liposome nanoparticles constructed in this invention exhibit primary NIR-II fluorescence enhancement during liposome assembly and secondary NIR-II fluorescence enhancement in the serum environment. They also possess excellent photostability, penetration depth, and loading stability. This enables long-cycle, high-contrast, and high-resolution fluorescence imaging within the near-infrared imaging window. Modifying these lipid nanoparticles with a targeting peptide (M2pep peptide) allows for precise imaging and surgical navigation of bladder tumors in mice at the far-end near-infrared (≥1300 nm) range, expanding the clinical application of ICG and providing new technologies and methods for NIR-II fluorescence imaging of target sites such as blood vessels, lymph nodes, and tumors. Attached Figure Description

[0016] Figure 1 For example, ICG-C-Sp in Embodiment 1 of the present invention 1 H NMR spectrum.

[0017] Figure 2 For example, ICG-S-Sp in Embodiment 2 of the present invention 1 H NMR spectrum.

[0018] Figure 3 This is a comparison of the fluorescence emission of ICG-C-Sp LP in Example 1 and ICG LP in Comparative Example 1.

[0019] Figure 4 This is a comparison of the fluorescence emission of ICG-C-Sp LP in PBS and serum in Example 1 of the present invention.

[0020] Figure 5 This is a comparison of NIR-II fluorescence imaging of ICG-C-Sp LP in Example 1 and ICG LP in Comparative Example 1 in PBS, BSA and serum environments.

[0021] Figure 6 The results show the leakage of ICG in serum from ICG-C-Sp LP in Example 1 and ICG LP in Comparative Example 1.

[0022] Figure 7 The pharmacokinetic results of ICG-C-Sp LP in Example 1 and ICG LP in Comparative Example 1 in normal mice are shown.

[0023] Figure 8NIR-II imaging of abdominal blood vessels in mice 30 min after ICG-C-Sp LP was injected into the tail vein in Example 1 of this invention.

[0024] Figure 9 NIR-II fluorescence imaging of hind limb lymph nodes of mice at different time points after ICG-C-Sp LP in Example 1 and ICG LP in Comparative Example 1 were injected into the footplates of normal mice.

[0025] Figure 10 NIR-II imaging of bladder tumor mice at different time points after tail vein injection of ICG-C-Sp LP in Example 1, M2pep-ICG-C-Sp LP in Example 3, and ICG LP in Comparative Example 1 (white dashed circles represent tumors). Detailed Implementation

[0026] 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.

[0027] 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.

[0028] The specific technical solution of the present invention is as follows: A sphingosine-modified fluorescent dye conjugate is synthesized by simple coupling of a fluorescent dye and sphingosine via an amide bond. The fluorescent dye includes indocyanine green derivatives, cyanine dyes (Cy dyes), near-infrared dyes (IR dyes), or FD dyes. The indocyanine green derivative is ICG-R-COOH, where ICG stands for indocyanine green and R stands for -(CH2). n H·[I - ], n=1~3, or R is -(CH2)4SO3 - The structural formula of cyanine dye is one of formulas (1) to (3), the structural formula of near-infrared dye is formula (4) or (5), and the structural formula of FD dye is formula (6). Sphingosine is D-sphingosine (Sp) or D-dihydrosphingosine (DiSp).

[0029] The structural formula of the indocyanine green derivative (ICG-R-COOH) is as follows: ; The structural formula of cyanine dyes is as follows: ; The structural formula of the near-infrared dye is as follows: ; The structural formula of FD dye is as follows: .

[0030] Preferably, the indocyanine green derivative is ICG-CH3·[I - ]-COOH.

[0031] The structural formula of sphingosine is as follows: .

[0032] Preferably, the sphingosine is D-sphingosine.

[0033] In this invention, the preferred structural formula of the indocyanine green and sphingosine conjugate is as follows: .

[0034] The method for synthesizing the sphingosine-modified fluorescent dye conjugate includes: dispersing 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate (HBTU), N,N-diisopropylethylamine (DIPEA), a fluorescent dye, and sphingosine in a solvent, reacting the mixture, concentrating it, redispersing and washing it, then extracting, drying and concentrating it, and eluting it by chromatography to obtain the sphingosine-modified fluorescent dye conjugate.

