Application of near-infrared ii organic fluorescent compound in preparation of biological imaging contrast agent and in blood vessel fluorescence imaging

Abstract

The application of a near-infrared II organic fluorescent compound in preparation of a biological imaging contrast agent and in blood vessel fluorescence imaging. A near-infrared II organic fluorescent compound with a structure shown in formula I has a high molar extinction coefficient and a high quantum yield, and can realize high-resolution imaging of blood vessels under white-light excitation; in addition, the near-infrared II organic fluorescent compound with the structure shown in formula I, when using white light as an excitation light source during imaging, can effectively avoid problems such as limited photon absorption, laser-induced biological damage and non-uniform irradiation caused by a single-wavelength excitation light source. Therefore, the near-infrared II organic fluorescent compound with the structure shown in formula I can be used for developing a high-performance biological imaging contrast agent, holding broad prospects in the fields of in-vivo imaging and fluorescence surgical navigation for non-diagnostic and non-therapeutic purposes.

Description

Application of a near-infrared second-region organic fluorescent compound in the preparation of biological imaging contrast agents and in vascular fluorescence imaging Technical Field

[0001] The invention belongs to the technical field of biochemical materials, and particularly relates to the application of a near-infrared second-zone organic fluorescent compound in the preparation of a biological imaging contrast agent and in vascular fluorescence imaging. Background Art

[0002] In vivo imaging technology uses imaging methods to qualitatively and quantitatively study biological processes in living organisms at the tissue, cellular, and molecular levels without damaging the animal. This technology enables non-invasive and intuitive observation of various biological processes in living animals, and is of great significance to fields such as life sciences and medicine. In vivo fluorescence imaging has garnered widespread attention due to its high sensitivity, high spatiotemporal resolution, and real-time monitoring capabilities. As the core of in vivo fluorescence imaging technology, high-performance fluorescent imaging reagents have long been a focus of research. Near-infrared II (NIR-II) organic fluorescent molecules, due to their deep tissue penetration and low autofluorescence interference, are ideal for high-performance fluorescent imaging reagents. However, in vivo fluorescence imaging using NIR-II organic nanoimaging reagents requires the use of expensive, specific-wavelength lasers as excitation sources. This is due to the long absorption wavelength, low quantum yield, and limited molar extinction coefficient of NIR-II organic fluorescent molecules. However, the use of single-wavelength lasers also presents challenges such as limited photon absorption and laser-induced biological damage. In addition, single-wavelength lasers cannot provide uniform irradiation, and the energy they provide will decay from the center of the spot to the surrounding areas, which will also affect the imaging quality of in vivo imaging.

[0003] In contrast, white light, which includes ordinary lighting, surgical shadowless lamps, and laparoscopic light sources, is a safe and visible excitation light with a wide continuous spectrum, low cost, and easy availability. It is an ideal choice for excitation light sources for in vivo fluorescence imaging. However, the application of white light as an excitation light source in NIR-II fluorescence imaging has not been reported. This requires NIR-II organic nanoimaging agents with high quantum yields and molar extinction coefficients. Therefore, the development of high-performance NIR-II organic nanoimaging agents excited by white light is urgently needed, but faces great challenges.

[0004] Summary of the Invention

[0005] The purpose of the present invention is to provide a near-infrared zone II organic fluorescent compound for use in the preparation of biological imaging contrast agents and in vascular fluorescence imaging. The near-infrared zone II organic fluorescent compound provided by the present invention has good biocompatibility and biostability; and has a high molar extinction coefficient and quantum yield, and can achieve high-resolution imaging of blood vessels under white light excitation; the near-infrared zone II organic fluorescent compound described in the present invention can be used to construct high-performance biological imaging contrast agents, and has broad prospects in the fields of in vivo imaging for non-therapeutic purposes and fluorescence surgical navigation.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a use of a near-infrared region II organic fluorescent compound in the preparation of a biological imaging contrast agent. The near-infrared region II organic fluorescent compound has a structure shown in Formula I:

[0008] In formula I, R1 and R2 are branched or straight chain alkyl groups;

[0009] R3 is X is one or more of H, F and Cl.

[0010] Preferably, R1 and R2 are C1-11 branched or straight chain alkyl groups.

[0011] Preferably, the near-infrared second region organic fluorescent compound has a structure shown in Formula Ia, Formula Ib or Formula Ic:

[0012] C in the structure shown in Formula Ia, Formula Ib or Formula Ic 11 H 23 It is a straight chain alkyl group.

[0013] The present invention provides an application of a near-infrared region II organic fluorescent compound in non-diagnostic and non-therapeutic vascular fluorescence imaging; the near-infrared region II organic fluorescent compound has a structure shown in Formula I:

[0014] In formula I, R1 and R2 are branched or straight chain alkyl groups;

[0015] R3 is X is one or more of H, F and Cl.

[0016] The present invention provides a use of a nano-imaging agent in the preparation of a biological imaging contrast agent, wherein the nano-imaging agent comprises a near-infrared second-region organic fluorescent compound having a structure represented by Formula I, and an organic coating agent coated on the surface of the near-infrared second-region organic fluorescent compound having a structure represented by Formula I;

[0017] In formula I, R1 and R2 are branched or straight chain alkyl groups;

[0018] R3 is X in R3 is one or more of H, F and Cl.

[0019] Preferably, the organic coating agent includes one or more of methoxypolyethylene glycol amine, distearoylphosphatidylethanolamine-polyethylene glycol, phosphatidylethanolamine-polyethylene glycol-maleimide, distearoylphosphatidylethanolamine-polyethylene glycol-folic acid, distearoylphosphatidylethanolamine-polyethylene glycol-sulfhydryl, distearoylphosphatidylacetamide-polyethylene glycol-carboxylic acid, distearoylphosphatidylethanolamine-polyethylene glycol-azide, distearoylethanolamine-polyethylene glycol-biotin, 1-palmitoyl-2-oleoylethanolamine, 1-stearoyl-2-oleoyl lecithin, dipalmitoylphosphatidylethanolamine-polyethylene glycol, polystyrene-g-polyethylene glycol, methoxy PEG polylactic acid-glycolic acid copolymer and poloxamer F127.

[0020] Preferably, the preparation method of the nano-imaging agent comprises the following steps:

[0021] Mixing an organic coating agent, a near-infrared second-zone organic fluorescent compound with a structure represented by Formula I, and an organic solvent to obtain a mixed solution; mixing the mixed solution with water and performing ultrasonic assembly to obtain an assembly liquid;

[0022] The assembly material solution is placed into a dialysis bag for dialysis to obtain a purified assembly material;

[0023] After concentrating the purified assembly material, a solution of the nano-imaging agent is obtained.

