Highly specific in situ lipoprotein-binding near-infrared II dyes, their preparation methods and applications

Abstract

The present application relates to the technical field of near-infrared imaging probe, and particularly relates to a high-specificity in-situ lipoprotein binding near-infrared two-region dye, a preparation method and application thereof, and the near-infrared two-region dye has a structure shown in formula I. The dye compound provided by the present application can form a stable complex with endogenous lipoprotein in-situ rapidly in a complex physiological environment in vitro or in vivo with high specificity, and the brightness of the dye can be restored and the biocompatibility can be enhanced, the binding kinetics with the lipoprotein can be regulated systematically to realize controllable vascular imaging time window; and the abnormal lipid metabolism diseases such as fatty liver, atherosclerosis and the like can be monitored and traced early.

Description

Technical Field

[0001] This invention relates to the field of near-infrared imaging probe technology, and in particular to a highly specific in-situ lipoprotein-binding near-infrared II dye, its preparation method, and its application. Background Technology

[0002] Lipid metabolism abnormalities and chronic inflammation are core mechanisms driving the development of metabolic syndrome-related diseases such as atherosclerosis, non-alcoholic fatty liver disease, and cardiovascular disease. Accurate identification and characterization of lipid-rich lesions in these diseases (such as atherosclerotic plaques and intrahepatic lipid deposits) are crucial for early intervention and assessment of treatment efficacy. However, current imaging techniques commonly used in clinical practice and research, such as ultrasound, computed tomography (CT), and magnetic resonance imaging (MRI), while providing important information in disease monitoring, have significant limitations: they lack sufficient sensitivity and specificity in the early stages of disease, making it difficult to accurately capture subtle lipid accumulation and inflammatory changes; their analysis is highly dependent on operator experience; and some of these techniques use ionizing radiation, posing additional risks to patients / animal models. Therefore, the development of next-generation molecular imaging probes or functional imaging agents is particularly urgent. These contrast agents should be safe, non-invasive, highly specific, and directly visualize lipid-related pathological changes, which is of great value for understanding disease mechanisms, especially suitable for imaging needs in laboratory small animal models.

[0003] Near-infrared II (NIR-II, 900-17000 nm) imaging technology, with its superior tissue penetration and extremely low autofluorescence, has become an innovative technique for achieving high-resolution visualization of deep in vivo tissues. In recent years, antibody-conjugated NIR-II probes have successfully achieved targeted detection of atherosclerotic plaques in animal models. However, these probes have significant limitations, such as instability in body fluid environments and non-specific uptake by the immune system triggered by immunogenicity, leading to strong non-targeted signals, reduced imaging signal-to-noise ratio, and severely limiting their application potential.

[0004] Lipoproteins, as the primary carriers of lipid transport, directly participate in numerous physiological and pathological processes, playing a crucial role, especially in cardiovascular diseases such as atherosclerosis. The hydrophilic phospholipid monolayer shell and hydrophobic core of lipoproteins make them ideal platforms for delivering hydrophobic drugs and contrast agents. Furthermore, the various apolipoproteins on the lipoprotein surface enable them to recognize lipoprotein receptors in multiple tissues and organs. These receptors are widely expressed in various tissues, particularly exhibiting overexpression in some tumor and non-tumor diseases. Currently, research on lipoproteins for drug delivery and therapy mainly focuses on purifying lipoproteins isolated from human plasma and then binding them to drugs in a non-physiological environment (recombinant lipoproteins); or designing synthetic nanoparticles with lipoprotein-like structures (synthetic lipoproteins). However, these methods face significant challenges: 1. The isolation and purification process of natural lipoproteins is complex and costly, and the effective modification of the whole lipoprotein or the selective extraction of specific components is technically difficult. 2. Synthetic lipoproteins cannot completely replicate the precise structure and biocompatibility of natural lipoproteins. These production complexities and high costs severely hinder their large-scale industrial production and clinical application. Given that most clinically used imaging probes are small molecules, developing small molecule probes that can directly and specifically target different classes of lipoproteins has become crucial in this field. Such probes offer significant advantages: 1. Their targeting ability is independent of large molecule antibodies, effectively avoiding their inherent immunogenicity and poor biodistribution; 2. They enable real-time, dynamic visualization and tracking of lipoprotein metabolic dynamics in vivo; and can intuitively reveal lipoprotein transport abnormalities directly related to the formation of atherosclerotic plaques and the pathogenesis of other cardiovascular diseases, providing a new and powerful tool for early diagnosis and efficacy assessment. Therefore, developing near-infrared II dyes suitable for in vivo in situ lipoprotein targeting, enabling visualization of lipid metabolism disorders such as fatty liver and atherosclerosis, has significant basic research and clinical implications. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a highly specific in situ lipoprotein-binding near-infrared II dye, its preparation method and application.