[0035] 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, fluorescent dye, and sphingosine is (0.1~5):(0.1~10):1:(0.1~3); the reaction is carried out by stirring, and the reaction time is 0.5~12 h.

[0036] 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.

[0037] 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.

[0038] The present invention also provides a lipid nanoparticle comprising a sphingosine-modified fluorescent dye conjugate, phospholipids, PEGylated lipids and cholesterol as described above, which is assembled by thin-film ultrasonication.

[0039] For example, 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC) as the matrix lipid, 1,2-distearate phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2k) as the functional lipid, and cholesterol (Chol) for regulating membrane fluidity were dissolved in chloroform in a molar ratio of 60:5:35. Then, a sphingosine-modified fluorescent dye conjugate was added, and the mixture was rotary evaporated and dried to form a membrane. After hydration with PBS, sonication, and extrusion through a polycarbonate membrane, liposomes with uniform particle size were obtained.

[0040] The present invention also provides an application of the above-mentioned lipid nanoparticles in a far-end near-infrared imaging window, wherein the near-infrared wavelength is 850~1800 nm.

[0041] Preferably, the wavelength of the near-infrared light is 1300~1500 nm.

[0042] Furthermore, the molar concentration of the sphingosine-modified fluorescent dye conjugate in the lipid nanoparticles is 0.1~1.6%; preferably, the molar concentration is 0.4%.

[0043] Furthermore, the application of lipid nanoparticles in near-infrared imaging offers advantages such as strong photostability, large penetration depth, strong NIR-II fluorescence, and long cycle time. For example, liposome nanoparticles in near-infrared vascular imaging hold promise for clinical diagnosis of blood supply during surgery; liposome nanoparticles in near-infrared lymph node imaging hold promise for clinical applications in lymph node metastases and lymphatic system diseases; and liposome nanoparticles in near-infrared tumor imaging enable clinical application in defining tumor contours during surgery.

[0044] Furthermore, lipid nanoparticles exhibit excellent photostability, deep penetration, and strong NIR-II fluorescence in in vitro environments (such as PBS).

[0045] Furthermore, lipid nanoparticles can co-assemble with serum proteins in the in vivo environment (such as serum) to achieve secondary fluorescence enhancement.

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

[0047] Example 1: Synthesis and liposome preparation of indocyanine green and D-sphingosine conjugate (ICG-C-Sp). Synthesis of the indocyanine green-D-sphingosine conjugate (ICG-C-Sp): HBTU, DIPEA, ICG-CH3·[I - ICG-C-Sp was dispersed in 10 mL of DMF at a molar ratio of 2:3:1:1 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, column chromatography with CH2Cl2 and MeOH was used as the elution agent to obtain the product, which was ICG-C-Sp. The ICG-C-Sp synthesized in this example... 1 H NMR such as Figure 1 As shown, ICG-C-Sp was successfully synthesized.

[0048] Preparation of ICG-C-Sp liposomes: 1,2-Dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC), 1,2-distearate phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2k), and cholesterol (Chol) were dissolved in chloroform at a molar ratio of 60:5:35. Then, ICG-C-Sp with different molar concentrations (0.1%, 0.2%, 0.4%, 0.8%, and 1.6% of the total concentration of all other lipids) was added. The mixtures were then dried by rotary evaporation to form films, hydrated with PBS, sonicated, and extruded through a polycarbonate membrane to obtain ICG-C-Sp liposomes (ICG-C-Sp LP) with uniform particle size.

[0049] Example 2: Synthesis and liposome preparation of indocyanine green and D-sphingosine conjugate (ICG-S-Sp). Synthesis of the indocyanine green-D-sphingosine conjugate (ICG-S-Sp): HBTU, DIPEA, ICG-(CH2)4SO3 - -COOH and D-sphingosine were dispersed in 10 mL DMF at a molar ratio of 2:3:1:1 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, column chromatography with CH2Cl2 and MeOH was used as the elution agent to obtain the product, which was ICG-S-Sp. The ICG-S-Sp synthesized in this example... 1 H NMR such as Figure 2 As shown, ICG-S-Sp was successfully synthesized.