[0024] Preferably, the mass ratio of the organic capping agent to the near-infrared second region organic fluorescent compound having the structure shown in Formula I is (3-8):1;

[0025] The molecular weight cut-off of the dialysis bag is 3500; and the dialysis time is 48 to 72 hours.

[0026] The present invention provides a use of a nano-imaging agent in non-diagnostic and non-therapeutic vascular fluorescence imaging; the nano-imaging agent comprises a near-infrared second-region organic fluorescent compound having a structure represented by Formula I, and an organic coating agent coated on the surface of the near-infrared second-region organic fluorescent compound having a structure represented by Formula I;

[0027] In formula I, R1 and R2 are branched or straight chain alkyl groups;

[0028] R3 is X in R3 is one or more of H, F and Cl.

[0029] Preferably, the vascular fluorescence imaging is performed under white light excitation conditions, and the wavelength of the white light is 400-800 nm.

[0030] The present invention provides an application of a near-infrared zone II organic fluorescent compound in the preparation of a biological imaging contrast agent, wherein the near-infrared zone II organic fluorescent compound has a structure shown in Formula I. The near-infrared zone II organic fluorescent compound with a structure shown in Formula I provided by the present invention has a high molar extinction coefficient and quantum yield, and can achieve high-resolution imaging of blood vessels under white light excitation; further, the near-infrared zone II organic fluorescent compound with a structure shown in Formula I can monitor the process of liver ischemia reperfusion and visualize the process of kidney transplantation; in addition, the near-infrared zone II organic fluorescent compound with a structure shown in Formula I provided by the present invention can effectively avoid problems such as limited photon absorption, laser-induced biological damage, and uneven irradiation caused by a single-wavelength excitation light source by using white light as an excitation light source during the imaging process; therefore, the near-infrared zone II organic fluorescent compound with a structure shown in Formula I described by the present invention can be used to construct a high-performance biological imaging contrast agent, and has broad prospects in the fields of in vivo imaging and fluorescence surgical navigation for non-diagnostic and non-therapeutic purposes.

[0031] The present invention provides a nano-imaging agent for use in preparing a bioimaging contrast agent. The nano-imaging agent comprises a near-infrared zone II organic fluorescent compound having a structure represented by Formula I, and an organic coating agent coated on the surface of the near-infrared zone II organic fluorescent compound having a structure represented by Formula I. The present invention obtains a nano-imaging agent from the near-infrared zone II organic fluorescent compound and the organic coating agent. The nano-imaging agent has good biocompatibility and biostability. The nano-imaging agent of the present invention has a high molar extinction coefficient and quantum yield, and can achieve high-resolution imaging of blood vessels under white light excitation. Furthermore, the nano-imaging agent can monitor the process of liver ischemia-reperfusion and visualize the process of kidney transplantation. In addition, the use of white light as an excitation light source during the imaging process can effectively avoid problems such as limited photon absorption, laser-induced biological damage, and uneven irradiation caused by a single-wavelength excitation light source. Therefore, the nano-imaging agent of the present invention can be used to construct a high-performance bioimaging contrast agent and has broad prospects in the fields of in vivo imaging for non-therapeutic purposes and fluorescence surgical navigation.

[0032] The results of the examples demonstrate that the nano-imaging reagent of the present invention exhibits uniform size, good stability, minimal toxic side effects, and fluorescence emission in the near-infrared region II with a high quantum yield. This can effectively increase the penetration depth of biological tissues, reduce the interference of tissue autofluorescence, and improve the signal-to-noise ratio and imaging resolution. When applied to imaging abdominal vascular vessels in mice, the nano-imaging reagent can achieve rapid and high-resolution imaging of the mouse abdominal vessels. Furthermore, the nano-imaging reagent of the present invention was applied to monitor liver ischemia-reperfusion in mice. Experimental results demonstrated that the nano-imaging reagent can rapidly and in real time image this process with high resolution. Furthermore, the nano-imaging reagent of the present invention can also be used to monitor kidney transplantation in New Zealand white rabbits, providing real-time, high-resolution imaging of changes in renal vasculature during the transplantation process. Notably, due to the excellent luminescence properties and imaging capabilities of the nano-imaging reagent of the present invention, all excitation light sources used in in vivo fluorescence imaging applications are white light sources. White light sources are not only more affordable and economical, but also effectively avoid the problems of limited photon absorption, laser-induced biological damage, and uneven irradiation caused by single-wavelength excitation light sources. Therefore, the nano-imaging agent of the present invention can be used to construct a high-performance biological imaging contrast agent, and has broad prospects in the field of in vivo imaging for non-therapeutic and non-diagnostic purposes. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 shows the particle size of the nano-imaging agents HY6-NPs, FY6-NPs, and Y6CT-NPs. The inset in Figure 1 is a transmission electron microscope image of the nano-imaging agent Y6CT-NPs.

[0034] Figure 2 shows the absorption and emission spectra of the nano-imaging agents HY6-NPs, FY6-NPs, and Y6CT-NPs in aqueous solution; Figure 2 A shows the absorption spectra of the nano-imaging agents HY6-NPs, FY6-NPs, and Y6CT-NPs in aqueous solution; Figure 2 B shows the emission spectra of the nano-imaging agents HY6-NPs, FY6-NPs, and Y6CT-NPs in aqueous solution;

[0035] FIG3 is a comparison of the photostability of the nano-imaging agent Y6CT-NPs and the commercial imaging agent indocyanine green (ICG);

[0036] Figure 4 shows the relative quantum yield test results of nano-imaging agents FY6-NPs, Y6CT-NPs, and HY6-NPs using the commercial dye IR26 as a reference;

[0037] Figure 5 shows the dark toxicity and phototoxicity of different concentrations of nano-imaging reagent Y6CT-NPs on normal human liver cells (LO2 cells);

[0038] Figure 6 shows the dark toxicity and phototoxicity of different concentrations of nano-imaging reagent Y6CT-NPs on mouse embryonic fibroblasts (NIH 3T3 cells);

[0039] Figure 7 shows NIR-II fluorescence imaging of the abdominal blood vessels of normal mice by the nano-imaging agent Y6CT-NPs under different filter conditions and resolution analysis diagrams under different filter conditions. Figure 7A shows NIR-II fluorescence imaging of the abdominal blood vessels of normal mice by the nano-imaging agent Y6CT-NPs under different filter conditions; Figure 7B, Figure 7C, and Figure 7D show resolution analysis diagrams under different filter conditions.