[0006] To achieve the above objectives, the present invention provides a highly specific in-situ lipoprotein-binding near-infrared II dye having the structure shown in Formula I:

[0007]

[0008] Formula I;

[0009] Wherein, R is selected from the structure shown in Equation II or Equation III:

[0010]

[0011] Formula II Formula III

[0012] n is any integer from 2 to 45;

[0013] R1 is a hydroxyl group or is linked to an amino acid or peptide;

[0014] R2 is It may be linked to amino acids or peptides.

[0015] In this invention, when the R structure is of formula II, the dye compound is denoted as FlavX, where X=n; when the R structure is of formula III, the dye compound is denoted as FlavA, where A is an abbreviation for a modifying group (amino acid or peptide).

[0016] In Formula II, n represents the number of polyethylene glycol repeating units. In some specific embodiments, n is 2, 4, 6, 8, 10, 12, 13, 14, 20, 30, 40 or 45, or any of the above values ​​as the upper or lower limit.

[0017] In some specific embodiments, A is a cyclic peptide c (RGDfC), and based on this, the dye compound has the following structure:

[0018] .

[0019] This invention also provides a method for preparing the above-mentioned highly specific in-situ lipoprotein-bound near-infrared II dye, comprising:

[0020] S1) Compound a undergoes a condensation reaction with mPEGn-NH2 to give compound b;

[0021] S2) Compound b reacts with Flav to obtain the near-infrared II dye compound shown in Formula I;

[0022]

[0023]

[0024] In Equation I, R is selected from the structure shown in Equation II;

[0025] In mPEGn-NH2 and compound b, n represents the number of polyethylene glycol repeating units, preferably any integer from 2 to 45; in some specific embodiments, n is 2, 4, 6, 8, 10, 12, 13, 14, 20, 30, 40 or 45, or a range of values ​​with the above values ​​as the upper or lower limit.

[0026] Optionally, in the condensation reaction of S1), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) is used as the condensation reagent.

[0027] Optionally, in the condensation reaction of S1), N,N-diisopropylethylamine (DIPEA) is used as a catalyst.

[0028] Optionally, in the condensation reaction of S1), tetrahydrofuran is used as the reaction solvent.

[0029] Optionally, in the reaction of S2), DIPEA is used as a catalyst.

[0030] Optionally, in the reaction described in S2), dimethyl sulfoxide (DMSO) is used as the reaction solvent;

[0031] Optionally, in the reaction of S2), the reaction temperature is 60~80℃, preferably 70℃; and the reaction time is 0.5~1h.

[0032] In some specific embodiments, the above preparation method is as follows:

[0033] Compound a and HATU were dissolved in tetrahydrofuran and reacted thoroughly with stirring at room temperature. DIPEA and mPEGn-NH2 were then dissolved in tetrahydrofuran and added dropwise to the reaction system. The mixture was stirred overnight at room temperature, and after purification, compound b was obtained. Compound b and Flav were dissolved in dimethyl sulfoxide and, after thorough dissolution, DIPEA was added. The reaction mixture was then heated to 70°C. o The reaction was stirred at C for 0.5-1 hour, and the product FlavX was obtained after purification.

[0034] When R is selected from the structure shown in Formula II, the preparation method of the highly specific in-situ lipoprotein-bound near-infrared II dye may further include:

[0035] SS1) Compound a reacts with Flav to give the intermediate FlavCOOH;

[0036] The intermediate FlavCOOH and mPEGn-NH2 undergo a condensation reaction to obtain the near-infrared II dye compound shown in Formula I;

[0037]

[0038] .

[0039] In mPEGn-NH2, n represents the number of polyethylene glycol repeating units, preferably any integer from 2 to 45; in some specific embodiments, n is 2, 4, 6, 8, 10, 12, 13, 14, 20, 30, 40 or 45, or any of the above values ​​as the upper or lower limit.