[0050] Preparation of ICG-S-Sp liposomes: DPPC, DSPE-PEG2k, and Chol were dissolved in chloroform at a molar ratio of 60:5:35. Then, 0.4% ICG-S-Sp 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-Sp liposomes with uniform particle size.

[0051] It should be noted that other fluorescent dyes, such as Cy dyes, IR dyes, FD dyes, or other indocyanine green derivatives, can be obtained through commercial channels or by simple synthesis. D-dihydrosphingosine can also be purchased through commercial channels. The sphingosine-modified fluorescent dye conjugates constructed from the above-mentioned fluorescent dyes and sphingosine exhibit similar properties to the preferred molecule ICG-C-Sp because their mechanism of regulating the hydrophilicity / hydrophobicity and structural symmetry of the fluorescent dyes is consistent.

[0052] Example 3: This embodiment, based on Example 1, further modifies the M2pep peptide to achieve precise imaging and surgical navigation of bladder tumor mice under far-infrared spectroscopy, including: Preparation of M2pep-ICG-C-Sp liposomes The preparation of M2pep-ICG-C-Sp liposomes is the same as the preparation process of ICG-C-Sp LP in Example 2, except that the addition of DSPE-PEG2k is replaced with the addition of DSPE-PEG2k-M2pep, thus obtaining M2pep-ICG-C-Sp liposomes, namely M2pep-ICG-C-Sp LP.

[0053] Comparative Example 1 To facilitate comparison of the properties of ICG-C-Sp LP, this embodiment also uses commercially prepared liposomes with the same preparation process parameters as described above, namely ICG LP, as a control group. Specifically: Preparation of ICG liposomes The preparation of ICG liposomes is the same as the preparation process of ICG-C-Sp LP in Example 1, except that ICG is replaced by ICG instead of ICG-C-Sp to obtain ICG LP.

[0054] Application Example 1: Application of Examples 1-3 in far-end near-infrared imaging, including: NIR-II in vivo fluorescence imaging 1: Vascular imaging 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.

[0055] NIR-II in vivo fluorescence imaging 2: Lymph node imaging After injecting the liposome nanoparticles (ICG = 50 μM, 25 μL) of the above-described embodiment into normal mice via the footplate, imaging was performed using an 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.

[0056] NIR-II in vivo fluorescence imaging 3: Tumor imaging 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.

[0057] 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: Lymph node imaging 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.

[0058] NIR-II live fluorescence imaging 2: tumor imaging 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.

[0059] The imaging performance study of the liposome nanoparticles prepared in the above embodiments and comparative examples includes: 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.

[0060] Serum loading stability: The liposome nanoparticles prepared in Example 1 and Comparative Example 1 were co-incubated with serum, and then the free ICG-CC-Sp in the serum was quantitatively analyzed by fluorescence spectroscopy.

[0061] 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. 2 The exposure time was 800 ms, and the filter was a 1300 nm long-pass filter.

[0062] In vivo pharmacokinetics: Normal mice were injected via tail vein with the liposome nanoparticles (ICG = 0.2 mM, 0.1 mL) described above. Blood samples were collected from the tail tip at different time points and quantified using an NIR-II in vivo imaging system. Imaging conditions: 808 nm laser light source, 37.5 mW / cm² laser power. 2 The exposure time was 100 ms, and the filter was an 1100nm long-pass filter.

[0063] See Figure 3 Fluorescence emission spectroscopy results showed that the fluorescence intensity of ICG-C-Sp in liposomes was significantly higher than that of ICG liposomes, and the emission peak was red-shifted, indicating that the interaction between ICG-C-Sp and phospholipids was stronger than that of ICG. See Figure 4 Further fluorescence emission spectroscopy results showed that the fluorescence emission intensity of ICG-C-Sp liposomes in serum was significantly higher than that of ICG-C-Sp liposomes in PBS, indicating that the assembly of ICG-C-Sp liposomes with serum proteins further enhanced the NIR-II fluorescence performance.

[0064] See Figure 5 NIR-II fluorescence imaging results also showed that in PBS, the fluorescence intensity of ICG-C-Sp LP was significantly higher than that of ICG LP, with a significant enhancement in fluorescence. Furthermore, the NIR-II fluorescence of ICG LP and ICG-C-Sp LP was further enhanced in BSA and serum.