[0040] FIG8 is a real-time NIR-II fluorescence imaging image of the nano-imaging reagent Y6CT-NPs during the ischemia-reperfusion process in mice;

[0041] Figure 9 shows the fluorescence intensity analysis of different regions during the process of liver ischemia-reperfusion monitored by Y6CT-NPs in mice;

[0042] FIG10 is a real-time NIR-II fluorescence imaging image of the donor kidney region of a New Zealand white rabbit using the nano-imaging agent Y6CT-NPs;

[0043] FIG11 is a real-time monitoring image of the renal vascular anastomosis during kidney transplantation in New Zealand white rabbits using the nano-imaging reagent Y6CT-NPs;

[0044] Figure 12 is a real-time monitoring image of the blood supply to the transplanted kidney ureter during kidney transplantation in New Zealand white rabbits using the nano-imaging reagent Y6CT-NPs. DETAILED DESCRIPTION

[0045] The present invention provides a use of a near-infrared region II organic fluorescent compound in the preparation of a biological imaging contrast agent. The near-infrared region II organic fluorescent compound has a structure shown in Formula I:

[0046] In formula I, R1 and R2 are branched or straight chain alkyl groups;

[0047] R3 is X is one or more of H, F and Cl.

[0048] In the present invention, unless otherwise specified, all preparation raw materials / components are commercially available products well known to those skilled in the art.

[0049] In the present invention, R1 and R2 are C1-11 branched or linear alkyl groups, more preferably C5-11 branched or linear alkyl groups, and most preferably C11 branched or linear alkyl groups.

[0050] In the present invention, R3 is

[0051] In the present invention, R3 is X is H, or X is H and F.

[0052] In the present invention, the near-infrared second region organic fluorescent compound preferably has a structure shown in Formula Ia, Formula Ib or Formula Ic:

[0053] C in the structure shown in Formula Ia, Formula Ib or Formula Ic 11 H 23 It is a straight chain alkyl group.

[0054] In a specific embodiment of the present invention, the near-infrared II organic fluorescent compound of the structure described in Formula Ia and the near-infrared II organic fluorescent compound of the structure described in Formula Ib are preferably prepared with reference to “Fluorination Enhances NIR-II Emission and Photothermal Conversion Efficiency of Phototheranostic Agents for Imaging-Guided Cancer Therapy” (Chunbin Li, Guoyu Jiang, Jia Yu, Weiwei Ji, Lingxiu Liu, Pengfei Zhang, Jian Du, Chuanlang Zhan, Jianguo Wang, and Ben Zhong Tang., Advanced Materials, 2023, 35, 2208229-2208239).

[0055] In a specific embodiment of the present invention, the near-infrared second-zone organic fluorescent compound of the structure described in Formula Ic is preferably prepared with reference to “A new non-fullerene acceptor based on the combination of a heptacyclic benzothiadiazole unit and a thiophene-fused end group achieving over 13% efficiency” (Yunqiang Zhang, Fangfang Cai, Jun Yuan, Qingya Wei, Liuyang Zhou, Beibei Qiu, Yunbin Hu, Yongfang Li, Hongjian Peng and Yingping Zou., Phys. Chem. Chem. Phys., 2019, 21, 26557--26563).

[0056] The present invention provides an application of a near-infrared region II organic fluorescent compound in non-diagnostic and non-therapeutic vascular fluorescence imaging; the near-infrared region II organic fluorescent compound has a structure shown in Formula I:

[0057] In formula I, R1 and R2 are branched or straight chain alkyl groups;

[0058] R3 is X is one or more of H, F and Cl.

[0059] The specific implementation of the near-infrared second-zone organic fluorescent compound with the structure shown in Formula 1 is as described above and will not be repeated here.

[0060] The present invention provides a use of a nano-imaging agent in the preparation of a biological imaging contrast agent, wherein the nano-imaging agent comprises a near-infrared second-region organic fluorescent compound having a structure represented by Formula I, and an organic coating agent coated on the surface of the near-infrared second-region organic fluorescent compound having a structure represented by Formula I;

[0061] In formula I, R1 and R2 are branched or straight chain alkyl groups;

[0062] R3 is X in R3 is one or more of H, F and Cl.

[0063] The specific implementation of the near-infrared second-zone organic fluorescent compound with the structure shown in Formula 1 is as described above and will not be repeated here.

[0064] In the present invention, the bioimaging contrast agent is preferably a bioimaging contrast agent for monitoring vascular imaging during liver ischemia-reperfusion, or a bioimaging contrast agent for monitoring vascular imaging during kidney transplantation, or a bioimaging contrast agent for vascular imaging in fluorescence imaging-guided surgery.

[0065] In the present invention, the organic coating agent preferably includes one or more of methoxypolyethylene glycol amine, distearoylphosphatidylethanolamine-polyethylene glycol, phosphatidylethanolamine-polyethylene glycol-maleimide, distearoylphosphatidylethanolamine-polyethylene glycol-folic acid, distearoylphosphatidylethanolamine-polyethylene glycol-sulfhydryl, distearoylphosphatidylacetamide-polyethylene glycol-carboxylic acid, distearoylphosphatidylethanolamine-polyethylene glycol-azide, distearoylethanolamine-polyethylene glycol-biotin, 1-palmitoyl-2-oleoylethanolamine, 1-stearoyl-2-oleoyl lecithin, dipalmitoylphosphatidylethanolamine-polyethylene glycol, polystyrene-g-polyethylene glycol, methoxy PEG polylactic acid-glycolic acid copolymer and poloxamer F127.

[0066] In the present invention, the preparation method of the nano-imaging agent preferably comprises the following steps:

[0067] Mixing an organic coating agent, a near-infrared second-zone organic fluorescent compound with a structure represented by Formula I, and an organic solvent to obtain a mixed solution; mixing the mixed solution with water and performing ultrasonic assembly to obtain an assembly liquid;

[0068] The assembly material solution is placed into a dialysis bag for dialysis to obtain a purified assembly material;

[0069] After the purified assembly material is concentrated to a desired concentration, a solution of the nano-imaging agent is obtained.

[0070] The present invention mixes an organic coating agent, a near-infrared zone II organic fluorescent compound with a structure shown in Formula I, and an organic solvent to obtain a mixed liquid; the mixed liquid is mixed with water, and ultrasonic assembly is performed to obtain an assembly liquid. In the present invention, the organic solvent is preferably tetrahydrofuran. The mass ratio of the organic coating agent and the near-infrared zone II organic fluorescent compound with a structure shown in Formula I is preferably (3-8):1, more preferably (4-7):1. The volume ratio of the organic solvent and the water is preferably 1:10. The present invention has no special requirements for the amount of the organic solvent, as long as the organic coating agent and the near-infrared zone II organic fluorescent compound with a structure shown in Formula I are completely dissolved. In the present invention, the ultrasonic power of the ultrasonic assembly is preferably 100-200W, more preferably 150W, and the time is preferably 3-10min, more preferably 5min.

