Near-infrared ii organic fluorescent probe, and preparation method therefor and application thereof

Abstract

Provided are a near-infrared II organic fluorescent probe, and a preparation method therefor and the application thereof. The fluorescent probe and an organic coating agent are mixed to prepare a nano-imaging reagent, which has good biocompatibility and biological stability; and the nano-imaging reagent has a high molar extinction coefficient and a high quantum yield, and has bright near-infrared II fluorescence under white-light excitation. Furthermore, the nano-imaging reagent can implement high-resolution imaging of blood vessels under white-light excitation. In addition, white light is used as an excitation light source during imaging, such that problems such as limited photon absorption, laser-induced biological damage and non-uniform irradiation caused by a single-wavelength excitation light source can be effectively avoided. Moreover, a laser light source is costly, while a white light source is cheaper and more readily available. Therefore, the nano-imaging reagent can be used for developing a high-performance biological imaging contrast agent, holding broad prospects in the field of in-vivo imaging for non-diagnostic and non-therapeutic purposes.

Description

A near-infrared second-region organic fluorescent probe and its preparation method and application Technical Field

[0001] The present invention belongs to the technical field of biochemical materials, and in particular relates to a near-infrared second-region organic fluorescent probe and a preparation method and application thereof. Background Art

[0002] In vivo imaging technology uses imaging methods to qualitatively and quantitatively study biological processes in vivo at the tissue, cellular, and molecular levels without damaging the animal. This technology allows for non-invasive and intuitive observation of various biological processes in living animals. Its high sensitivity, high spatiotemporal resolution, and real-time monitoring capabilities have garnered widespread attention and are of great significance to fields such as life sciences and medicine. 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 candidates for high-performance fluorescent imaging reagents. However, NIR-II organic fluorescent molecules have long absorption and emission wavelengths, small band gaps, and readily decay their excited-state energy through nonradiative transitions, resulting in low luminescence efficiency. Therefore, the development of highly efficient NIR-II organic nanoimaging reagents is of great significance.

[0003] On the other hand, due to the long absorption wavelength, low quantum yield, and limited molar extinction coefficient of NIR-II organic fluorescent molecules, in vivo fluorescence imaging using NIR-II organic nanoimaging agents requires the use of expensive, specific-wavelength lasers as excitation sources. However, the use of single-wavelength lasers also presents challenges such as limited photon absorption and laser-induced biological damage. Furthermore, single-wavelength lasers lack uniform irradiation, with energy decaying from the center of the spot, which also compromises the quality of in vivo imaging. In contrast, white light, a safe and visible excitation light source, including general lighting, surgical shadowless lamps, and laparoscopic light sources, offers a wide continuous spectrum, is inexpensive, and readily available, making it an ideal excitation source for in vivo fluorescence imaging. However, the application of white light as an excitation source in NIR-II fluorescence imaging has not yet been reported, requiring 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 significant challenges.

[0004] Summary of the Invention

[0005] The present invention aims to provide a method for preparing a white-light-excited ADA-type near-infrared region II organic fluorescent probe and its application. The fluorescent probe provided by the present invention is mixed with an organic capping agent to prepare a nano-imaging agent with good biocompatibility and biostability. It also exhibits a high molar extinction coefficient and quantum yield, and bright near-infrared region II fluorescence under white-light excitation. This agent can be used to construct high-performance bioimaging contrast agents, with promising applications in non-therapeutic in vivo imaging, imaging-guided surgery, and other fields.

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

[0007] The present invention provides a near-infrared second-region organic fluorescent probe having a structure shown in Formula I:

[0008] In formula I, R1 and R2 are independently C 1～16 branched or straight chain alkyl groups;

[0009] The R3 is X in R3 is H and / or halogen.

[0010] Preferably, R1 and R2 are independently C 1～11 Branched or straight chain alkyl.

[0011] Preferably, X in R3 is one or more of H, F, Cl and Br.

[0012] Preferably, it has the structure shown in Formula Ia, Ib or Ic;

[0013] The present invention provides a method for preparing the near-infrared second region organic fluorescent probe described in the above technical solution, comprising the following steps:

[0014] Mixing a compound represented by Formula II, a compound represented by Formula III, a compound represented by Formula IV, or a compound represented by Formula V, an organic base catalyst, and an organic solvent, and performing a Brainerd reaction to obtain a near-infrared second-region organic fluorescent probe having a structure represented by Formula I;

[0015] Preferably, the molar ratio of the compound of formula II, the compound of formula III and the compound of formula IV or the compound of formula V is 1:1:1;

[0016] The organic base catalyst is pyridine; the molar ratio of the compound represented by the structure of formula II to the organic base catalyst is 1:3-10;

[0017] The temperature of the brain wenge reaction is 25 to 70° C., and the time is 8 to 24 hours.