[0040] Optionally, in the reaction of SS1), dimethyl sulfoxide is used as the reaction solvent;

[0041] Optionally, in the reaction of SS1), the reaction temperature is 60~80℃, preferably 70℃; the reaction time is 0.5~1h.

[0042] Optionally, HATU is used as the condensation reagent in the condensation reaction of SS2.

[0043] Optionally, in the reaction of SS2), DIPEA is used as a catalyst.

[0044] Optionally, in the reaction of SS2), tetrahydrofuran is used as the reaction solvent.

[0045] In some specific embodiments, the above preparation method is as follows:

[0046] Compound a and Flav dissolved in DMSO, 70 o The reaction was stirred at C for 0.5-1 hours, and the product FlavCOOH was obtained after purification. FlavCOOH and HATU were dissolved in tetrahydrofuran and stirred at room temperature to react fully. DIPEA and mPEGn-NH2 were dissolved in tetrahydrofuran and added dropwise to the reaction system. The reaction was stirred at room temperature overnight, and the dye FlavX was obtained after purification.

[0047] When R is selected from the structure shown in Formula III, the preparation method of the highly specific in-situ lipoprotein-bound near-infrared II dye includes:

[0048] Compound c and compound Flav undergo a condensation reaction to obtain the near-infrared II dye compound shown in Formula I.

[0049] .

[0050] R1 is a hydroxyl group or is linked to an amino acid or peptide;

[0051] R2 is It may be linked to amino acids or peptides.

[0052] Optionally, the condensation reaction uses dimethyl sulfoxide as the reaction solvent;

[0053] The reaction temperature is 60~80℃, preferably 70℃; the reaction time is 0.5~1h.

[0054] In some specific embodiments, the above preparation method is as follows:

[0055] Compound c and compound Flav were dissolved in DMSO and reacted with stirring at 70°C for 0.5-1 hour. After purification, the product FlavA was obtained, where A represents amino acids or short peptides containing thiol groups.

[0056] This invention also provides the application of the above-mentioned highly specific in situ lipoprotein-binding near-infrared II dye in the preparation of fluorescent probes.

[0057] Optionally, the fluorescent probe may be a reagent for binding purified lipoproteins in vitro or for binding endogenous lipoproteins in situ with high specificity in vivo, and / or a reagent for regulating lipoprotein binding kinetics and vascular imaging time window, and / or a reagent for monitoring the early progression of fatty liver, and / or a reagent for in situ imaging of atherosclerotic plaques and long-term plaque labeling.

[0058] The dye described in this invention can bind to purified lipoproteins in vitro or bind to endogenous lipoproteins in situ in vivo with high specificity.

[0059] The dye described in this invention can regulate the in vivo and in vitro lipoprotein binding dynamics and vascular imaging time window by changing the hydrophilicity of its hydrophilic fragments; when the hydrophilicity of the dye is enhanced, the dye binding efficiency and binding speed with lipoproteins become faster, the blood vessels light up quickly and are cleared from the blood more rapidly.

[0060] The experimental results show that when R is one of the optional groups mentioned above, the selectivity of the dye binding to lipoprotein is not affected by the change in the R structure; however, when the R group is another non-hydrophilic group (-Cl, -Br, -CH3, etc.), the dye does not have the property of binding to lipoprotein.

[0061] When R is one of the optional groups mentioned above, the optical properties of the dye are basically unaffected by changes in the structure of R.

[0062] The affinity and binding efficiency of dyes to lipoproteins are related to the type of R. When the R structure is I, the affinity and binding efficiency of dye molecules to lipoproteins increase with the increase of PEG chain length (increase in hydrophilicity). When the R structure is II, the affinity and binding efficiency of dye molecules to lipoproteins increase with the increase of hydrophilicity of the R structure, but the increase is not significant.

[0063] This invention uses methyl polyethylene glycol derivatives, amino acids, or short peptides with thiol groups as nucleophilic substitution reaction sites. The presence of S heteroatoms does not cause a blue shift in the absorption and emission peaks of the molecules.

[0064] This invention relates to dyes that can bind to plasma lipoproteins in vitro or in situ in vivo to form complexes. These complexes exhibit significantly enhanced luminescence, increased photostability, higher resolution bioimaging capabilities, and excellent biocompatibility compared to single small molecules. Furthermore, the imaging time window can be adjusted based on the different binding rates between dyes with different structures and lipoproteins. Therefore, the highly specific lipoprotein-binding dyes described in this invention can be used in the preparation of high-resolution angiography agents with adjustable imaging time windows, including but not limited to high-resolution imaging of mouse hind limb vessels.