[0071] After obtaining the assembly solution, the present invention places the assembly solution into a dialysis bag and dialyzes it to obtain a purified assembly material. In the present invention, the molecular weight cutoff of the dialysis bag is preferably 3500. The dialysis is preferably performed by immersing the dialysis bag containing the assembly solution in water. The dialysis temperature is preferably room temperature, and the duration is preferably 48 to 72 hours.

[0072] After obtaining the purified assembly material, the present invention concentrates the purified assembly material to a desired concentration to obtain a solution of the nano-imaging agent. In the present invention, the concentration is preferably performed using polyethylene glycol for water absorption concentration, and the average molecular weight of the polyethylene glycol is preferably 100,000. The concentration yields a concentrated solution, which is preferably filtered to remove impurities using a syringe filter to obtain a solution of the nano-imaging agent.

[0073] The present invention provides a use of a nano-imaging agent in non-diagnostic and non-therapeutic vascular fluorescence imaging; the nano-imaging agent comprises a near-infrared second-region organic fluorescent compound having a structure represented by Formula I, and an organic coating agent coated on the surface of the near-infrared second-region organic fluorescent compound having a structure represented by Formula I;

[0074] In formula I, R1 and R2 are branched or straight chain alkyl groups;

[0075] R3 is X in R3 is one or more of H, F and Cl.

[0076] The specific implementation of the near-infrared second-zone organic fluorescent compound with the structure shown in Formula 1 is as described above and will not be repeated here.

[0077] In a specific embodiment of the present invention, the specific implementation method of the use of the nano-imaging agent in non-diagnostic and non-therapeutic vascular fluorescence imaging preferably includes the following steps:

[0078] The nano-imaging reagent was injected into the mouse through the tail vein, and fluorescence imaging of the mouse abdominal blood vessels was performed under white light excitation.

[0079] In the present invention, the non-diagnostic and non-therapeutic vascular fluorescence imaging is preferably in vivo imaging.

[0080] In a specific embodiment of the present invention, the application of the vascular fluorescence imaging preferably includes vascular imaging during monitoring of liver ischemia-reperfusion, or vascular imaging during monitoring of kidney transplantation, or vascular imaging during surgery guided by fluorescence imaging.

[0081] In a specific embodiment of the present invention, the specific implementation method of the use of nano-imaging agents in monitoring hepatic ischemia-reperfusion for non-diagnostic and non-therapeutic purposes preferably includes the following steps:

[0082] The nano-imaging reagent was injected into the liver ischemia-reperfusion model mice through the tail vein, and the liver ischemia-reperfusion process of the mice was fluorescently imaged under white light excitation.

[0083] In a specific embodiment of the present invention, the use of nano-imaging agents in monitoring a kidney transplant process for non-diagnostic and non-therapeutic purposes comprises the following steps:

[0084] The nano-imaging reagent was injected into the kidney transplant model New Zealand white rabbit through the ear vein, and the kidney transplantation process of the rabbit was fluorescently imaged under white light excitation.

[0085] In the present invention, when vascular fluorescence imaging is used, the fluorescence imaging device used is preferably a near-infrared zone II small animal in vivo imager. Vascular fluorescence imaging is performed under white light excitation conditions, preferably with a wavelength of 400 to 800 nm. The white light excitation light source is the illumination lamp of the near-infrared zone II small animal in vivo imager or a laparoscope LED cold white light source, preferably with a wavelength range of 400 to 800 nm.

[0086] In the present invention, the effective concentration of the nano-imaging agent injected into the mouse during the vascular fluorescence imaging or the hepatic ischemia-reperfusion fluorescence imaging is preferably not less than 300 μmol L -1 , the volume is not less than 100 μL.

[0087] In the present invention, the effective concentration of the nano-imaging agent injected into the New Zealand white rabbit during the fluorescence imaging of the kidney transplantation process is preferably not less than 300 μmol L -1 , the volume is preferably not less than 2 mL.

[0088] In order to further illustrate the present invention, the technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0089] Example 1

[0090] The near-infrared second-zone organic fluorescent compound of the structure shown in Formula I used in this embodiment and excited by white light is specifically the structure shown in Formula Ia (HY6), the structure shown in Formula Ib (FY6) or the structure shown in Formula Ic (Y6CT).

[0091] 2 mg of near-infrared second-region organic fluorescent compound (HY6, FY6 or Y6CT) and 10 mg of DSPE-PEG 2000The nanoparticles were dissolved in 1 mL of tetrahydrofuran and then added to 10 mL of deionized water for ultrasonic assembly. After the nanoparticle solution was completed, it was transferred to a dialysis bag with a molecular weight cutoff of 3500 and dialyzed for purification for 72 hours. The dialyzed nanoparticle solution was concentrated with polyethylene glycol with an average molecular weight of 100,000 and then filtered with a syringe filter to remove impurities. Finally, the nano-imaging agents HY6-NPs, FY6-NPs, and Y6CT-NPs were prepared. The effective concentration of the nano-imaging agent was determined to be 500 μmol L-1 by the pre-established concentration curve. -1 .

[0092] Performance testing:

[0093] (1) Particle size test of nano-imaging reagents HY6-NPs, FY6-NPs, and Y6CT-NPs: 30 μL of nano-imaging reagent was added to 3 mL of deionized water, and then the particle size was measured using a dynamic light scattering instrument. The results are shown in Figure 1.

[0094] Figure 1 shows the particle size of the nanoimaging agents HY6-NPs, FY6-NPs, and Y6CT-NPs. As shown in Figure 1, the sizes of HY6-NPs, FY6-NPs, and Y6CT-NPs are 95 nm, 104 nm, and 176 nm, respectively. The transmission electron microscopy image in the inset further demonstrates the spherical morphology and uniform size of the Y6CT-NPs.

[0095] (2) Photophysical properties of nano-imaging agents HY6-NPs, FY6-NPs, and Y6CT-NPs: The absorption and emission spectra of Y6CT-NPs in aqueous solution were measured using a UV-visible spectrophotometer equipped with an integrating sphere module and a steady-state transient fluorescence spectrometer, respectively. The test concentration was 10 μmol L -1 , the results are shown in Figure 2.