[0018] The present invention provides a fluorescent probe-coated nanoparticle, comprising the near-infrared region II organic fluorescent probe described in the above technical solution or the near-infrared region II organic fluorescent probe prepared by the preparation method described in the above technical solution, and an organic coating agent coated on the surface of the near-infrared region II organic fluorescent probe.

[0019] The present invention provides a method for preparing fluorescent probe-coated nanoparticles according to the above technical solution, comprising the following steps:

[0020] Mixing the near-infrared second region organic fluorescent probe described in the above technical solution or the near-infrared second region organic fluorescent probe prepared by the preparation method described in the above technical solution, an organic coating agent, and an organic solvent to obtain a mixed solution;

[0021] The mixed solution is mixed with water and ultrasonic co-precipitation is performed to obtain fluorescent probe-coated nanoparticles.

[0022] The present invention provides the use of the near-infrared second region organic fluorescent probe described in the above technical solution, the near-infrared second region organic fluorescent probe prepared by the preparation method described in the above technical solution, the fluorescent probe-coated nanoparticles described in the above technical solution, or the fluorescent probe-coated nanoparticles prepared by the preparation method described in the above technical solution in the preparation of biological imaging contrast agents.

[0023] The present invention provides the use of the near-infrared second region organic fluorescent probe described in the above technical solution, the near-infrared second region organic fluorescent probe prepared by the preparation method described in the above technical solution, the fluorescent probe-coated nanoparticles described in the above technical solution, or the fluorescent probe-coated nanoparticles prepared by the preparation method described in the above technical solution in fluorescence imaging for non-diagnostic and non-therapeutic purposes.

[0024] The present invention provides a near-infrared second-zone organic fluorescent probe having a structure shown in Formula I. The fluorescent probe provided by the present invention has an ADA (acceptor-donor-acceptor structure) type large π conjugated planar structure. The fluorescent probe provided by the present invention and an organic capping agent are mixed to prepare nanoparticles for use in imaging agents, which have good biocompatibility and biostability. The fluorescent probe and nanoparticles provided by the present invention have high molar extinction coefficients and quantum yields as nano-imaging agents, and have bright near-infrared second-zone fluorescence under white light excitation. Furthermore, the fluorescent probe provided by the present invention can achieve high-resolution imaging of blood vessels under white light excitation; in addition, using 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 fluorescent probe with a structure shown in Formula I provided by the present invention can be used as a nano-imaging agent to construct a high-performance biological imaging contrast agent, and has broad prospects in the fields of in vivo imaging for non-therapeutic purposes, imaging-guided surgery, and the like.

[0025] Results from the examples demonstrate that the fluorescent probe molecule of Formula I provided by the present invention exhibits an acceptor-donor-acceptor structure, effectively extending the absorption and emission window even beyond the near-infrared window. It also possesses a high molar extinction coefficient and a broad absorption band, significantly enhancing the fluorescent probe's white light absorption capacity. The nano-imaging agent described herein emits bright near-infrared second-region fluorescence under white light excitation, with an emission wavelength extending up to 1400 nm, effectively avoiding self-absorption and reducing background interference.

[0026] The present invention provides a method for preparing the near-infrared second region organic fluorescent probe described in the above technical solution. The preparation method provided by the present invention is simple to synthesize, requiring only a one-step metal-free catalytic Naowenge reaction, is easy to operate, and is suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 shows the particle size diagrams of the nano-imaging agents HY6CT-NPs, FY6CT-NPs, and CY6CT-NPs, as well as the transmission electron micrographs of the nano-imaging agents HY6CT-NPs. Figure 1 A shows the transmission electron micrograph of the nano-imaging agents HY6CT-NPs; Figure 1 B, C, and D show the particle size diagrams of the nano-imaging agents HY6CT-NPs, FY6CT-NPs, and CY6CT-NPs, respectively.

[0028] Figure 2 shows the absorption spectra of nano-imaging agents HY6CT-NPs, FY6CT-NPs, and CY6CT-NPs in aqueous solution;

[0029] Figure 3 shows the emission spectra of aqueous solutions of nano-imaging reagents HY6CT-NPs, FY6CT-NPs, and CY6CT-NPs under white light excitation;

[0030] Figure 4 shows the relative quantum yield test results of nano-imaging agents HY6CT-NPs, FY6CT-NPs, and CY6CT-NPs using the commercial dye IR26 as a reference;

[0031] Figure 5 shows NIR-II imaging images of HY6CT-NPs, FY6CT-NPs, CY6CT-NPs and ICG aqueous solution at the same concentration under white light excitation;