[0065] This invention relates to dyes that selectively bind to lipoproteins in complex physiological environments (such as plasma), unaffected by other proteins, and exhibit significantly enhanced fluorescence after binding to lipoproteins, thus possessing the ability to target imaging and in vivo tracing of lipoprotein metabolic diseases. Therefore, the highly specific lipoprotein in situ binding dyes described in this invention can be used in the preparation of reagents for monitoring lipoprotein metabolic diseases, including but not limited to precise imaging of early fatty liver in mice and atherosclerotic plaques in the aorta.

[0066] Compared with the prior art, the beneficial effects of the present invention are:

[0067] 1. The dye provided by this invention achieves selective binding of lipoproteins under complex physiological conditions by modifying existing dyes, and regulates the binding kinetics of the dye and lipoproteins by adjusting the size of the hydrophilic fragment, thereby achieving highly selective, rapid and efficient binding of endogenous lipoproteins in vitro and in the complex internal environment of organisms.

[0068] 2. The complex formed by the dye and lipoprotein provided by the present invention has extremely strong fluorescence enhancement (compared to the fluorescence brightness of the dye in the aqueous phase) and effectively increases the photostability of the dye, while reducing the biotoxicity of the dye.

[0069] 3. The dye provided by this invention can monitor the early development of fatty liver in mice by binding to endogenous lipoproteins in situ with high specificity.

[0070] 4. The dye provided by this invention can achieve long-term labeling (greater than 10 days) of atherosclerotic plaques in mice by binding to endogenous lipoproteins in situ with high specificity. Attached Figure Description

[0071] Figure 1 Figure 1 shows the synthetic routes of dyes FlavX and FlavA in Example 1; Figure 2 shows the synthetic route of dye FlavX; Figure 3 shows the synthetic route of dye FlavA; Figure 4 shows the synthetic route of dye FlavAc.

[0072] Figure 2 For the compound FlavCOOH 1 H-NMR spectrum;

[0073] Figure 3 For compound Flav1 1 H-NMR spectrum;

[0074] Figure 4 For compound Flav3 1 H-NMR spectrum;

[0075] Figure 5 For compound Flav7 1 H-NMR spectrum;

[0076] Figure 6 For compound Flav9 1 H-NMR spectrum;

[0077] Figure 7 For compound Flav12 1 H-NMR spectrum;

[0078] Figure 8 For compound Flav45 1 H-NMR spectrum;

[0079] Figure 9 For the compound FlavAc 1 H-NMR spectrum;

[0080] Figure 10 For the compound FlavRGD 1 H-NMR spectrum;

[0081] Figure 11 The Maldi-Tof mass spectrum of compound Flav1;

[0082] Figure 12 This is the Maldi-Tof mass spectrum of compound Flav3;

[0083] Figure 13 The Maldi-Tof mass spectrum of compound Flav7;

[0084] Figure 14 The Maldi-Tof mass spectrum of compound Flav9;

[0085] Figure 15 The Maldi-Tof mass spectrum of compound Flav12;

[0086] Figure 16 The Maldi-Tof mass spectrum of compound Flav45;

[0087] Figure 17 This is the high-resolution mass spectrum of the compound FlavAc;

[0088] Figure 18 This is the high-resolution mass spectrum of the compound FlavRGD;

[0089] Figure 19 The absorption and emission spectra of the dye in DMSO and PBS;

[0090] Figure 20The fluorescence retention rates of some dyes in different solvents are shown; fluorescence retention rate = (fluorescence intensity of dye in a certain solvent / fluorescence intensity of dye in DMSO) * 100%; the dye concentration is 10 μM.

[0091] Figure 21 Example 3 shows the dye's high specificity targeting of plasma lipoproteins; where a represents the dye being uniformly mixed with different blood components at 50... o The fluorescence intensity after incubation at C for 2 hours and the separation results of different dye-labeled lipoprotein components by agarose gel electrophoresis after Sudan Black staining; b shows the results of the dye being thoroughly mixed with serum from different species and incubated at 50°C. o The fluorescence intensity after incubation at C for 2 hours and the separation results of agarose gel electrophoresis after Sudan Black staining of different species serum labeled with dye; the dye concentration was 10 μM.