[0096] Figure 2A shows the absorption spectra of the nano-imaging agents HY6-NPs, FY6-NPs, and Y6CT-NPs in aqueous solution. As can be seen from Figure 2A, the three nano-imaging agents all exhibit strong absorption in the range of 400-1000 nm. The molar extinction coefficients of HY6-NPs, FY6-NPs, and Y6CT-NPs at the maximum absorption peak are 2.68×10 4 L mol -1 cm -1 (763nm)7.76×10 4 L mol -1 cm -1 (811nm) and 8.24×10 4 L mol -1 cm -1(798nm). Clearly, FY6-NPs and Y6CT-NPs exhibit higher absorptivity and red-shifted absorption wavelengths than HY6-NPs, which facilitates the absorption of white light energy. Under white light excitation, all three nanoimaging agents exhibit fluorescence emission in the NIR-II region. In contrast, Y6CT-NPs exhibit the highest fluorescence intensity under white light excitation (laparoscopic LED cold light source), with emission peaks at 947nm and 1030nm, extending to 1400nm, facilitating their application in white light-excited bioimaging.

[0097] (3) Photostability test of nano-imaging reagent Y6CT-NPs: The commercial imaging reagent indocyanine green (ICG) was selected as a comparison. -2 Y6CT-NPs and ICG were irradiated with white light (laparoscopic LED cold light source) for 40 minutes respectively. The fluorescence intensity of the two was recorded at different time points and the ratio was made with the initial fluorescence intensity to compare the attenuation of the fluorescence intensity of the two and their photostability. The results are shown in Figure 3.

[0098] Figure 3 compares the photostability of the nano-imaging reagent Y6CT-NPs and the commercial imaging reagent indocyanine green (ICG). As shown in Figure 3, after 40 minutes of continuous illumination, the fluorescence intensity of Y6CT-NPs decreases slightly, while the fluorescence intensity of ICG decreases significantly, reaching only 30% of its initial fluorescence intensity at 40 minutes, demonstrating the excellent photostability of Y6CT-NPs.

[0099] (3) Relative quantum yield of nano-imaging agent Y6CT-NPs: The commercial near-infrared II (NIR-II) fluorescent dye IR26 was selected as a reference to test the relative quantum yields of FY6-NPs, Y6CT-NPs, and HY6-NPs. IR26 was prepared as a dichloroethane (DCE) solution. First, the absorption spectra of FY6-NPs, Y6CT-NPs, HY6-NPs and IR26-DCE were tested respectively, and the corresponding concentrations when the absorbance values ​​at 808 nm were 0.02, 0.04, 0.06, 0.08 and 0.10 were determined. Then, the fluorescence spectra of the corresponding concentrations when excited at 808 nm were tested respectively. The area integration of the fluorescence spectrum in the emission wavelength range of 850-1400 nm was performed as the ordinate, and the slope was obtained by linear regression analysis with the absorbance value as the abscissa. Then, the relative quantum yields of FY6-NPs, Y6CT-NPs and HY6-NPs were calculated using the following formula 1.

[0100] The refractive index of water described in Formula 1 is 1.333, and the refractive index of ethylene dichloride is 1.4448.

[0101] Figure 4 shows the relative quantum yields of the nanoimaging agents FY6-NPs (A), Y6CT-NPs (C), and HY6-NPs (B) using IR26 as a reference. Figure 4 shows that the relative quantum yields of FY6-NPs, Y6CT-NPs, and HY6-NPs are 4.08%, 18.72%, and 16.12%, respectively.

[0102] (4) Cytotoxicity test of nano-imaging reagent Y6CT-NPs: Cells in the logarithmic growth phase were taken and 5×10 3 Cells were seeded in a 96-well plate at a density of 100 cells / well and cultured in a carbon dioxide incubator (37°C, 5% CO2) for 24 h. Subsequently, different concentrations of Y6CT-NPs were added. After a further incubation of 20 h, the cells were placed under white light (laparoscopic LED cold light source) (50 mW cm -2 ) for 30 min (light group) or 30 min (dark group) under dark conditions, and then incubated for 4 h. After incubation, 10 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (5 mg mL -1 ) solution and wait for another 4 hours; then remove the MTT solution, add 100 μL of DMSO to each well, and measure the absorbance of the product at a wavelength of 490 nm using a microplate reader. The results are expressed as the percentage of viable cells in the treated cells relative to the untreated control cells. Relative cell viability was calculated using the following formula 2: Cell viability (%) = (OD sample -OD background ) / (OD control -OD background )×100% Formula 2.

[0103] The results are shown in Figures 5 and 6.

[0104] Figure 5 shows the dark toxicity and phototoxicity of the nanoimaging reagent Y6CT-NPs at different concentrations on normal human liver cells (LO2 cells), and Figure 6 shows the dark toxicity and phototoxicity of the nanoimaging reagent Y6CT-NPs at different concentrations on mouse embryonic fibroblasts (NIH3T3 cells). As can be seen from Figures 5 and 6, Y6CT-NPs showed no significant dark toxicity or phototoxicity to either LO2 or NIH 3T3 cells, indicating that Y6CT-NPs have minimal toxic side effects and good biocompatibility.

[0105] Example 2

[0106] The high-resolution NIR-II fluorescence imaging capability of the nano-imaging agent Y6CT-NPs on the abdominal blood vessels of mice was tested: 100 μL of Y6CT-NPs with a concentration of 300 μmol / L was injected into BALBC / b mice through the tail vein. The mice were then imaged with a near-infrared II small animal living imager. The excitation light source was the illumination light source of the near-infrared II small animal living imager (16.5 mW cm -2 ), filters of different wavelengths were adjusted to collect fluorescence images, and the results are shown in Figure 7A. The imaging resolution under different filter conditions was analyzed by imaging software, and the results are shown in Figure 7B, Figure 7C, and Figure 7D.

[0107] Figure 7A shows NIR-II fluorescence imaging of normal mouse abdominal vessels using the nanoimaging reagent Y6CT-NPs under different filter conditions. As shown in Figure 7A, after tail vein injection of Y6CT-NPs, the mouse vessels rapidly "light up," with a fluorescent signal appearing in the NIR-II window. Adjusting the filter from 900 nm to 1100 nm gradually improves the clarity of the fluorescence image. At the 1100 nm filter, the abdominal vessels are clearly visible and distinct from surrounding tissue. Figures 7B, 7C, and 7D show resolution analysis under different filter conditions. As shown in Figures 7B, 7C, and 7D, the signal-to-noise ratio (SBR) of the images increases with the gradual adjustment of the filter conditions, reaching 1.79 at the 1100 nm filter, while the half-peak width decreases to 0.2738 mm. These results demonstrate that Y6CT-NPs possess excellent capability for high-resolution NIR-II fluorescence imaging of mouse abdominal vessels under white light excitation.

[0108] Example 3

[0109] The high-resolution NIR-II fluorescence imaging capability of the nano-imaging agent Y6CT-NPs during the ischemia-reperfusion process in mice was tested: 100 μL of 300 μmol / L Y6CT-NPs were injected into BALBC / b mice via the tail vein. The liver of the mice was then imaged using a near-infrared II small animal living imager. The excitation light source was the near-infrared II small animal living imager illumination light source (16.5 mW cm -2 ), the results are shown in Figure 8A, Figure 8B, Figure 8C and Figure 8D, and the fluorescence signal intensity analysis was performed on different areas, and the results are shown in Figure 9.