[0032] Figure 6 shows the NIR-II fluorescence imaging images of the abdominal blood vessels of normal mice using the nano-imaging agent FY6CT-NPs under different filter conditions and the resolution analysis graphs under different filter conditions; Figure 6 A shows the NIR-II fluorescence imaging images of the abdominal blood vessels of normal mice using the nano-imaging agent Y6CT-NPs under different filter conditions; Figure 6 B, C and D are the resolution analysis graphs under different filter conditions. DETAILED DESCRIPTION

[0033] The present invention provides a near-infrared second-region organic fluorescent probe having a structure shown in Formula I:

[0034] In formula I, R1 and R2 are independently C 1～16 branched or straight chain alkyl groups;

[0035] The R3 is X in R3 is H and / or halogen.

[0036] In the present invention, R1 is preferably C 1～11 Branched or straight chain alkyl, more preferably C 3～11 Branched or straight chain alkyl, more preferably C 5～11 Branched or straight chain alkyl.

[0037] In a specific embodiment of the present invention, R1 is preferably C 11 H 23 Straight chain alkyl.

[0038] In the present invention, R2 is preferably C 1～11 Branched or straight chain alkyl, more preferably C 3～11 Branched or straight chain alkyl, more preferably C 5～8 Branched or straight chain alkyl.

[0039] In a specific embodiment of the present invention, R2 is preferably

[0040] In the present invention, the R3 is preferably

[0041] In the present invention, X in R3 is preferably one or more of H, F, Cl and Br, more preferably one or more of H, F and Cl.

[0042] In a specific embodiment of the present invention, X in R3 is preferably H, or H and F, or H and Cl.

[0043] In the present invention, the near-infrared second region organic fluorescent probe having the structure shown in Formula I preferably has the structure shown in Formula Ia, Ib or Formula Ic;

[0044] R1 in Formula Ia, Ib or Ic is C 11 H 23 of a straight chain alkyl group.

[0045] The present invention provides a method for preparing the near-infrared second region organic fluorescent probe described in the above technical solution, comprising the following steps:

[0046] Mixing a compound represented by Formula II, a compound represented by Formula III, a compound represented by Formula IV, or a compound represented by Formula V, an organic base catalyst, and an organic solvent, and performing a Brainerd reaction to obtain a near-infrared second-region organic fluorescent probe having a structure represented by Formula I;

[0047] In the present invention, unless otherwise specified, all preparation raw materials / components are commercially available products well known to those skilled in the art.

[0048] In the present invention, the compound of the structure represented by formula IV is preferably a dicyanoindanone compound represented by formula IV-a, a dicyanoindanone compound represented by formula IV-b, or a dicyanoindanone compound represented by formula IV-c;

[0049] In the present invention, the molar ratio of the compound represented by formula II, the compound represented by formula III, and the compound represented by formula IV or the compound represented by formula V is preferably 1:1:1.

[0050] In the present invention, the organic base catalyst is preferably pyridine. The molar ratio of the compound represented by formula II to the organic base catalyst is preferably 1:3 to 10, more preferably 1:5.

[0051] In the present invention, the organic solvent is preferably chloroform. The present invention has no particular limitation on the amount of the organic solvent used, as long as it allows the reaction to proceed smoothly. The present invention has no particular requirements on the specific implementation of the mixing.

[0052] In the present invention, the temperature of the Novo-Wenge reaction is preferably 25 to 70° C., more preferably 40 to 60° C. In the present invention, the time of the Novo-Wenge reaction is preferably 8 to 24 hours, more preferably 12 to 24 hours.

[0053] In the present invention, the Novo-Wenge reaction is preferably carried out under a protective gas atmosphere, preferably nitrogen or argon. In the present invention, a TLC plate (thin layer chromatography plate) is preferably used to monitor the reaction.

[0054] After the Novowegian reaction, the present invention preferably performs post-treatment on the obtained Novowegian reaction solution, and the post-treatment preferably includes the following steps:

[0055] The Naowenge reaction solution is extracted and concentrated to obtain a concentrated solution.

[0056] The concentrated solution is subjected to column chromatography separation and recrystallization to obtain a pure product of a near-infrared second-region organic fluorescent probe having a structure shown in Formula I.

[0057] In the present invention, the extraction preferably comprises the following steps: after the reaction system has cooled to room temperature, water is added to the reaction solution, and the aqueous phase is extracted multiple times with dichloromethane. The present invention further concentrates the organic phase obtained by the extraction. The present invention has no particular requirements for the concentration method; any concentration method familiar to those skilled in the art may be used.