[0092] Figure 22 The dyes and different types of lipoproteins in the in vitro environment and mouse serum 37 in Example 4 o Changes in fluorescence intensity at different time points after incubation at C; the dye concentration was 10 μM for all conditions.

[0093] Figure 23 This section describes the ability of the dye to form complexes with endogenous lipoproteins in situ and the regulation of the vascular imaging time window in Example 4; where a is an image of the optimal imaging time point for the hind limb vessels of mice after tail vein injection of different dyes; b is a change in the fluorescence intensity of mouse blood over time after tail vein injection of different dyes.

[0094] Figure 24 The images show fluorescence images of the livers of control mice and mice fed a high-fat diet for different durations 48 hours after tail vein injection of dye in Example 5.

[0095] Figure 25 This is an in situ imaging and long-term labeling of atherosclerotic plaques by the dye in Example 6; where a is an in situ imaging image of aortic arch plaques in mice 24 hours after dye injection; b is an in vitro imaging image of the mouse aorta at different times after dye injection and statistical analysis of fluorescence signals in normal tissues and plaques; Detailed Implementation

[0096] To further illustrate the present invention, a detailed description is provided below with reference to embodiments. However, it should be understood that these descriptions are merely for further illustrating the features and advantages of the present invention, and not for limiting the scope of the claims.

[0097] There are no particular restrictions on the source of any raw materials used in this invention; they can be purchased from the market or prepared using conventional methods known to those skilled in the art.

[0098] Example 1: Synthesis of the dye FlavX

[0099] The synthetic route of dye FlavX is as follows Figure 1 As shown in Figure a, the preparation method specifically includes the following steps:

[0100] (1) Compound a (1 mmol) and mPEG-NH2 (1 mmol) were completely dissolved in tetrahydrofuran (0.1 M) and cooled to 0. o Stirring at C for half an hour, EDC and DMAP were uniformly dispersed in tetrahydrofuran (0.1M) and added to the reaction system. After stirring for another half hour, the mixture was brought back to room temperature and the reaction continued for 4 hours. After the reaction was completed, the solvent was removed by rotary evaporation to obtain the crude product compound b, which was directly used for the next reaction without purification.

[0101] (2) Compound b (1.2 mmol) and Flav (1 mmol) were dissolved in anhydrous DMSO (0.1 M) and then heated at 70 °C. o The reaction proceeds at C for 0.5–1 hour. After removing the reaction solvent, the crude product is purified by silica gel column chromatography (dichloromethane / methanol) to obtain the purple product FlavX (yield approximately 40%).

[0102] Synthetic route two for dye FlavX Figure 1 As shown in Figure b, the preparation method specifically includes the following steps:

[0103] (1) Compound Flav was obtained based on the method proposed in the invention patent with authorization announcement number CN117164845B. Compound a (3 mmol) and diisopropylethylamine (522 μL, 3 mmol) were added to a dry flask, and anhydrous DMSO (0.1 M) was added. The mixture was stirred at room temperature for 30 minutes. Then Flav (1 mmol) was added to the mixture, and the reaction was continued to be carried out under inert gas protection at 70°C for 0.5-1 hours. The crude product was purified by silica gel column chromatography (dichloromethane / methanol) to give the purple product FlavCOOH (yield of about 50%).

[0104] (2) FlavCOOH (1 mmol) and HATU (1 mmol) were added to a dry flask, followed by 0.1 M tetrahydrofuran solvent, and stirred at room temperature for 30 minutes. mPEG-NH2 and DIPEA were completely dissolved in tetrahydrofuran (0.1 M) and then slowly added dropwise to the reaction system. The reaction was carried out overnight at room temperature under inert gas protection. The crude product was purified by silica gel column chromatography (dichloromethane / methanol) to give the purple product FlavX (yield approximately 80%).

[0105] Example 2 Synthesis of dye FlavA (A refers to thiol-containing amino acids or short peptides)

[0106] Taking the dye FlavAc as an example, the synthetic route is as follows: Figure 1As shown in Figure c, the preparation method specifically includes the following steps:

[0107] Compound Flav (1 mmol) and acetylated cysteine ​​(Ac, 1 mmol) were dissolved in 0.1 M anhydrous DMSO at 70 °C. o The reaction proceeds for 0.5-1 hour. After the reaction is complete, the solvent is removed, and the crude product is purified by silica gel column chromatography (dichloromethane / methanol) to obtain the purple product FlavAc (yield approximately 50%).