[0110] Figures 8A, 8B, 8C, and 8D are real-time NIR-II fluorescence imaging images of the nano-imaging reagent Y6CT-NPs during the ischemia-reperfusion process in mice. Figure 9 is an analysis of the fluorescence intensity of different regions during the ischemia-reperfusion process of the mouse liver monitored by Y6CT-NPs. Combining Figures 8B and 9, it can be seen that after the tail vein injection of Y6CT-NPs, the healthy part of the liver (rectangular area) can be seen with obvious NIR-II fluorescence signals (fluorescence intensity is recorded as I R1 ), while the left lower lobe (ischemic area, elliptical area) has negligible fluorescence signal (0.03I) because the portal vein is blocked by hemostatic clamps. R1 ). The hemostatic clamp was removed 1 hour after injection, and the NIR-II fluorescence signal in the elliptical area gradually increased to 0.33I R1 , indicating that blood flow was restored to the ischemic part (C in Figure 8), but its fluorescence intensity was much lower than that of the normal liver, which was related to the metabolism of Y6CT-NPs. After the injection of Y6CT-NPs again, the fluorescence intensity of the elliptical area increased from 0.33I R1 Rapidly rise to 0.90I R1 , which is similar to the fluorescence signal changes of healthy liver (1.19I R1 to 1.74I R1 ), further demonstrating that the liver's blood supply function has been restored. These results fully demonstrate that Y6CT-NPs can achieve real-time high-resolution NIR-II fluorescence imaging of the mouse liver ischemia-reperfusion process under white light excitation.

[0111] Example 4

[0112] The high-resolution NIR-II fluorescence imaging capability test of the nano-imaging agent Y6CT-NPs in the New Zealand white rabbit kidney transplantation process: The test of the Y6CT-NPs' fluorescence real-time monitoring capability of the kidney transplantation process is divided into three parts, namely, the fluorescence imaging capability test of the blood vessels in the kidney donor area, the real-time monitoring of the renal vascular anastomosis during kidney transplantation, and the real-time monitoring of the blood supply to the transplanted kidney ureter. Models were established to perform fluorescence imaging of these processes.

[0113] (1) Test of the real-time NIR-II fluorescence imaging capability of the nano-imaging agent Y6CT-NPs on the kidney donor area of ​​New Zealand white rabbits: 2 mL of Y6CT-NPs with a concentration of 300 μmol / L were injected into the New Zealand white rabbits through the ear vein. Then, the near-infrared zone II small animal living imager was used to perform NIR-II fluorescence imaging of the kidney donor area of ​​the New Zealand white rabbits. The excitation light source was a laparoscope LED cold white light source (20 mW cm -2 ), the results are shown in Figure 10.

[0114] Figure 10 shows real-time NIR-II fluorescence imaging images of the donor kidney area of ​​New Zealand white rabbits using the nano-imaging agent Y6CT-NPs. As can be seen from Figures 10C and 10D, with the injection of the nano-imaging agent Y6CT-NPs, the donor renal artery (hollow triangle arrow) and vein (solid triangle arrow) were immediately "lit up", and clear NIR-II fluorescence signals appeared. As time went on, the boundaries of the renal arteries and veins gradually became clear, and the renal fluorescence gradually increased (Figure 10E). When the kidney was rotated 180° counterclockwise around the renal arteries and veins, the boundaries of the renal arteries and veins remained clear and easy to identify (Figure 10F). These results fully demonstrate that Y6CT-NPs can perform high-resolution NIR-II fluorescence imaging of renal arteries and veins in different orientations under white light excitation.

[0115] (2) Testing the real-time monitoring ability of the nano-imaging agent Y6CT-NPs on renal vascular anastomosis during kidney transplantation: Whether the renal vascular anastomosis during kidney transplantation is successful is related to whether the kidney transplantation is successful. Therefore, monitoring the renal vascular anastomosis during kidney transplantation is very important. Four models were established to evaluate the real-time monitoring ability of Y6CT-NPs on renal vascular anastomosis during kidney transplantation: normal anastomosis of the transplanted renal vascular anastomosis, stenosis of the transplanted renal artery anastomosis (complete occlusion), stenosis of the transplanted renal vein anastomosis, and torsion of the transplanted renal vein anastomosis. The results are shown in Figure 11.

[0116] ① Normal anastomosis of transplanted kidney blood vessels

[0117] A normal anastomosis model of the transplanted renal vessels was constructed by anastomosing the donor renal artery and vein with the recipient renal artery and vein using a sliding thread. 2 mL of 300 μmol / L Y6CT-NPs were injected into New Zealand white rabbits through the ear vein. NIR-II fluorescence imaging of the transplanted kidney region of the New Zealand white rabbits was then performed using a near-infrared zone II small animal in vivo imaging device. The excitation light source was a laparoscopic white light source (20 mW cm -2 ), the result is shown in A of Figure 11.

[0118] Figure 11A shows real-time NIR-II fluorescence imaging of a transplanted kidney region with normal renal vascular anastomosis using the nanoimaging agent Y6CT-NPs. As shown in Figure 11A, after the nanoimaging agent Y6CT-NPs were injected and the arteriovenous hemostatic clamps were released, the transplanted renal artery first "lit up" ((II) in Figure 11A, hollow triangle arrow), displaying a clear NIR-II fluorescence signal. Subsequently, NIR-II fluorescence also appeared in the kidney and renal vein ((III) in Figure 11A, solid triangle arrow). When the kidney was rotated 180° counterclockwise around the renal artery and vein, the outlines of the renal artery and vein remained clearly visible. These changes in NIR-II fluorescence signals indicate that the transplanted renal vascular anastomosis was normal, with no bleeding at the anastomosis site. The transplanted renal vessels were unobstructed, with no stenosis of the anastomosis site.

[0119] ② Stenosis of the transplanted renal artery anastomosis (complete occlusion)

[0120] A model of renal artery anastomosis stenosis (complete occlusion) was constructed by anastomosing the donor renal artery and vein with the recipient renal artery and vein using a sliding thread. 2 mL of 300 μmol / L Y6CT-NPs were injected into New Zealand white rabbits via the ear vein. Then, NIR-II fluorescence imaging of the transplanted kidney region of the New Zealand white rabbits was performed using a near-infrared zone II small animal in vivo imager. The excitation light source was a laparoscopic white light source (20 mW cm -2 ), the result is shown in B of Figure 11.