[0058] In the present invention, the eluent for column chromatography separation is preferably a mixture of dichloromethane and petroleum ether, wherein the volume ratio of dichloromethane to petroleum ether is preferably 2:1. After the column chromatography is completed, the present invention preferably removes the solvent from the column chromatography product. The present invention is not particularly limited to the method for removing the solvent, and conventional solvent removal methods such as rotary evaporation can be used.

[0059] In the present invention, the solvent for recrystallization is preferably dichloromethane and n-hexane. The volume ratio of dichloromethane to n-hexane is preferably 1:3-6. A specific embodiment of the recrystallization preferably includes dissolving the crude product separated by column chromatography in dichloromethane, slowly adding n-hexane under ultrasonic conditions, and filtering after the target product precipitates. The resulting solid is the target product.

[0060] The present invention provides a fluorescent probe-coated nanoparticle, comprising the near-infrared region II organic fluorescent probe described in the above technical solution or the near-infrared region II organic fluorescent probe prepared by the preparation method described in the above technical solution, and an organic coating agent coated on the surface of the near-infrared region II organic fluorescent probe.

[0061] In the present invention, the organic coating agent is preferably 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.

[0062] In the present invention, the particle size of the fluorescent probe-coated nanoparticles is preferably 50 to 200 nm, more preferably 100 to 200 nm.

[0063] The present invention provides a method for preparing fluorescent probe-coated nanoparticles according to the above technical solution, comprising the following steps:

[0064] Mixing the near-infrared second region organic fluorescent probe described in the above technical solution or the near-infrared second region organic fluorescent probe prepared by the preparation method described in the above technical solution, an organic coating agent, and an organic solvent to obtain a mixed solution;

[0065] The mixed solution is mixed with water and ultrasonic co-precipitation is performed to obtain fluorescent probe-coated nanoparticles.

[0066] The present invention comprises mixing the near-infrared region II organic fluorescent probe described in the above technical solution or the near-infrared region II organic fluorescent probe prepared by the preparation method described in the above technical solution, an organic capping agent, and an organic solvent to produce a mixed solution. In the present invention, the mass ratio of the fluorescent probe to the organic capping agent is preferably 1:3-8, more preferably 1:5-6. The organic solvent is tetrahydrofuran. The present invention has no particular requirements for the amount of the organic solvent used.

[0067] After obtaining the mixed solution, the present invention mixes the mixed solution with water and performs ultrasonic coprecipitation to obtain fluorescent probe-coated nanoparticles. In the present invention, the power of the ultrasonic coprecipitation is preferably 100-200W, more preferably 150W, and the time is preferably 3-10 minutes, more preferably 5 minutes.

[0068] In the present invention, the assembly liquid is directly obtained after the ultrasonic coprecipitation. The present invention preferably further comprises: placing the assembly liquid into a dialysis bag for dialysis to obtain a purified assembly material; and concentrating the purified assembly material to obtain a solution of the nano-imaging agent. In the present invention, the molecular weight cut-off of the dialysis bag is preferably 3500; and the dialysis time is preferably 48 to 72 hours. 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 obtains a concentrated solution, and the present invention preferably uses a syringe filter to filter impurities from the concentrated solution to obtain a solution of the nano-imaging agent.

[0069] The present invention prepares the fluorescent probe into water-dispersible nanoparticles, which is conducive to imaging blood vessels for non-therapeutic and non-diagnostic purposes under white light excitation.

[0070] The present invention provides the use of the near-infrared second region organic fluorescent probe described in the above technical solution or the near-infrared second region organic fluorescent probe prepared by the preparation method described in the above technical solution or the fluorescent probe-coated nanoparticles described in the above technical solution or the fluorescent probe-coated nanoparticles prepared by the preparation method described in the above technical solution in the preparation of biological imaging contrast agents.

[0071] 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.

[0072] The present invention provides the use of the near-infrared second region organic fluorescent probe described in the above technical solution, the near-infrared second region organic fluorescent probe prepared by the preparation method described in the above technical solution, the fluorescent probe-coated nanoparticles described in the above technical solution, or the fluorescent probe-coated nanoparticles prepared by the preparation method described in the above technical solution in fluorescence imaging for non-therapeutic but diagnostic purposes.

[0073] In the present invention, the non-diagnostic and non-therapeutic fluorescence imaging is preferably vascular fluorescence imaging.

[0074] In the present invention, the non-diagnostic and non-therapeutic fluorescence imaging is preferably in vivo imaging.

[0075] In a specific embodiment of the present invention, the application of the fluorescence imaging preferably includes vascular fluorescence imaging during monitoring of liver ischemia-reperfusion, or vascular fluorescence imaging during monitoring of kidney transplantation, or vascular fluorescence imaging during fluorescence imaging-guided surgery.

[0076] In the present invention, the device for vascular imaging is a near-infrared two-zone in vivo imager.