[0108] The proton NMR spectra of the lipoprotein selective in situ binding dyes (FlavX, FlavA) prepared in this invention ( 1 H-NMR and mass spectrometry data are shown in [reference]. Figures 2-18 .

[0109] Example 3 Optical Properties

[0110] like Figure 19 As shown, the dyes involved in this invention have absorption and emission peaks in the near-infrared II region in DMSO and phosphate buffered solution (PBS), and the changes in the different R structures hardly alter the spectral properties of the dyes.

[0111] like Figure 20 As shown, the dyes involved in this invention exhibited different fluorescence retention rates in DMSO, PBS, bovine serum albumin (BSA), high-density lipoprotein (HDL), low-density lipoprotein (LDL), and mouse serum (MS) (with the fluorescence intensity of the dye in DMSO as a reference). Specifically, the dyes involved in this invention showed a fluorescence retention rate of 30% to 50% in HDL, LDL, and MS, while almost no fluorescence retention was observed in PBS and BSA. As a control, Flav showed no fluorescence retention in any system.

[0112] Example 4: Validation of the high specificity of dye targeting plasma lipoproteins

[0113] This embodiment uses FlavAc as an example to illustrate that the dye involved in this invention can bind lipoproteins with high specificity in an in vitro environment, and bind lipoproteins with high selectivity in a complex environment of multi-species serum.

[0114] Experiment 1: FlavAc was dissolved in DMSO to prepare a 2mM stock solution. The stock solution was then added to equal volumes of DMSO, PBS, whole blood, blood cells, plasma, serum, mouse albumin (MSA), mouse immunoglobulin G (IgG), HDL, LDL, and a mixture of HDL and LDL, and mixed thoroughly. The final dye concentration for all solutions was 10μM. To ensure complete reaction, all systems were kept at 50°C. oThe reaction was carried out at C for 2 hours. After the reaction, the serum and different lipoprotein systems were separated by agarose gel electrophoresis.

[0115] The fluorescence intensity of each system was obtained using a near-infrared camera, such as... Figure 21 As shown in Figure a, the dye involved in this invention exhibits high fluorescence enhancement in whole blood, plasma, serum, and various lipoproteins. Electrophoretic separation results show that the dye-labeled fluorescent bands highly overlap with the Sudan Black-labeled lipoprotein bands.

[0116] Experiment 2: FlavAc was dissolved in DMSO to prepare a 2mM stock solution. The dye stock solution was then added to DMSO, mouse serum, rat serum, pig serum, rabbit serum, and fetal bovine serum, respectively, and mixed thoroughly. The final dye concentration for each solution was 10μM. To ensure reaction reproducibility, all the above systems were kept at 50°C. o The reaction was carried out at C for 2 hours. Separation was performed by agarose gel electrophoresis.

[0117] The fluorescence intensity of each system was obtained using a near-infrared camera, such as... Figure 21 As shown in Figure b, the dye involved in this invention exhibits high fluorescence enhancement in the serum of different species. Electrophoretic separation results show that the dye-labeled fluorescent bands highly overlap with the Sudan Black-labeled lipoprotein bands.

[0118] The above experiments demonstrate that the dye involved in this invention can specifically bind to lipoproteins, and this binding process is accompanied by significant fluorescence enhancement. In complex physiological environments (such as blood), the dye exhibits high selectivity for lipoproteins, and its binding behavior is not affected by other components in the blood. Furthermore, the binding of this dye to lipoproteins is cross-species applicable, and no significant interspecies differences were observed, indicating that its binding ability maintains stable performance across different species.

[0119] Example 5: Regulation of the time window for in vivo vascular imaging by controlling the binding kinetics of dyes to lipoproteins in vitro and by binding endogenous lipoproteins in situ in vivo.

[0120] Figure 22 The present invention provides dyes involved in the invention that react with HDL, LDL and MS at 37°C. o The fluorescence intensity curve changes with incubation time at C, and the final dye concentration in the system is 10 μM. In vitro studies confirm that the enhanced hydrophilicity of the hydrophilic fragment of this dye effectively improves the binding efficiency of the dye to single lipoproteins and serum lipoproteins.