[0121] Figure 11B shows real-time NIR-II fluorescence imaging of a transplanted kidney with renal artery anastomotic stenosis (complete occlusion) using the nanoimaging agent Y6CT-NPs. As shown in Figure 11B, after the Y6CT-NPs were injected and the arteriovenous hemostatic clamps were released, only the distal renal artery ((II) in Figure 11B, hollow triangle arrow) was illuminated by NIR-II fluorescence. However, no fluorescence signal was observed in the transplanted renal artery ((II) in Figure 11B, directional arrow) due to the stenosis of the transplanted renal artery anastomosis, indicating that the arterial stenosis blocked blood flow to the kidney. Subsequently, blood flow to the stenotic anastomosis of the transplanted renal artery was restored by adjusting the position of the transplanted renal vessels. However, only the proximal transplanted renal artery emitted weak fluorescence ((IV) in Figure 11B, directional arrow), indicating complete occlusion of the renal artery. Furthermore, the transplanted renal vein exhibited NIR-II fluorescence ((III) in Figure 11B, solid triangle arrow), indicating reflux of the inferior vena cava. These changes in NIR-II fluorescence signals indicate that the anastomosis of the transplanted renal artery is narrowed, resulting in complete occlusion of the arterial vessel.

[0122] ③ Stenosis of the transplanted renal vein anastomosis

[0123] A stenosis model of the transplanted renal vein anastomosis was constructed by anastomosing the donor renal artery and vein with the recipient renal artery and vein using a sliding thread. 2 mL of 300 μmol / L Y6CT-NPs were injected into New Zealand white rabbits via the ear vein. Then, NIR-II fluorescence imaging of the transplanted kidney region of the New Zealand white rabbits was performed using a near-infrared zone II small animal in vivo imaging device. The excitation light source was a laparoscopic white light source (20 mW cm -2 ), the result is shown in C in Figure 11.

[0124] Figure 11, C, shows real-time NIR-II fluorescence imaging of the transplanted kidney region with renal vein anastomosis stenosis using the nano-imaging agent Y6CT-NPs. As can be seen in Figure 11, C, after the nano-imaging agent Y6CT-NPs were injected, the arteriovenous hemostatic clamps were released, and the distal transplanted renal artery first emitted fluorescence ((II) in Figure 11, C, hollow triangle arrow), followed by the proximal transplanted renal artery ((III) in Figure 11, C, directional arrow), and finally the transplanted kidney emitted a weak fluorescence. Because the transplanted renal vein anastomosis was narrow, blood flow was difficult to return to the inferior vena cava through the transplanted renal vein anastomosis, and no fluorescence signal appeared in the transplanted renal vein in the early stage ((II) in Figure 11, C, solid triangle arrow, hollow triangle arrow). Subsequently, the transplanted kidney blood vessels were adjusted to open the narrowed blood flow at the transplanted renal vein anastomosis. As shown in (III) in C of Figure 11, blood flow from the transplanted kidney slowly flows back through the venous anastomosis to the distal end of the transplanted renal vein and the inferior vena cava. The transplanted renal vein emits a weak fluorescence ((III) in C of Figure 11, directional arrow), and the NIR-II fluorescence of the transplanted kidney is slightly enhanced. The kidney is then rotated 180° counterclockwise around the renal artery and vein, and the transplanted kidney and the transplanted renal artery and vein show consistent fluorescence signals ((IV) in C of Figure 11). These changes in NIR-II fluorescence signals indicate that the transplanted renal vein anastomosis is partially occluded, but not completely occluded. After adjusting the renal blood vessels, part of the transplanted renal vein is recanalized, and the blood perfusion of the transplanted kidney increases compared to before, but it still has not reached a normal state.

[0125] ④Twisted anastomosis of transplanted renal vein

[0126] A sliding thread was used to anastomose the donor renal artery and vein with the recipient renal artery and vein to construct a torsion model of the transplanted renal vein anastomosis. 2 mL of 300 μmol / L Y6CT-NPs were injected into New Zealand white rabbits through the ear vein. NIR-II fluorescence imaging of the transplanted kidney region of the New Zealand white rabbits was then performed using a near-infrared zone II small animal in vivo imaging device. The excitation light source was a laparoscopic white light source (20 mW cm -2 ), the result is shown in D of Figure 11.

[0127] Figure 11(D) shows real-time NIR-II fluorescence imaging of a renal transplant with a twisted renal vein anastomosis using the nanoimaging agent Y6CT-NPs. As can be seen in Figure 11(D), after the nanoimaging agent Y6CT-NPs were injected and the arteriovenous hemostatic clamps were released, the distal renal artery emitted a fluorescent signal ((II) in Figure 11(D), hollow triangle arrow), followed by the proximal renal artery ((II) in Figure 11(D), directional arrow), and finally, the renal transplant emitted a faint fluorescence signal. Because the renal vein anastomosis was twisted, blood flow had difficulty returning to the inferior vena cava through the renal vein anastomosis, and no fluorescent signal appeared in the renal vein in the early stages ((II) in Figure 11(D), solid triangle arrow and diamond arrow). Subsequently, the transplanted renal blood vessels were adjusted to open the stenotic blood flow at the transplanted renal vein anastomosis. As shown in FIG11D-III, blood flow out of the transplanted kidney quickly returned to the distal end of the transplanted renal vein and the inferior vena cava through the venous anastomosis. The transplanted renal vein emitted fluorescence ((III) in FIG11D, directional arrow), and the transplanted kidney NIR-II fluorescence increased to normal. The kidney was then rotated 180° clockwise around the renal artery and vein, and the transplanted kidney and the transplanted renal artery and vein showed consistent fluorescence signals ((IV) in FIG11D). These changes in NIR-II fluorescence signals indicate that the transplanted renal vein anastomosis was twisted. After adjusting the direction of the transplanted renal blood vessels, the transplanted renal vein was recanalized, and the transplanted kidney blood perfusion returned to normal.

[0128] By establishing a model of different renal vascular anastomosis conditions during kidney transplantation, the real-time monitoring capability of the nanoimaging agent Y6CT-NPs was evaluated. The above experimental results fully demonstrate that Y6CT-NPs can perform real-time high-resolution NIR-II fluorescence imaging monitoring of different renal vascular anastomosis conditions during kidney transplantation under white light excitation.