[0077] In the present invention, the fluorescence imaging is performed under white light excitation conditions, and the white light excitation light source is a near-infrared second-zone in vivo imaging instrument lighting lamp with a wavelength range of 400 to 800 nm.

[0078] In the present invention, the effective concentration of the nano-imaging agent injected into the mouse during the vascular fluorescence imaging is not less than 500 μmol L -1 , the volume is not less than 100 μL.

[0079] 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.

[0080] Example 1

[0081] Prepared according to the following reaction scheme:

[0082] TPB-CHO (103 mg, 0.1 mmol), CPT30 (20 mg, 0.1 mmol), and HIC (20 mg, 0.1 mmol) were dissolved in 10 mL of chloroform. Pyridine (40 mg, 0.5 mmol) was added dropwise to the solution, and the mixture was reacted at 50°C for 12 h. After the solution was cooled to room temperature, the solvent was evaporated in vacuo, and the residue was dissolved in dichloromethane (30 mL) and extracted with water (30 mL). The organic phases were combined, dried over anhydrous Na2SO4, and purified by silica gel column chromatography using petroleum ether / dichloromethane 2:1 as the eluent. The crude product was dissolved in dichloromethane and recrystallized by slowly adding n-hexane under ultrasonic conditions. The precipitated solid was filtered to obtain HY6CT (yield: 78 mg, 55%) as a brown-black powder.

[0083] 1 H NMR(600MHz,Chloroform-d)δ9.11(s,1H),9.00(s,1H),8.66(d,J=7.0Hz,1H),8.33(s,1H),7.98-7.96(m,2H),7.78-7.74(m,2H),4.80(t,J=8.7 Hz,4H),3.35-3.10(m,4H),2.21-2.17(m,2H),1.87-1.84(m,4H),1.51- 1.47(m,4H),1.38-0.96(m,44H),0.86-0.80(m,12H),0.71-0.65(m,6H).

[0084] 13C NMR(151MHz,Chloroform-d)δ188.53,181.68,156.66,153.23,153.01,147.32,145.03,144.93,142.60,1 42.34,140.04,137.84,136.89,136.17,135.29,135.04,134.28,133.60,133.32,133.25,127.32,126.98, 125.18,123.63,120.86,115.35,113.63,113.50,68.28,55.67,40.38,40.32,31.91,31.14,29.82,29.64,29.62,29.52,29.50,29.33,27.63,27.55,23.29,22.89,22.84,22.67,14.10,13.75,13.71,10.38,10.34.

[0085] Example 2

[0086] Prepared according to the following reaction scheme:

[0087] TPB-CHO (103 mg, 0.1 mmol), CPT30 (20 mg, 0.1 mmol), and FIC (23 mg, 0.1 mmol) were dissolved in 10 mL of chloroform. Pyridine (40 mg, 0.5 mmol) was added dropwise to the solution, and the mixture was reacted at 50°C for 12 h. After the solution was cooled to room temperature, the solvent was evaporated in vacuo, and the residue was dissolved in dichloromethane (30 mL) and extracted with water (30 mL). The organic phases were combined, dried over anhydrous Na2SO4, and purified by silica gel column chromatography using petroleum ether / dichloromethane 2:1 as the eluent. The crude product was dissolved in dichloromethane and recrystallized by slowly adding n-hexane under ultrasonic conditions. The precipitated solid was filtered to obtain FY6CT (yield: 82 mg, 58%) as a brown-black powder.

[0088] 1H NMR(600MHz,Chloroform-d)δ9.16(s,1H),9.09(s,1H),8.57(s,1H),8.40(s,1H),7.97(s,1H),7.71(t,J=7.2,1H),4.79-4.75(m,4H), 3.24-3.20(m,4H),2.17-2.09(m,2H),1.88-1.84(m,4H),1.52-1.48(m,4H),1.37-0.96(m,44H),0.87-0.74(m,12H),0.67-0.64(m,6H).

[0089] 13 C NMR(151MHz,Chloroform-d)δ186.09,181.60,158.48,153.84,153.09,147.24,145.12,138.00,137.78 ,136.05,136.01,135.74,133.34,133.28,133.16,130.33,129.79,127.02,125.29,119.72,114.96,11 4.51,113.77,113.57,66.49,55.74,40.42,40.35,31.90,31.18,31.10,29.81,29.71,29.63,29.61,29.51,29.46,29.32,27.58,23.35,23.27,22.93,22.85,22.67,14.09,13.78,13.71,13.68,10.44,10.36.