[0121] like Figure 23As shown, after tail vein injection of FlavX (200 μL, 600 μM), the blood vessels of the mouse hind limb were imaged at a wavelength of 1200 nm using a 980 nm laser excitation. The fluorescence intensity of the blood at different time points after injection (excited by a 980 nm laser) was also obtained. This demonstrates that the dyes involved in this invention all possess excellent vascular imaging capabilities, and that the circulation time of the dye in the blood is affected by the hydrophilicity of the dye's hydrophilic fragments. With increasing hydrophilicity, the dye rapidly binds to endogenous lipoproteins in the organism and is quickly cleared from the blood.

[0122] Example 6: Highly specific in-situ binding of dyes to lipoproteins enables early monitoring of fatty liver.

[0123] Based on the fact that the dye involved in this invention can achieve rapid and highly selective binding to endogenous lipoproteins in organisms, this dye can be used to monitor the early development of fatty liver in mice.

[0124] A mouse model of early fatty liver was established: C57 mice (8-10 weeks old, with an average weight of 20g) were randomly divided into four groups. One group served as the control group and was fed a normal diet. The other three groups were fed a high-fat diet for 2, 4, and 6 weeks, respectively. Fat accumulation in the mouse liver was confirmed by monitoring mouse body weight and staining of isolated liver sections.

[0125] Figure 24 The imaging schematic and fluorescence accumulation in the livers of mice in different groups 48 hours after FlavAc injection are provided, confirming that the dye accumulates with increasing fat content in the mouse liver, accompanied by an enhancement of fluorescence signal.

[0126] Example 7: Highly specific in-situ binding of dyes to lipoproteins enables in-situ targeted imaging and high-resolution long-term labeling of atherosclerotic plaques.

[0127] Establishing a mouse model of atherosclerosis: ApoE - / - Mice (8-10 weeks old), with an average weight of 20g, were fed a high-fat diet for 8-12 weeks. The occurrence of atherosclerotic plaques was confirmed by staining of mouse aortic arch tissue.

[0128] Figure 25 Figure a shows an in situ image of the plaque at the aortic arch of a mouse 24 hours after dye injection. The plaque is clearly visible. The mice were euthanized and given adequate cardiac perfusion before imaging. Figure 25 Figure b in the middle section provides in vitro bright-field imaging and NIR-II imaging of mouse aortic arch tissue at different time points after dye injection, and provides statistical analysis of the fluorescence intensity of plaques and normal tissues, demonstrating that the present invention has the ability of dyes to label plaques for a long time.

[0129] The above experimental results show that the dye compounds provided by this invention can form stable complexes with endogenous lipoproteins in situ rapidly and with high specificity in complex physiological environments in vitro or in vivo, thereby restoring dye brightness and enhancing biocompatibility. They can also systematically regulate the binding kinetics with lipoproteins to achieve controllable vascular imaging time windows, and can be used for early monitoring and tracing of lipid metabolism disorders such as fatty liver and atherosclerosis.

[0130] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent.

Claims

1. A high specificity in situ lipoprotein binding near-infrared two-region dye having a structure shown in Formula I: Formula I; R is selected from a structure shown in Formula III: Formula III R1 is hydroxyl.

2. A preparation method of the high specificity in situ lipoprotein binding near-infrared two-region dye according to claim 1, comprising: performing a condensation reaction of compound c and compound Flav to obtain a near-infrared two-region dye compound shown in Formula I; in Formula I, R is selected from a structure shown in Formula III; R1 is hydroxyl; the condensation reaction uses dimethyl sulfoxide as a reaction solvent; the reaction temperature is 60-80°C, and the reaction time is 0.5-1h. wherein 4. Application of the high specificity in situ lipoprotein binding near-infrared two-region dye according to claim 1 in preparation of a fluorescent probe. The fluorescent probe is a reagent for binding to purified lipoprotein in vitro or for high specificity in situ binding to endogenous lipoprotein in vivo, and / or a reagent for regulating lipoprotein binding kinetics and vascular imaging time window, and / or a reagent for monitoring early fatty liver development process, and / or a reagent for in situ imaging of atherosclerotic plaque and long-term labeling of the plaque. ​ R2 is . ​ ​ ​ ​ R2 is .

3. The preparation method according to claim 2, characterized in that, ​ ​ ​ 5. Use according to claim 4, characterized in that, ​

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