[0129] (3) Test of the ability of nano-imaging reagent Y6CT-NPs to monitor the blood supply of transplanted kidney ureter: The blood supply of transplanted kidney ureter is weak. If the blood supply of the terminal ureter of transplanted kidney is poor or even zero, it will lead to the failure of the transplanted kidney uretero-vesical anastomosis to heal, and urine will flow into the pelvic cavity to cause infection, which may lead to the death of the patient in severe cases. Therefore, it is very important to monitor the blood supply of transplanted kidney ureter in real time. Two models of transplanted kidney normal ureter and injured ureter were established to evaluate the real-time monitoring ability of Y6CT-NPs on the blood supply of transplanted kidney ureter. 2mL of Y6CT-NPs with a concentration of 300μmol / L were injected into New Zealand white rabbits through the ear vein, and then NIR-II fluorescence imaging of the donor kidney area of ​​New Zealand white rabbits was performed using a near-infrared zone II small animal living imager. The excitation light source was a laparoscopic white light source (20mW cm -2 ), the results are shown in Figure 12.

[0130] Figure 12 is a real-time monitoring image of the blood supply to the transplanted kidney ureter during kidney transplantation in New Zealand white rabbits using the nano-imaging agent Y6CT-NPs. As can be seen from Figure 12, after the injection of the nano-imaging agent Y6CT-NPs, fluorescent signals quickly appeared in the bilateral kidneys and arteries and veins, followed by gradual fluorescent signals in the ureters on both sides (D in Figure 12). The right ureter is a damaged ureter, and the left ureter is a normal ureter. It can be seen that the fluorescence signal of the left ureter is normal (D in Figure 12, four-pointed asterisk), and the right ureter only has a fluorescent signal at the proximal end due to the presence of a vascular clamp, and no fluorescent signal is seen at the distal end (D in Figure 12, asterisk), which indicates that the distal end of the right ureteral vascular clamp has no blood supply. After loosening the hemostatic clamp, the blood supply to the distal end of the right ureteral vascular clamp did not recover, because long-term ischemia caused necrosis of the ureteral blood vessels in this part. After trimming off the necrotic part, the blood supply to the distal end of the right ureter was restored. These results fully demonstrate that Y6CT-NPs can perform real-time high-resolution NIR-II fluorescence imaging monitoring of the blood supply of the transplanted kidney ureter under white light excitation.

[0131] Through tests in different model experiments, it was shown that Y6CT-NPs can perform high-resolution NIR-II fluorescence imaging of the kidney transplantation process in New Zealand white rabbits.

[0132] The test results of compounds HY6-NPs and FY6-NPs are basically consistent with the test results of the above-mentioned compound Y6CT-NPs.

[0133] As can be seen from the above examples, the preparation method of the white light-excited near-infrared II zone organic nano-imaging agent for vascular imaging provided by the present invention is simple, has excellent luminescence properties, can achieve high-resolution NIR-II imaging of mouse blood vessels under white light excitation, and has good application effect.

[0134] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. Other embodiments can be obtained based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. Application of a near-infrared second region organic fluorescent compound in the preparation of a biological imaging contrast agent, characterized in that: The near-infrared second region organic fluorescent compound has a structure shown in Formula I: In Formula I, R1 and R2 are branched or straight-chain alkyl groups; R3 is X is one or more of H, F, and Cl.

2. The application according to claim 1, wherein R1 and R2 are C1-C11 branched or straight-chain alkyl groups.

3. The application according to claim 1, wherein R3 is X is H, or X is H and F.

4. The application according to any one of claims 1 to 3, characterized in that, The near-infrared second region organic fluorescent compound has a structure shown in Formula Ia, Formula Ib or Formula Ic: C in the structure represented by Formula I-a, Formula I-b or Formula I-c 11 H 23 is a straight-chain alkyl group.

5. Use of a near-infrared second region organic fluorescent compound in non-diagnostic and non-therapeutic vascular fluorescence imaging; characterized in that: The near-infrared second region organic fluorescent compound has a structure shown in Formula I: In Formula I, R1 and R2 are branched or straight-chain alkyl groups; R3 is X is one or more of H, F, and Cl.

6. Use of a nanoimaging reagent in the preparation of a bioimaging contrast agent, characterized in that, The nanoimaging reagent includes a near-infrared second-region organic fluorescent compound having a structure shown in Formula I, and an organic coating agent coated on the surface of the near-infrared second-region organic fluorescent compound having the structure shown in Formula I; In Formula I, R1 and R2 are branched or straight-chain alkyl groups; R3 is X in R3 is one or more of H, F, and Cl.

7. The application according to claim 6, characterized in that, The organic coating agent includes one or more of methoxypolyethylene glycol amine, distearoyl phosphatidylethanolamine-polyethylene glycol, phosphatidylethanolamine-polyethylene glycol-maleimide, distearoyl phosphatidylethanolamine-polyethylene glycol-folic acid, distearoyl phosphatidylethanolamine-polyethylene glycol-thiol, distearoyl phosphatidylethanolamide-polyethylene glycol-carboxylic acid, distearoyl phosphatidylethanolamine-polyethylene glycol-azide, distearoyl ethanolamine-polyethylene glycol-biotin, 1-palmitoyl-2-oleoyl ethanolamine, 1-stearoyl-2-oleoyl lecithin, dipalmitoyl phosphatidylethanolamine-polyethylene glycol, polystyrene-g-polyethylene glycol, methoxy PEG poly(lactic-co-glycolic acid) copolymer, and poloxamer F127.

8. The application according to claim 6 or 7, characterized in that, The preparation method of the nanoimaging reagent includes the following steps: Mix an organic coating agent, a near-infrared second-region organic fluorescent compound having the structure shown in Formula I, and an organic solvent to obtain a mixed solution; mix the mixed solution with water and perform ultrasonic assembly to obtain an assembled liquid material; Load the assembled liquid material into a dialysis bag for dialysis to obtain a purified assembled material; After concentrating the purified assembled material, obtain a solution of the nanoimaging reagent.

9. The application according to claim 8, characterized in that, The mass ratio of the organic coating agent to the near-infrared second-region organic fluorescent compound having the structure shown in Formula I is (3-8):1; The cut-off molecular weight of the dialysis bag is 3500; the dialysis time is 48-72 h.

10. The application according to claim 8 or 9, characterized in that, The ultrasonic power of the ultrasonic assembly is 100-200 W, and the time is 3-10 min.

11. Use of a nanoimaging reagent in vascular fluorescence imaging for non-diagnostic and non-therapeutic purposes; characterized in that, The nano-imaging agent comprises a near-infrared second-region organic fluorescent compound having a structure shown in Formula I, and an organic coating agent coated on the surface of the near-infrared second-region organic fluorescent compound having a structure shown in Formula I; In Formula I, R1 and R2 are branched or straight-chain alkyl groups; R3 is X in R3 is one or more of H, F, and Cl.

12. The application according to claim 11, wherein The vascular fluorescence imaging is performed under white light excitation conditions, and the wavelength of the white light is 400-800 nm.

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