[0090] Example 3

[0091] Prepared according to the following reaction scheme:

[0092] TPB-CHO (103 mg, 0.1 mmol), CPT30 (20 mg, 0.1 mmol), and CIC (26 mg, 0.1 mmol) were dissolved in 10 mL of chloroform. Pyridine (40 mg, 0.5 mmol) was added dropwise to the solution, and the mixture was reacted at 50°C for 12 h. After the solution was cooled to room temperature, the solvent was evaporated in vacuo, and the residue was dissolved in dichloromethane (30 mL) and extracted with water (30 mL). The organic phases were combined and dried over anhydrous Na2SO4 and purified by silica gel column chromatography using petroleum ether / dichloromethane 2:1 as eluent. The crude product was dissolved in dichloromethane and recrystallized by slowly adding n-hexane under ultrasonic conditions. The precipitated solid was filtered to obtain a brown-black powder CY6CT (yield: 75 mg, 52%).

[0093] 1 HNMR(600MHz,Chloroform-d)δ9.07(s,1H),9.00(s,1H),8.70(s,1H),8.33(s,1H),7.97(d,J=12.1Hz,2H),4.81(t,J=10.6Hz,4H),3. 20-3.14(m,4H),2.20-2.17(m,2H),1.86-1.84(m,4H),1.49-1.46(m,4H),1.35-0.96(m,44H),0.87-0.80(m,12H),0.70-0.65(m,6H).

[0094] 13 C NMR(151MHz,Chloroform-d)δ186.12,181.59,153.12,147.39,145.16,142.60,139.47,139.12,138.72,138.04,1 36.41,136.11,135.53,134.03,133.50,133.44,133.35,130.82,129.98,127.34,127.17,126.80,125.20,124.93 ,119.73,115.05,114.96,114.55,113.54,55.80,40.46,40.42,31.89,31.16,31.08,29.80,29.71,29.62,29.59,29.49,29.46,29.43,29.30,27.73,27.68,23.42,23.34,22.85,22.81,22.65,14.05,13.71,13.67,10.37,10.31.

[0095] Example 4

[0096] Prepared according to the following reaction scheme:

[0097] 2 mg of fluorescent probes (HY6CT prepared in Example 1, FY6CT prepared in Example 2, and CY6CT prepared in Example 3) were mixed with 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 impurities were filtered out with a syringe filter to finally obtain fluorescent probe-coated nanoparticles (nanoimaging agents, respectively denoted as HY6CT-NPs, FY6CT-NPs, and CY6CT-NPs). The effective concentration of the nanoimaging agent was determined to be 500 μmol L-1 by the pre-established concentration curve. -1 .

[0098] Performance testing:

[0099] (1) Particle size test of fluorescent probe-coated nanoparticles HY6CT-NPs, FY6CT-NPs, and CY6CT-NPs: 30 μL of nanofluorescent probe was added to 3 mL of deionized water, and then the particle size was measured using transmission electron microscopy and dynamic light scattering, respectively. The results are shown in Figure 1.

[0100] Figure 1 (A) shows a transmission electron micrograph of the nanofluorescent probe HY6CT-NPs, while Figures 1 (B), (C), and (D) show the particle size of HY6CT-NPs, FY6CT-NPs, and CY6CT-NPs, respectively. As shown in Figure 1, the sizes of HY6CT-NPs, FY6CT-NPs, and CY6CT-NPs are 130 nm, 149 nm, and 141 nm, respectively.

[0101] (2) Absorption spectrum test of nanofluorescent probes: The absorption spectra of HY6CT-NPs, FY6CT-NPs, CY6CT-NPs, and ICG in aqueous solution were measured using a UV-visible spectrophotometer equipped with an integrating sphere module. The test concentration was 10 μmol L -1 , the results are shown in Figure 2.

[0102] Figure 2 shows the absorption spectra of fluorescent probe-coated nanoparticles HY6CT-NPs, FY6CT-NPs, and CY6CT-NPs in aqueous solution. As can be seen from Figure 2, the three fluorescent probe-coated nanoparticles HY6CT-NPs, FY6CT-NPs, and CY6CT-NPs all exhibit high absorptivity in the range of 400-1000 nm. The molar extinction coefficients of HY6CT-NPs, FY6CT-NPs, and CY6CT-NPs at the maximum absorption point are 6.35×10 4 (780nm), 8.38×10 4 (806nm) and 7.85×10 4 Lmol -1 cm -1(803nm). Compared with ICG, the nanofluorescent probe exhibits a wider absorption in the white light region (400-800nm).

[0103] (3) Emission spectrum test of fluorescent probe-coated nanoparticles: The emission spectra of HY6CT-NPs, FY6CT-NPs, and CY6CT-NPs in aqueous solution were measured using a steady-state transient fluorescence spectrometer. The test concentration was 10 μmol L -1 , the results are shown in Figure 3.