[0065] See Figure 6 In serum, ICG rapidly leaks from liposomes, while ICG-C-Sp is relatively stable. Figure 4 and Figure 5The results indicate that the enhanced fluorescence of ICG LP in serum is due to the binding of ICG to serum proteins after leakage, while the secondary fluorescence enhancement of ICG-C-Sp LP is due to the direct co-assembly of ICG-C-Sp LP with serum proteins in the form of liposome nanoparticles.

[0066] See Figure 7 The pharmacokinetic half-life of ICG in ICG LP is ~5 min, while the pharmacokinetic half-life of ICG-C-Sp in ICG-C-Sp LP is significantly prolonged to ~95 min. This further indicates that the secondary fluorescence enhancement of ICG-C-Sp LP is due to the direct co-assembly of ICG-C-Sp LP with serum proteins in the form of liposome nanoparticles.

[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 8 Vascular imaging results showed that ICG-C-Sp LP can achieve high-contrast and clear vascular imaging under NIR-II.

[0068] See Figure 9 Lymph node imaging results showed that the contrast and clarity of ICG-C-Sp LP lymph node imaging under NIR-II were superior to those of ICG LP.

[0069] See Figure 10 Tumor imaging results showed that M2pep-ICG-C-Sp LP could achieve precise NIR-II imaging of bladder tumor mice, and the mean fluorescence intensity of the tumor and the tumor / normal tissue ratio were significantly better than those of ICG LP.

[0070] Experimental results show that the liposomes constructed from the conjugate of indocyanine green and D-sphingosine have strong photostability, large penetration depth, and strong NIR-II fluorescence. They have a long circulation time in vivo and their interaction with serum proteins leads to further enhancement of NIR-II fluorescence. In particular, they can achieve high-contrast and high-resolution fluorescence imaging of ICG in the far-end near-infrared (≥1300 nm).

[0071] 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 sphingosine-modified fluorescent dye conjugate, characterized in that, It is formed by simple coupling of fluorescent dye and sphingosine via amide bonds; The fluorescent dyes include indocyanine green derivatives, anthocyanin dyes, near-infrared dyes, or FD dyes. The indocyanine green derivative is ICG-R-COOH, where ICG stands for indocyanine green and R stands for -(CH2). n H·[I - ], n=1~3, or R is -(CH2)4SO3 - The structural formula of the cyanine dye is one of formulas (1) to (3), the structural formula of the near-infrared dye is formula (4) or (5), and the structural formula of the FD dye is formula (6). The sphingosine is D-sphingosine or D-dihydrosphingosine; ， ， 。 2. The sphingosine-modified fluorescent dye conjugate as described in claim 1, characterized in that, The fluorescent dye is ICG-CH3·[I - ]-COOH, wherein the sphingosine is D-sphingosine.

3. A synthesis of a sphingosine-modified fluorescent dye 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, fluorescent dye, and sphingosine were dispersed in a solvent and reacted. The mixture was then concentrated, redispersed, washed, extracted, dried, and concentrated, followed by chromatographic elution to obtain a sphingosine-modified fluorescent dye conjugate.

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

5. The application of the liposome nanoparticles as described in claim 4 in the preparation of near-infrared imaging reagents, wherein the near-infrared wavelength is 850~1800 nm.

6. The application as described in claim 5, wherein the near-infrared wavelength is 1300~1500 nm.

7. The application as described in claim 5, characterized in that, When the near-infrared imaging reagent is a near-infrared in vivo imaging reagent, the liposome nanoparticles can assemble with proteins in in vivo serum to form secondary NIR-II fluorescence enhancement.

8. The application as described in claim 5, characterized in that, When the near-infrared imaging reagent is a near-infrared in vivo imaging reagent, the pharmacokinetic half-life of the sphingosine-modified fluorescent dye conjugate is extended to 60-120 min, and the imaging time is 0.1-96 h, preferably 12-96 h.

9. An application as described in claim 5, wherein the molar concentration of the sphingosine-containing fluorescent dye conjugate in the liposome nanoparticles is 0.1-1.6%; preferably, the molar concentration is 0.4%.

10. The application as described in claim 5, 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.

Images