[0104] Notably, the three fluorescent probe-coated nanoparticles exhibited NIR-II fluorescence emission extending to 1400 nm under white light excitation. The maximum emission peaks for HY6CT-NPs, FY6CT-NPs, and CY6CT-NPs were 947 nm, 948 nm, and 954 nm, respectively. These results indicate that HY6CT-NPs, FY6CT-NPs, and CY6CT-NPs can emit NIR-II fluorescence under white light excitation. In contrast, the spectrum of ICG was barely detectable at the same concentration.

[0105] (4) Relative quantum yield of fluorescent probe-coated nanoparticles HY6CT-NPs, FY6CT-NPs, and CY6CT-NPs in aqueous solution: The NIR-II commercial fluorescent dye IR26 was selected as a reference to test the relative quantum yield of HY6CT-NPs, FY6CT-NPs, and CY6CT-NPs. IR26 was prepared as a dichloroethane solution. First, the absorption spectra of HY6CT-NPs, FY6CT-NPs, CY6CT-NPs, and IR26-DCE were tested respectively, and the corresponding concentrations when their absorbance values ​​at 808 nm were 0.02, 0.04, 0.06, 0.08, and 0.10, respectively, were determined. Then, the fluorescence spectra of the corresponding concentrations were tested when excited at 808 nm. The area integration of the fluorescence spectra in the emission wavelength range of 850-1400 nm was used as the ordinate, and linear regression analysis was performed with the absorbance value as the abscissa to obtain the slope. The relative quantum yields of HY6CT-NPs, FY6CT-NPs, and CY6CT-NPs were then calculated using the equation described in Formula 1.

[0106] The refractive index of water described in Formula 1 is 1.333, and the refractive index of dichloroethane is 1.4448.

[0107] Figure 4 shows the relative quantum yields of the nanoimaging agents HY6CT-NPs, FY6CT-NPs, and CY6CT-NPs using IR26 as a reference. As shown in Figure 4, the relative quantum yields of HY6CT-NPs, FY6CT-NPs, and CY6CT-NPs are 25.22%, 18.85%, and 9.78%, respectively.

[0108] Figure 5 shows the NIR-II fluorescence images of the nano-imaging reagents HY6CT-NPs, FY6CT-NPs, CY6CT-NPs and ICG aqueous solution at the same concentration under white light excitation (white light illumination). The PE tubes filled with the nano-imaging probes at the same concentration were imaged using the NIR-II small animal imager. The test concentration was 10 μmol L -1 , the results are shown in Figure 5.

[0109] As can be seen from Figure 5, the three imaging probes can monitor obvious NIR-II fluorescence signals under white light excitation. In contrast, ICG, as a NIR-II commercial dye, has no fluorescence signal detected under white light excitation.

[0110] Example 5

[0111] The ability of fluorescent probe-coated nanoparticles FY6CT-NPs to perform high-resolution NIR-II fluorescence imaging of mouse abdominal vessels was tested: 100 μL of 500 μmol / L FY6CT-NPs was injected into BALBC / b mice via the tail vein. The mice were then imaged using a near-infrared II in vivo imaging system. The excitation light source was a near-infrared II in vivo imaging light source (16.5 mW cm -2 ), filters of different wavelengths were adjusted to collect fluorescence images, and the results are shown in Figure 6A. The imaging resolution under different filter conditions was analyzed by imaging software, and the results are shown in Figure 6B, Figure 6C, and Figure 6D.

[0112] Figure 6A shows NIR-II fluorescence imaging of normal mouse abdominal vessels using fluorescent probe-coated nanoparticles (FY6CT-NPs) under different filter conditions. As shown in Figure 6A, after tail vein injection of FY6CT-NPs, the mouse vessels rapidly "illuminate," 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 1100 nm, the abdominal vessels are clearly visible and distinct from surrounding tissue. Figures 6B, 6C, and 6D show resolution analysis under different filter conditions. As shown in Figures 6B, 6C, and 6D, the signal-to-noise ratio (SBR) of the images increases with increasing filter conditions, reaching 2.94 at 1100 nm, while the full width at half maximum decreases to 0.1440 mm. These results demonstrate that FY6CT-NPs possess excellent capabilities for high-resolution NIR-II fluorescence imaging of mouse abdominal vessels under white light excitation.

[0113] The high-resolution NIR-II fluorescence imaging capabilities of HY6CT-NPs and CY6CT-NPs on mouse abdominal blood vessels under white light excitation were similar to those of FY6CT-NPs.

[0114] 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.

[0115] 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. A near-infrared second-region organic fluorescent probe, characterized in that, It has the structure shown in Formula I: In Formula I, the R1 and R2 are independently C 1～16 branched or straight-chain alkyl; The R3 is X in R3 is H and / or halogen.

2. The near-infrared second-region organic fluorescent probe according to claim 1, wherein R1 and R2 are independently C 1～11 branched or straight-chain alkyl groups.

3. The near-infrared region II organic fluorescent probe according to claim 1, wherein Multiple Xs in the said R3 are one or more of H, F, Cl and Br.

4. The near-infrared II organic fluorescent probe according to any one of claims 1 to 3, characterized in that, Having the structure shown in Formula I-a, I-b or Formula I-c; 5. The preparation method of the near-infrared II organic fluorescent probe according to any one of claims 1 to 4, characterized in that, Comprising the following steps: Mix the compound with the structure shown in Formula II, the compound with the structure shown in Formula III, the compound with the structure shown in Formula IV or the compound with the structure shown in Formula V, an organic base catalyst and an organic solvent, and carry out the Knoevenagel reaction to obtain a near-infrared second-region organic fluorescent probe with the structure shown in Formula I; 6. The preparation method according to claim 5, characterized in that, The molar ratio of the compound of the structure shown in formula II, the compound of the structure shown in formula III and the compound of the structure shown in formula IV or the compound of the structure shown in formula V is 1:1:1; The organic base catalyst is pyridine; the molar ratio of the compound of the structure shown in formula II to the organic base catalyst is 1:3 - 10; The temperature of the Knoevenagel reaction is 25 - 70 °C, and the time is 8 - 24 h.

7. The preparation method according to claim 5, characterized in that, After the Knoevenagel reaction, post-treatment is carried out on the obtained Knoevenagel reaction solution, and the post-treatment comprises the following steps: The Knoevenagel reaction solution is extracted and concentrated to obtain a concentrated solution; Column chromatography separation and recrystallization are carried out on the concentrated solution to obtain a pure product of the near-infrared second-region organic fluorescent probe with the structure shown in formula I; the eluent for the column chromatography separation is a mixed solution of dichloromethane and petroleum ether, and the volume ratio of dichloromethane to petroleum ether is 2:1; the solvent for the recrystallization is dichloromethane and n-hexane, and the volume ratio of dichloromethane to n-hexane is 1:3 - 6.

8. A fluorescent probe-coated nanoparticle, characterized in that, Comprising the near-infrared second-region organic fluorescent probe described in any one of claims 1 - 4 or the near-infrared second-region organic fluorescent probe prepared by the preparation method described in any one of claims 5 - 7, and an organic coating agent coated on the surface of the near-infrared second-region organic fluorescent probe.

9. The fluorescent probe-coated nanoparticles according to claim 8, wherein The organic coating agent is 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 phosphatidylethanamide-polyethylene glycol-carboxylic acid, distearoyl phosphatidylethanolamine-polyethylene glycol-azide, distearoylethanolamine-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.

10. The fluorescent probe-coated nanoparticles according to claim 8 or 9, characterized in that, The particle size of the fluorescent probe-coated nanoparticles is 50 - 200 nm.

11. The preparation method of the fluorescent probe-coated nanoparticles according to any one of claims 8 to 10, characterized in that, Comprising the following steps: The near-infrared second-region organic fluorescent probe described in any one of claims 1 - 4 or the near-infrared second-region organic fluorescent probe prepared by the preparation method described in any one of claims 5 - 7, an organic coating agent and an organic solvent are mixed to obtain a mixed solution; The mixed solution is mixed with water and subjected to ultrasonic co-precipitation to obtain fluorescent probe-coated nanoparticles.

12. The preparation method according to claim 11, wherein, The power of the ultrasonic co-precipitation is 100 - 200 W, and the time is 3 - 10 min.

13. Use of the near-infrared second-region organic fluorescent probe described in any one of claims 1 - 4 or the near-infrared second-region organic fluorescent probe prepared by the preparation method described in any one of claims 5 - 7 or the fluorescent probe-coated nanoparticles described in any one of claims 8 - 10 or the fluorescent probe-coated nanoparticles prepared by the preparation method described in claim 11 or 12 in the preparation of a biological imaging contrast agent.

14. Use of the near-infrared II organic fluorescent probe according to any one of claims 1 to 4, or the near-infrared II organic fluorescent probe prepared by the preparation method according to any one of claims 5 to 7, or the fluorescent probe-coated nanoparticle according to any one of claims 8 to 10, or the fluorescent probe-coated nanoparticle prepared by the preparation method according to claim 11 or 12 in fluorescence imaging for non-diagnostic and non-therapeutic purposes.

15. The application according to claim 14, wherein The fluorescence imaging is carried out under white light excitation, and the wavelength range of the white light excitation is 400-800 nm.

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